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Characterization of the specific ligand-receptor interactions between rod outer segments and retinal… Laird, Dale W. 1988

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CHARACTERIZATION OF THE SPECIFIC LIGAND-RECEPTOR INTERACTIONS BETWEEN ROD OUTER SEGMENTS AND RETINAL PIGMENT EPITHELIAL CELLS by Dale W. L a i r d B . S c , U n i v e r s i t y o f P r i n c e Edward I s l a n d , 1982 M . S c , U n i v e r s i t y o f B r i t i s h Columbia , 1984 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY i n THE FACULTY OF GRADUATE STUDIES DEPARTMENT OF BIOCHEMISTRY FACULTY OF MEDICINE UNIVERSITY OF BRITISH COLUMBIA We accept t h i s t h e s i s as conforming to the r e q u i r e d s tandard THE UNIVERSITY OF BRITISH COLUMBIA A p r i l , 1988 © Dale W. L a i r d In presenting this thesis in partial fulfilment of the requirements for an advanced degree at the University of British Columbia, I agree that the Library shall make it freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the head of my department or by his or her representatives. It is understood that copying or publication of this thesis for financial gain shall not be allowed without my written permission. Department of V^jXdh The University of British Columbia 1956 Main Mall Vancouver, Canada V6T 1Y3 Date jrfty s/tf DE-6(3/81) ABSTRACT An i n v i t r o phagocytos i s assay system was developed and c h a r a c t e r i z e d f o r s t u d y i n g the s p e c i f i c receptor-media ted phagocytos i s o f bovine ROS by bov ine RPE c e l l s . The phagocytos i s o f ROS was de tec ted q u a l i t a t i v e l y by e l e c t r o n microscopy and q u a n t i t a t i v e l y by t r e a t i n g RPE c e l l s w i t h r a d i o i o d i n a t e d ROS or by p r o b i n g ROS-treated RPE c e l l s w i t h a r a d i o l a b e l e d a n t i r h o d o p s i n monoclonal a n t i b o d y . The b i n d i n g s i t e s f o r v a r i o u s a n t i r h o d o p s i n monoclonal a n t i b o d i e s were l o c a l i z e d as an e s s e n t i a l s tep i n t h e i r a p p l i c a t i o n as immunochemical probes f o r a n a l y s i s o f the s t r u c t u r e and f u n c t i o n o f r h o d o p s i n . F i v e monoclonal a n t i b o d i e s r a i s e d aga i n s t rhodops in have been shown to be d i r e c t e d aga in s t the N - t e r m i n a l r eg ions on the b a s i s o f t h e i r r e a c t i v i t y to an immunoaf f in i ty p u r i f i e d 2-39 g l y c o p e p t i d e , a 2-16 t r y p t i c g l y c o p e p t i d e and a 1-16 s y n t h e t i c pep t ide as measured by radioimmune c o m p e t i t i o n assays . L i m i t e d p r o t e o l y s i s , immunogold-dextran l a b e l i n g and c o m p e t i t i v e i n h i b i t i o n s t u d i e s i d e n t i f i e d two a n t i r h o d o p s i n monoclonal a n t i b o d i e s which bound to i n t e r n a l c y t o p l a s m i c loop reg ions o f r h o d o p s i n . F i n a l l y , the b i n d i n g s i t e s f o r these and other C-t e r m i n a l s p e c i f i c a n t i r h o d o p s i n monoclonal a n t i b o d i e s were used to e l u c i d a t e the proposed transmembrane h e l i c a l model o f r h o d o p s i n . An a n t i r h o d o p s i n monoclonal ant ibody (rho 4D2), which bound to rhodops in i n g l u t a r a l d e h y d e - f i x e d ROS plasma membranes, was employed as an immunocytochemical probe i n s t u d y i n g the p o s s i b l e r o l e o f rhodops in i n the b i n d i n g and phagocytos i s o f rod outer segments. An immunoaf f in i ty p u r i f i e d 2-39 N - t e r m i n a l rhodops in g l y c o p e p t i d e , a s y n t h e t i c 1-16 pept ide analogue of rhodops in and p h o s p h o l i p i d v e s i c l e s r e c o n s t i t u t e d w i t h rhodops in were a l l 125 found to be i n e f f e c t i v e i n i n h i b i t i n g the phagocytos i s o f I - l a b e l e d ROS - i i i -by RPE c e l l s . I n essence, these r e s u l t s p r o v i d e d c o m p e l l i n g evidence t h a t r h o d o p s i n i n the ROS plasma membrane does not f u n c t i o n as the l i g a n d f o r r e c o g n i t i o n by RPE c e l l s . The m o l e c u l a r p r o p e r t i e s o f the ROS c e l l sur face l i g a n d ( s ) , which are i n v o l v e d i n r e c o g n i t i o n by bovine RPE c e l l s , were s t u d i e d by l i m i t e d -p r o t e o l y t i c d i g e s t i o n i n c o n j u n c t i o n w i t h q u a n t i t a t i v e phagocytos i s assays . M i l d l y t r y p s i n - t r e a t e d ROS were found to be l e s s e f f e c t i v e l y phagocyt i zed than u n t r e a t e d ROS by bovine RPE c e l l s . Moreover, the g l y c o p o l y p e p t i d e s (34kD and 24kD) r e l e a s e d from the ROS c e l l sur face by t r y p s i n were capable o f i n h i b i t i n g ROS p h a g o c y t o s i s . The ROS plasma membrane s p e c i f i c , r i c i n -b i n d i n g , 230kD g l y c o p r o t e i n was observed by SDS-gel e l e c t r o p h o r e s i s and western b l o t t i n g to be h i g h l y t r y p s i n s e n s i t i v e under these c o n d i t i o n s . Hence, r i c i n a f f i n i t y chromatography and immunoaf f in i ty chromatography were employed i n an attempt to p u r i f y t h i s 230kD g l y c o p r o t e i n from ROS membranes. E n r i c h e d p r e p a r a t i o n s o f the 230kD g l y c o p r o t e i n were r e c o n s t i t u t e d i n t o p h o s p h o l i p i d v e s i c l e s and e f f e c t i v e l y used to i n h i b i t the phagocytos i s o f ROS by RPE c e l l s . I n summary, a ROS plasma membrane s p e c i f i c , 230kD g l y c o p r o t e i n has been i d e n t i f i e d and i s o l a t e d ; t h i s p r o t e i n may ac t as a l i g a n d i n s p e c i f i c l i g a n d - r e c e p t o r i n t e r a c t i o n s between ROS and RPE c e l l s . - i v -LIST OF ABBREVIATIONS BSA bovine serum albumin CHAPS 3- [ (3-cho lamidopropy l ) dimethylammonio]-1-propanesulfonate Con A c o n c a n a v a l i n A DMSO d i m e t h y l s u l f o x i d e dPBS Dulbecco ' s phosphate-buf fered s a l i n e EDTA - e thylenediamine t e t r a a c e t a t e FCS f e t a l c a l f serum IMDM I s c o v e ' s m o d i f i e d Dulbecco ' s medium I g immunoglobulin M r e l a t i v e molecu la r weight PBS -- phosphate-buf fered s a l i n e PMSF - phenylmethyl s u l f o n y l f l u o r i d e RIA radioimmune assay RCA R i c i n u s communis a g g l u t i n i n ROS rod outer segments RPE r e t i n a l pigment e p i t h e l i u m S .D. s tandard d e v i a t i o n SDS sodium dodecyl s u l f a t e SEM scanning e l e c t r o n microscopy STEM scanning t r a n s m i s s i o n e l e c t r o n microscopy TBS T r i s - b u f f e r e d s a l i n e TEM t r a n s m i s s i o n e l e c t r o n microscopy TRIS t r i s (hydroxymethyl) aminomethane -v-TABLE OF CONTENTS Page ABSTRACT i i LIST OF ABBREVIATIONS i v TABLE OF CONTENTS v LIST OF TABLES x LIST OF FIGURES x i ACKNOWLEDGEMENTS x v i CHAPTER 1 INTRODUCTION 1. R e t i n a 1 2. Photoreceptor c e l l s 1 3. S t r u c t u r e o f rod outer segments 4 4 . Rod outer segment d i s c renewal and d i s c shedding 6 5 . Rhodopsin a) . L o c a l i z a t i o n and b i o s y n t h e s i s 8 b) . P u r i f i c a t i o n and molecu la r p r o p e r t i e s 9 c) . P o s t - t r a n s l a t i o n a l m o d i f i c a t i o n s o f rhodops in 10 d) . Rhodopsin f u n c t i o n i n v i s u a l t r a n s d u c t i o n 11 e) . Probes o f rhodops in s t r u c t u r e and topography 11 f ) . A model o f rhodops in s t r u c t u r e i n d i s c membrane 14 6. R e t i n a l pigment e p i t h e l i a l c e l l s t r u c t u r e and f u n c t i o n 17 7. C u l t u r i n g o f RPE c e l l s 17 8. Process o f phagocytos i s 21 9. RPE phagocytos i s assays 22 10. Phagocyt i c de fec t s i n the RPE c e l l s 24 11. The r o l e o f the c y t o s k e l e t a l system i n phagocytos i s 25 - v i -12. The r o l e o f carbohydrates i n ROS/RPE i n t e r a c t i o n s 26 13. Irranunocytochemical l a b e l i n g s t u d i e s 28 14. Thes i s i n v e s t i g a t i o n 28 CHAPTER 2 MATERIALS AND METHODS MATERIALS 31 METHODS 1. P r o t e i n assays 32 2. P r e p a r a t i o n s o f ROS membranes 32 3. P r o t e o l y s i s o f ROS d i s c membranes 33 4 . SDS-gel e l e c t r o p h o r e s i s and g e l t r a n s f e r 34 5. Monoclonal ant ibody techniques a) . Immunizat ion and c e l l f u s i o n 35 b) . Hybridoma c l o n i n g , f r e e z i n g and a s c i t e s f o r m a t i o n 35 c) . Monoclonal ant ibody p u r i f i c a t i o n 36 d) . P r e p a r a t i o n and i o d i n a t i o n o f a n t i b o d i e s and l e c t i n s 37 e) . Standard radioimmune assay 37 f ) . So l id-phase radioimmune c o m p e t i t i o n assays 38 6. P o l y p e p t i d e d e t e c t i o n by monoclonal a n t i b o d i e s 39 7. P r e p a r a t i o n o f monoclonal antibody-Sepharose a f f i n i t y columns . . 40 8. Cyanogen bromide cleavage o f rhodops in 40 9. P u r i f i c a t i o n o f rhodops in pept ides and rhodops in 41 10. A n a l y s i s o f rhodops in pept ides by HPLC and amino a c i d a n a l y s i s . 42 11 . Immunocytochemical l a b e l i n g o f ROS membranes a) . P r e p a r a t i o n o f p r o t e i n g o l d - d e x t r a n conjugates 43 b) . Pre-embedding l a b e l i n g o f ROS membranes 44 c) . P r e p a r a t i o n o f membrane samples f o r TEM 45 - v i i -RESULTS 1. A n t i r h o d o p s i n monoclonal a n t i b o d i e s 46 2. I d e n t i f i c a t i o n o f rhodops in N - t e r m i n a l monoclonal a n t i b o d i e s . . . 47 3. I s o l a t i o n o f N - t e r m i n a l rhodops in pept ide s 50 4 . L o c a l i z a t i o n o f N - t e r m i n a l a n t i r h o d o p s i n ant ibody b i n d i n g s i t e s . 53 5 . E f f e c t o f b l e a c h i n g on monoclonal ant ibody b i n d i n g 57 6. E f f e c t o f f i x a t i o n on rho 4D2 b i n d i n g to ROS membranes 58 7. Immunocytochemical l a b e l i n g o f ROS membranes 61 8. Monoclonal a n t i b o d i e s aga ins t i n t e r n a l segments o f rhodops in . . . 63 9. P u r i f i c a t i o n o f the l ' - 3 1 ' C - t e r m i n a l pep t ide o f rhodops in 67 125 10. I n h i b i t i o n o f I - l a b e l e d rho 3A6 b i n d i n g by a n t i b o d i e s 71 11. P u r i f i c a t i o n o f rhodops in 75 DISCUSSION 78 CHAPTER 3 MATERIALS AND METHODS MATERIALS 88 METHODS 1. RPE i s o l a t i o n and t i s s u e c u l t u r e 88 2. P r e p a r a t i o n o f plasma membrane f r ee d i s c membranes 89 3. R a d i o i o d i n a t i o n o f ROS 90 4. A n t i r h o d o p s i n rho 4D2 and i t s FCab ' )^ fragment 90 5. Monoclonal ant ibody l a b e l i n g o f ROS 91 6. Phagocytos i s assay 92 7. E l e c t r o n microscopy 93 RESULTS 1. Morphology o f c u l t u r e d bovine RPE c e l l s 94 - v i i i -2. Phagocytos i s o f ROS by c u l t u r e d RPE c e l l s 94 3. E f f e c t s o f pept ides on b i n d i n g and phagocytos i s 98 4. B i n d i n g and phagocytos i s o f ant ibody l a b e l e d ROS 101 DISCUSSION 107 CHAPTER 4 MATERIALS AND METHODS MATERIALS I l l METHODS 1. Monoclonal a n t i b o d i e s I l l 2. Neuraminidase and t r y p s i n treatments o f ROS membranes 112 3. SDS-gel e l e c t r o p h o r e s i s and immunoblott ing 113 4. D i r e c t an t ibody b i n d i n g s t u d i e s 114 5. A f f i n i t y chromatography a) . RCA I a f f i n i t y chromatography 114 b) . Immunoaf f in i ty chromatography 116 6. R e c o n s t i t u t i o n s t u d i e s 117 7. Phagocytos i s assays a) . D i r e c t r a d i o i o d i n a t i o n o f ROS method 118 b) . A n t i b o d y l a b e l i n g method 118 RESULTS 1. Phagocytos i s assays 121 2. K i n e t i c s t u d i e s on ROS phagocytos i s by bovine RPE c e l l s 122 3. E f f e c t o f b l e a c h i n g and temperature on ROS b i n d i n g 126 4. E f f e c t o f t r y p s i n and neuraminidase on ROS phagocytos i s 126 5. A n a l y s i s of enzyme-treated ROS by SDS-gel e l e c t r o p h o r e s i s and western b l o t t i n g 129 6. A n a l y s i s o f t r y p s i n and neuraminidase treatments o f ROS by TEM . 133 - i x -7. I n h i b i t i o n o f phagocytos i s by t r y p s i n - t r e a t e d ROS supernatants . 135 8. RCA I a f f i n i t y chromatography 139 9. P r o t e i n r e c o n s t i t u t i o n s t u d i e s 139 10. ROS F i c o l l d i s c membranes a) . R i c i n b i n d i n g a n a l y s i s 141 b) . I n h i b i t i o n o f ROS phagocytos i s by ROS F i c o l l d i s c membranes 145 11. I o d i n a t i o n and t r y p s i n i z a t i o n o f i n t a c t ROS 148 12. ROS 1B3 monoclonal an t ibody 154 13. ROS 1B3 immunoaff in i ty chromatography 157 DISCUSSION 163 THESIS SUMMARY 174 REFERENCES 182 - X -LIST OF TABLES Table Page I . Amino a c i d compos i t ion o f the immunoaf f in i ty p u r i f i e d 2-39 N - t e r m i n a l CNBr fragment o f bovine rhodops in 52 I I . C o n c e n t r a t i o n (uM) o f pep t ide s or rhodops in r e q u i r e d to i n h i b i t by 50% ant ibody b i n d i n g to i m m o b i l i z e d rhodops in 56 I I I . Amino a c i d compos i t ion o f the immunoaf f in i ty p u r i f i e d l ' - 3 1 ' C - t e r m i n a l CNBr fragment o f bovine rhodops in 72 I V . P r o p e r t i e s o f a n t i r h o d o p s i n monoclonal a n t i b o d i e s 79 125 V . The e f f e c t of T r i t o n X-100 treatment on I - l a b e l e d rho 4D2 ant ibody b i n d i n g to f i x e d ROS 123 125 V I . The e f f e c t o f neuraminidase and t r y p s i n on I - l a b e l e d rho 4D2 ant ibody b i n d i n g to f i x e d ROS 131 - x i -LIST OF FIGURES F i g u r e Page 1. Diagram i l l u s t r a t i n g the l a y e r s o f the r e t i n a 2 2. Schematic diagram o f a rod photoreceptor c e l l 3 3. Schematic r e p r e s e n t a t i o n o f the membrane topology o f ROS 5 4 . Diagram i l l u s t r a t i n g the renewal o f p r o t e i n i n r o d photoreceptor c e l l s 7 5 . Schematic r e p r e s e n t a t i o n o f the components i n v o l v e d i n v i s u a l t r a n s d u c t i o n 12 6. A diagrammatic model o f bovine rhodops in i n d i s c membranes . . . . 16 7. A schematic r e p r e s e n t a t i o n o f ROS i n t e r d i g i t a t i n g w i t h a RPE c e l l 18 8. Schematic diagram showing the anatomica l r e l a t i o n s h i p between ROS and RPE c e l l s d u r i n g d i s s e c t i o n 20 9. T i t r a t i o n curves o f rho 5A3, rho 4D2 and rho 8A6 monoclonal ant ibody c u l t u r e f l u i d s aga ins t i m m o b i l i z e d rhodops in 48 10. A n a l y s i s o f p o l y p e p t i d e s from u n t r e a t e d and protease d i g e s t e d ROS d i s c s by monoclonal ant ibody b i n d i n g 49 11. HPLC chromatographs o f pept ides d e r i v e d from CNBr-c leaved r h o d o p s i n 51 12. Compet i t i ve i n h i b i t i o n o f rho 4A2 and rho 2B2 ant ibody b i n d i n g to rhodops in by CNBr pept ides o f rhodops in and rhodops in 54 13. Compet i t ive i n h i b i t i o n o f rho 4D2 ant ibody b i n d i n g to rhodops in by the 2-39 rhodops in g lycopept ide and s o l u b i l i z e d rhodops in . . 55 14. Compet i t ive i n h i b i t i o n o f ant ibody b i n d i n g to i m m o b i l i z e d rhodops in by b leached and unbleached rhodops in 59 125 15. Compet i t ive i n h i b i t i o n o f I - l a b e l e d rho 4D2 ant ibody b i n d i n g to rhodops in by ROS, s o l u b i l i z e d ROS and f ixed-ROS . . . . 60 - x i i -16. Transmi s s ion e l e c t r o n micrographs o f p r e - f i x e d r o d photo-r e c e p t o r c e l l s or u n f i x e d ROS l a b e l e d w i t h rho 4D2 monoclonal a n t i b o d y - g o l d - d e x t r a n conjugates 62 17. Transmi s s ion e l e c t r o n micrographs o f f i x e d ROS d i r e c t l y l a b e l e d w i t h a n t i r h o d o p s i n g o l d - d e x t r a n conjugates 64 18. Compet i t ive i n h i b i t i o n o f rho 4B4 ant ibody b i n d i n g to i m m o b i l i z e d rhodops in by ROS d i s c s and s y n t h e t i c pept ides 65 19. SDS-gel e l e c t r o p h o r e s i s and immunoblott ing o f ROS d i s c membranes t r e a t e d w i t h proteases 66 20. Transmi s s ion e l e c t r o n micrographs o f i s o l a t e d ROS d i s c membranes l a b e l e d w i t h immunogold-dextran markers 68 21. Compet i t ive i n h i b i t i o n o f rho 8A6 ant ibody b i n d i n g to rhodops in by d i s c membranes and a s y n t h e t i c pep t ide 69 22. HPLC chromatographs o f CNBr-c leaved rhodops in pept ide s 70 23. Compet i t ive i n h i b i t i o n o f rho 3A6 ant ibody b i n d i n g to rhodops in by s o l u b i l i z e d ROS d i s c s and rhodops in pept ide s 73 125 24. I n h i b i t i o n o f I - l a b e l e d rho 3A6 ant ibody b i n d i n g to rhodops in by monoclonal a n t i b o d i e s 74 125 25. Compet i t ive i n h i b i t i o n o f I - l a b e l e d rho 3A6 ant ibody b i n d i n g to rhodops in i n d i s c membranes by monoclonal a n t i b o d i e s 76 26. P u r i f i c a t i o n o f rhodops in by immunoaf f in i ty chromatography as observed by SDS-gel e l e c t r o p h o r e s i s 77 27. A diagrammatic model o f bovine rhodops in showing the approximate l o c a t i o n o f the a n t i g e n i c s i t e s f o r v a r i o u s monoclonal a n t i b o d i e s 80 28. A computer generated rhodops in composite graph i l l u s t r a t i n g the p r e d i c t e d pept ide segments o f g r e a t e s t a n t i g e n i c i t y 86 - x i i i -29. Scanning and scanning t r a n s m i s s i o n e l e c t r o n micrographs o f bovine RPE c e l l s 95 30. E l e c t r o n micrographs of the b i n d i n g and phagocytos i s o f f i x e d ROS by c u l t u r e d bovine RPE c e l l s 96 31. Transmi s s ion e l e c t r o n micrographs o f ROS-treated RPE c e l l s . . . . 97 125 32. I n h i b i t i o n o f I - l a b e l e d ROS b i n d i n g and phagocytos i s by p r e i n c u b a t i o n s w i t h u n l a b e l e d ROS 99 125 33. I n h i b i t i o n o f b i n d i n g and phagocytos i s o f I - l a b e l e d ROS by ROS membranes and pept ides o f rhodops in 100 34. The e f f e c t o f l a b e l i n g f i x e d ROS w i t h a n t i r h o d o p s i n a n t i b o d i e s on t h e i r a b i l i t y to be phagocyt i zed by bovine RPE c e l l s 102 35. E l e c t r o n micrographs o f f i x e d ROS l a b e l e d w i t h rho 4D2 a n t i b o d y - g o l d - d e x t r a n conjugates 103 36. T r a n s m i s s i o n e l e c t r o n micrographs i l l u s t r a t i n g the b i n d i n g and phagocytos i s o f rho 4D2 immunogold-dextran l a b e l e d ROS 105 37. E l e c t r o n micrographs i l l u s t r a t i n g the b i n d i n g and phagocytos i s o f immunogold-dextran l a b e l e d ROS by bovine RPE c e l l s 106 38. A schematic r e p r e s e n t a t i o n o f the assays used f o r the d e t e c t i o n o f ROS phagocytos i s by c u l t u r e d bovine RPE c e l l s 119 39. The e f f e c t o f i n c u b a t i o n time on the b i n d i n g and i n g e s t i o n o f ROS by c u l t u r e d bovine RPE c e l l s 124 40. The e f f e c t o f c o n c e n t r a t i o n on ROS b i n d i n g to RPE c e l l s 125 4 1 . The e f f e c t o f b l e a c h i n g and temperature on ROS b i n d i n g to c u l t u r e d RPE c e l l s 127 42. The e f f e c t o f t r y p s i n and neuraminidase on ROS b i n d i n g to RPE c e l l s 128 43 . The e f f e c t o f t r y p s i n on ROS b i n d i n g and i n g e s t i o n by bovine RPE c e l l s 130 - x i v -44. SDS-gel e l e c t r o p h o r e s i s and western b l o t a n a l y s i s o f neuraminidase and t r y p s i n t r e a t e d ROS 132 45. A n a l y s i s o f neuraminidase and t r y p s i n t r e a t e d ROS p o l y p e p t i d e s by Con A b i n d i n g 134 46. Transmi s s ion e l e c t r o n micrographs o f u n t r e a t e d and enzyme-t r e a t e d ROS 136 47. Transmi s s ion e l e c t r o n micrographs o f n e u r a m i n i d a s e - t r e a t e d ROS l a b e l e d w i t h r i c i n - g o l d - d e x t r a n conjugates 137 48. I n h i b i t i o n o f ROS b i n d i n g and i n g e s t i o n by t r y p s i n - t r e a t e d ROS supernatants 138 49. A n a l y s i s o f RCA I a f f i n i t y chromatography by SDS-gel e l e c t r o p h o r e s i s and western b l o t t i n g 140 50. I n h i b i t i o n o f ROS phagocytos i s by membrane v e s i c l e s r e c o n s t i t u t e d w i t h rhodops in and RCA I a f f i n i t y column i s o l a t e d p r o t e i n s 142 51. A n a l y s i s o f ROS and F i c o l l d i s c membrane p r e p a r a t i o n s by SDS-g e l e l e c t r o p h o r e s i s and r i c i n l a b e l i n g 144 52. The e f f e c t o f F i c o l l d i s c membranes on the phagocytos i s o f 125 I - l a b e l e d ROS by RPE c e l l s 146 125 53. I n h i b i t i o n o f I - l a b e l e d ROS phagocytos i s by F i c o l l d i s c and t r y p s i n - t r e a t e d F i c o l l d i s c membranes 147 125 54. A n a l y s i s o f neuraminidase and t r y p s i n t r e a t e d I - l a b e l e d ROS by SDS-gel e l e c t r o p h o r e s i s and autoradiography 149 55. S e p a r a t i o n o f g a l a c t o s e - c o n t a i n i n g , r a d i o i o d i n a t e d p o l y p e p t i d e s by RCA I a f f i n i t y chromatography 151 125 56. SDS-gel e l e c t r o p h o r e s i s and autoradiography o f I - l a b e l e d ROS and RCA I a f f i n i t y column f r a c t i o n s 152 - X V -57. I n h i b i t i o n o f ROS phagocytos i s by p o l y p e p t i d e s o b t a i n e d from a RCA I a f f i n i t y column 153 58. SDS-gel e l e c t r o p h o r e s i s and immunoblot a n a l y s i s o f ROS membranes t r e a t e d w i t h t r y p s i n and neuraminidase 155 59. Compet i t ive i n h i b i t i o n o f ROS 1B3 monoclonal ant ibody b i n d i n g to ROS 230kD g l y c o p r o t e i n by ROS membrane p r e p a r a t i o n s 156 125 60. I n h i b i t i o n o f I - l a b e l e d ROS phagocytos i s by monoclonal a n t i b o d i e s 158 61. I s o l a t i o n o f the 230kD g l y c o p r o t e i n by immunoaf f in i ty chromatography as determined by RIA 159 62. SDS-gel e l e c t r o p h o r e s i s and immunoblot a n a l y s i s o f ROS sub jec ted to immunoaf f in i ty chromatography 160 63. I n h i b i t i o n o f ROS phagocytos i s by membrane v e s i c l e s r e c o n s t i t u t e d w i t h immunoaf f in i ty i s o l a t e d ROS p r o t e i n s 162 64. S t r u c t u r e o f a r e p r e s e n t a t i v e a s p a r a g i n e - l i n k e d o l i g o s a c c h a r i d e from a complex g l y c o p r o t e i n 164 - x v i -ACKNOWLEDGEMENTS I would l i k e to thank D r . Robert Molday f o r h i s c o n t i n u a l suppor t , f i n a n c i a l a s s i s t a n c e , encouragement and e x c e l l e n t s u p e r v i s i o n throughout my graduate s t u d i e s . I would a l s o l i k e to extend my g r a t i t u d e and thanks t o : D r . Dav id H i c k s f o r h i s pa t i ence i n t e a c h i n g me many o f the monoclonal an t ibody and e l e c t r o n m i c r o s c o p i c techniques used i n t h i s s tudy , and a l s o f o r r a i s i n g the rho 4D2 a n t i r h o d o p s i n monoclonal a n t i b o d y ; Don MacKenzie f o r r a i s i n g many o f the a n t i r h o d o p s i n monoclonal a n t i b o d i e s employed i n t h i s r e p o r t ; L a u r i e Molday f o r her c o n t r i b u t i o n o f the r i c i n - g o l d - d e x t r a n l a b e l e d ROS; and Dr . D e l y t h R e i d f o r r a i s i n g the ROS 1B3 monoclonal a n t i b o d y . I would a l s o l i k e to thank D r . Simon Wong, Dr . F i o n a M i l l a r and Greg C o n n e l l f o r t h e i r h e l p f u l and en joyable rap s e s s i o n s . Much a p p r e c i a t i o n i s a l s o extended to my graduate s tudent a d v i s o r y committee (Dr . G e r a l d Weeks and Dr . Char le s Beer) f o r t h e i r i n t e r e s t , adv ice and l e t t e r s o f r e f e r e n c e . F i n a l l y , and most i m p o r t a n t l y , I would l i k e to thank my w i f e (Chery l ) and my f a m i l y f o r t h e i r uncompromising l o v e , support and encouragement. - 1 -CHAPTER 1 INTRODUCTION RETINA The v i s u a l r e t i n a i s a complex e x t e n s i o n o f the c e n t r a l nervous system. Located a long the p o s t e r i o r p a r t o f the e y e b a l l , the r e t i n a i s d i v i d e d i n t o the n e u r a l r e t i n a proper and the pigment e p i t h e l i u m . The n e u r a l r e t i n a i s r e s p o n s i b l e f o r the t r a n s m i s s i o n o f p h o t o s i g n a l s (Rodieck, 1973). L i g h t e n t e r i n g the r e t i n a passes through the o p t i c nerve f i b e r l a y e r , i n n e r p l e x i f o r m l a y e r , i n n e r n u c l e a r l a y e r , outer p l e x i f o r m l a y e r , outer n u c l e a r l a y e r and photoreceptor c e l l s , r e s p e c t i v e l y ( F i g . 1 ) . R e s i d u a l s c a t t e r e d l i g h t i s absorbed by the monolayer o f p o l a r i z e d pigment e p i t h e l i a l c e l l s l i n i n g the o p t i c cup (Leeson and Leeson, 1981). PHOTORECEPTOR CELLS V e r t e b r a t e photoreceptor c e l l s c o n s i s t o f m o d i f i e d neurons s u b c l a s s i f i e d i n t o rods and cones on the b a s i s o f morphology and v i s u a l pigments they c o n t a i n . Human cone photoreceptor c e l l s are 1-1.5 urn t h i c k and approx imate ly 75 um l o n g . Cone v i s u a l c e l l s are concent ra ted i n the macular r e g i o n o f the r e t i n a and are r e s p o n s i b l e f o r c o l o r v i s i o n ( S h i c h i , 1983). In c o n t r a s t human rod photoreceptor c e l l s (1-3 um t h i c k and 40-60 um long) are d i s t r i b u t e d i n the p e r i p h e r a l r e g i o n o f the r e t i n a and are r e s p o n s i b l e f o r v i s i o n i n dim l i g h t ( S h i c h i , 1983). The rod photoreceptor c e l l c o n s i s t s o f an outer segment connected by a c i l i u m to the i n n e r segment w h i c h , i n t u r n , i s a t t ached to the s y n a p t i c terminus ( F i g . 2 ) . The outer segment conta in s the v i s u a l pigment and metabo l i c enzymes r e q u i r e d f o r v i s u a l e x c i t a t i o n . The i n n e r segment c o n s i s t s o f c e l l u l a r and s u b c e l l u l a r o r g a n e l l e s . The s y n a p t i c Figure 1. Diagram to i l l u s t r a t e the l a y e r s of the r e t i n a . (1) pigment e p i t h e l i u m ; (2) l a y e r s o f rods and cones; (3) e x t e r n a l l i m i t i n g membrane; (4) ou te r n u c l e a r l a y e r ; (5) outer p l e x i f o r m l a y e r ; (6) i n n e r n u c l e a r l a y e r (7) i n n e r p l e x i f o r m l a y e r ; (8) g a n g l i o n c e l l l a y e r ; (9) o p t i c f i b e r l a y e r ; (10) i n t e r n a l l i m i t i n g membrane. The arrow i n d i c a t e s the d i r e c t i o n o f l i g h t (Leeson and Leeson, 1981). M = C e l l s o f M u l l e r - 3 -FiRure Schematic diagram of a rod photoreceptor c e l l . The r o d outer segment i s connected to the i n n e r segment by a c i l i u m (Diagram p r o v i d e d by D r . Robert Molday ) . - 4 -terminus r e l a y s messages to d e n d r i t e s o f b i p o l a r c e l l s and axons o f h o r i z o n t a l c e l l s i n the outer p l e x i f o r m l a y e r ( S h i c h i , 1983). STRUCTURE OF ROD OUTER SEGMENTS Rod outer segments (ROS) are composed o f s tacks o f h i g h l y - o r d e r e d , c l o s e d membrane u n i t s ( d i s c s ) surrounded by a plasma membrane ( F i g . 2 , 3 ) . The s t r u c t u r a l components o f t h i s e l abora te membrane system are g e n e r a l l y unknown a l though some recent advancements have been made. The b i o g e n e s i s o f ROS d i s c l a m e l l a r reg ions and ROS d i s c r i m reg ions have been proposed to develop by separate mechanisms ( S t e i n b e r g e t a l . , 1980). The l a m e l l a r r e g i o n o f the d i s c forms from an e v a g i n a t i o n o f the c i l i a r y plasma membrane. The d i s c r i m i s i n i t i a l l y s p e c i f i e d as a r e g i o n o f c i l i a r y membrane between ad jacent e q u a l l y spaced d i s c sur face evag ina t ions which grows b i l a t e r a l l y around the d i s c to form the completed d i s c ( S t e i n b e r g e t a l . , 1980). R e s u l t i n g d i s c membranes are d i s c o n t i n u o u s from the plasma membrane. D i s c membranes a l s o c o n t a i n i n c i s u r e s which are i n v a g i n a t i o n s o f the r i m s . Recent s t u d i e s showed t h a t proper d i s c fo rmat ion i s a l t e r e d when r e t i n a s are incubated i n the presence o f t u n i c a m y c i n , an i n h i b i t o r o f o l i g o s a c c h a r i d e b i o s y n t h e s i s , r e s u l t i n g i n the accumulat ion o f t u b u l o v e s i c u l a r m a t e r i a l i n the i n t e r s e g m e n t a l space ( F l i e s l e r e t a l . , 1985). T h i s l a c k o f p r o t e i n g l y c o s y l a t i o n r e s u l t s i n nascent d i s c f o r m a t i o n . The immunocytochemical l o c a l i z a t i o n o f a c t i n a t the d i s t a l c i l i u m o f f r o g and mammalian photoreceptor c e l l s , p r o v i d e d evidence t h a t d i s c morphogenesis may i n v o l v e an a c t i n mediated c o n t r a c t i l e mechanism ( C h a i t i n e t a l . , 1984; C h a i t i n and Bok, 1986). Recent s t u d i e s u s i n g monoclonal a n t i b o d i e s and immunocytochemical l a b e l i n g techniques have i d e n t i f i e d a d i s c r i m s p e c i f i c p r o t e i n c a l l e d p e r i p h e r i n (Molday et a l . , 1987). The s t r u c t u r e , o r g a n i z a t i o n and f u n c t i o n - 5 -disk. plasma membrane Figure 3. Schematic r e p r e s e n t a t i o n o f the membrane topo logy o f r o d outer segments. The most b a s a l rod outer segment d i s c s are shown to be cont inuous w i t h the plasma membrane w h i l e the m a j o r i t y o f the d i s c s are separate membrane domains ( F e i n and Szut s , 1982). - 6 -o f t h i s p r o t e i n i s c u r r e n t l y b e i n g i n v e s t i g a t e d at the p r o t e i n and DNA l e v e l s . Other s t u d i e s i n t o the s t r u c t u r a l components o f bov ine ROS have c h a r a c t e r i z e d a s p e c t r i n - l i k e 240kD p r o t e i n t h a t may be i n v o l v e d i n m a i n t a i n i n g the constant d i s t a n c e between ROS d i s c s and the ROS plasma membrane (Wong and Molday, 1986). The major 220kD g l y c o p r o t e i n , found i n bov ine d i s c membranes, appears to be analogous to the f r o g 290kD p r o t e i n which has been l o c a l i z e d by i m m u n o f e r r i t i n l a b e l i n g to the r i m r e g i o n o f f r o g d i s c membranes (Papermaster e t a l . , 1979; Converse, 1979). T h i s l a r g e transmembrane p r o t e i n has been suggested to be i n v o l v e d i n l i n k i n g ad jacent d i s c membranes ( C o r l e s s e t a l . , 1987). ROD OUTER SEGMENT DISC RENEWAL AND DISC SHEDDING Rod outer segments are c o n t i n u a l l y renewed (Young, 1967). P r o t e i n s are s y n t h e s i z e d i n the i n n e r segment of the photoreceptor c e l l p r i o r to b e i n g t r a n s p o r t e d through the c i l i u m to the rod outer segment. A d d i t i o n o f d i s c membrane a t the base o f the ROS i s synchron ized w i t h the removal o f d i s c m a t e r i a l a t the apex o f the c e l l thereby m a i n t a i n i n g a cons tant rod outer segment l e n g t h (Young, 1967; Young and Bok, 1969; Bok, 1985). Shed a p i c a l ROS packets are phagocy t i zed and degraded by the pigment e p i t h e l i u m (Young and Bok, 1969). E a r l y experiments monitored the m i g r a t i o n o f r a d i o a c t i v e amino a c i d s i n j e c t e d i n t o the v i t r e o u s o f f r o g eyes by autoradiography o f the r o d photoreceptor c e l l s ( F i g . 4 ) . A f t e r an i n i t i a l accumula t ion o f r a d i o a c t i v i t y i n the i n n e r segment and the i n c o r p o r a t i o n o f r a d i o a c t i v e amino a c i d s i n t o the rod outer segment d e s t i n e d p r o t e i n s , the newly l a b e l e d p r o t e i n s migra ted to the base o f the ROS and formed d i s c membranes. The newly formed d i s c membranes were s e q u e n t i a l l y d i s p l a c e d toward the d i s t a l end o f the rod p r i o r to b e i n g shed and phagocy t i zed by the RPE (Young, -7-Figure 4 . Diagram i l l u s t r a t i n g the renewal o f p r o t e i n i n r o d photoreceptor c e l l s . The i n c o r p o r a t i o n of r a d i o a c t i v e amino a c i d s i n t o ou ter segment d e s t i n e d p r o t e i n s was monitored over time by autorad iography (Young, 1976). P r o t e i n molecules are f i r s t s y n t h e s i z e d i n the myoid r e g i o n o f the i n n e r segment ( a ) . Many ROS d e s t i n e d p r o t e i n s are g l y c o s y l a t e d i n the G o l g i apparatus (b ) , p r i o r to b e i n g s o r t e d to the s i t e o f d i s c membrane fo rmat ion at the base of the r o d outer segment ( c ) . The repeated f o r m a t i o n o f new d i s c membrane d i s p l a c e s the r a d i o a c t i v e d i s c s a long the outer segment ( d ) . E v e n t u a l l y , the r a d i o a c t i v e d i s c s reach the a p i c a l t i p o f the r o d ( e ) , and are shed as plasma membrane encapsulated packets o f s t acked d i s c membranes. F i n a l l y , the shed d i s c s are phagocyt ized by the pigment e p i t h e l i u m ( f ) ( F e i n and Szut s , 1982). - 8 -1976). I t i s g e n e r a l l y b e l i e v e d t h a t the t i p s o f the ROS are shed i n t o the s u b r e t i n a l space as opposed to b e i n g p inched o f f by the m i c r o v i l l i o f the pigment e p i t h e l i u m (Besharse e t a l . , 1980; Besharse and Duni s , 1982; Besharse , 1982). A peak i n ROS d i s c shedding was observed to occur 30-120 min a f t e r l i g h t onset ( L a V a i l , 1976a). L a t e r the c i r c a d i a n nature o f ROS d i s c shedding was e s t a b l i s h e d ( L a V a i l , 1976b). The ba lance between d i s c shedding and d i s c assembly can be a l t e r e d by the l i g h t c y c l e . For example, i n Xenopus l a e v i s cons tant exposure to l i g h t g r e a t l y reduced d i s c shedding and l e d to r a p i d e l o n g a t i o n o f the ROS (Besharse e t a l . , 1977). However, d i s c shedding and d i s c assembly may not be d i r e c t l y coupled processes (Besharse e t a l . , 1977). RHODOPSIN A . L o c a l i z a t i o n and B i o s y n t h e s i s Rhodopsin , the major membrane p r o t e i n o f ROS d i s c s , has been shown by immunocytochemical l a b e l i n g s t u d i e s u s i n g bo th p o l y c l o n a l and monoclonal a n t i b o d i e s to be present i n s i g n i f i c a n t q u a n t i t i e s i n the ROS plasma membrane (Dewey e t a l . , 1969; Bas inger e t a l . , 1976; Papermaster e t a l . , 1978; H i c k s and Molday, 1986). Rhodopsin d i f f u s e s f r e e l y i n the membrane (Poo and Cone, 1974) and c o n s t i t u t e s approx imate ly 85% o f the t o t a l p r o t e i n found i n rod outer segment membranes (Papermaster and Dreyer , 1974). Bovine o p s i n i s s y n t h e s i z e d i n the rough endoplasmic r e t i c u l u m (RER) o f the rod i n n e r segment w i t h o u t a s i g n a l sequence (Schechter e t a l . , 1979). I t was concluded t h a t the o p s i n p o l y p e p t i d e c h a i n i n i t i a t e d at Met''" and t h a t an unc leaved i n t e r n a l s i g n a l sequence must enable o p s i n to t r a v e r s e the RER 2 (Schechter e t a l . , 1979). The o p s i n molecule i s g l y c o s y l a t e d on the Asn and A s n ^ re s idues i n the RER p r i o r to b e i n g t r a n s p o r t e d by a v e s i c u l a r - 9 -mechanism to the G o l g i apparatus where f u r t h e r p r o c e s s i n g by g l y c o s i d a s e s and t r a n s f e r a s e s o c c u r s . F u l l y processed membrane bound o p s i n v e s i c u l a t e s from the t r ans compartment o f the G o l g i apparatus and i s t r a n s p o r t e d through the c o n n e c t i n g c i l i u m to the ROS membrane (Papermaster e t a l . , 1978; Papermaster e t a l . , 1986). R e c e n t l y N - g l y c o s y l a t i o n has been shown not to be r e q u i r e d f o r t r a n s p o r t of o p s i n from i t s s i t e of s y n t h e s i s i n the i n n e r segment to the s i t e o f d i s c assembly i n the outer segment ( F l i e s l e r e t a l . , 1985). However, normal g l y c o s y l a t i o n o f ROS d e s t i n e d p r o t e i n s i s r e q u i r e d f o r proper d i s c morphogenesis ( F l i e s l e r e t a l . , 1985). H y p e r g l y c o s y l a t e d o p s i n a l s o undergoes normal i n t r a c e l l u l a r t r a n s p o r t w i t h normal d i s c f o r m a t i o n ( F l i e s l e r e t a l . , 1986). F i n a l l y , immunocytochemical s t u d i e s have i d e n t i f i e d rhodops in i n the rod i n n e r segment plasma membrane, as w e l l as i n ROS membranes (Jan and R e v e l , 1974). I t i s p o s s i b l e t h a t a d e f a u l t mechanism may e x i s t t h a t a l l o w s f o r low amounts o f rhodops in to be l o c a l i z e d i n the plasma membrane o f the rod i n n e r segment. B . P u r i f i c a t i o n and M o l e c u l a r P r o p e r t i e s The transmembrane g l y c o p r o t e i n rhodops in c o n t a i n s an 1 1 - c i s r e t i n a l group conjugated to l y s i n e - 2 9 6 o f the 348 amino a c i d p o l y p e p t i d e c h a i n (Hargrave, 1982). Rhodopsin can be p u r i f i e d from detergent s o l u b i l i z e d ROS d i s c membranes by g e l f i l t r a t i o n ( H e l l e r , 1968), c a l c i u m phosphate chromatography (Papermaster and Dreyer , 1974), i o n exchange chromatography (Zorn and Futterman, 1973), c o n c a n a v a l i n A a f f i n i t y chromatography (Steinemann and S t r y e r , 1973) o r , more r e c e n t l y , immunoaf f in i ty chromatography (Opr ian e t a l . , 1987). Bovine rhodops in has a molecu la r weight o f 38kD w i t h an apparent molecu la r weight on SDS- p o l y a c r y l a m i d e ge l s o f approx imate ly 34-37kD. Rhodopsin a b s o r p t i o n at 500 nm i s due to a p r o t e i n bound r e t i n a l . - 1 0 -Moreover, rhodops in has a molar e x t i n c t i o n c o e f f i c i e n t o f approx imate ly 40,000 (Wald and Brown, 1953). The absorbance r a t i o o f A o o n / A C A A i s o f t e n zoU DUU used as a measurement o f rhodops in p u r i t y where a low r a t i o i s i n d i c a t i v e of a more pure p r e p a r a t i o n . Upon b l e a c h i n g o f rhodops in there i s a subsequent s h i f t i n the absorbance peak from 500 nm to 380-400 nm as the bound 1 1 - c i s -r e t i n a l group i somer ize s to a l l - t r a n s - r e t i n a l and leaves the a c t i v e s i t e . Consequent ly , the r a t i o o f A ^ Q Q / A , _ Q Q i s o f t e n used as a measurement o f r h o d o p s i n b l e a c h i n g . C. P o s t - T r a n s l a t i o n a l M o d i f i c a t i o n s o f Rhodopsin Rhodopsin undergoes c o n s i d e r a b l e p o s t - t r a n s l a t i o n a l m o d i f i c a t i o n . The amino terminus o f rhodops in i s N - a c e t y l a t e d on the i n i t i a l Met r e s idue (Tsunasawa et a l . , 1980). A l s o two mannose and N - a c e t y l glucosamine-2 15 c o n t a i n i n g o l i g o s a c c h a r i d e chains are l i n k e d to rhodops in a t Asn and Asn (Hargrave e t a l . , 1984). Bovine rhodops in has been shown to c o n t a i n b i n d i n g s i t e s f o r c o n c a n a v a l i n A , s p e c i f i c f o r mannose r e s i d u e s , and wheat germ a g g l u t i n i n , s p e c i f i c f o r N - a c e t y l glucosamine re s idues (Molday and Molday, 1979). F u r t h e r p o s t - t r a n s l a t i o n a l p r o c e s s i n g i n v o l v e s the p h o s p h o r y l a t i o n o f r h o d o p s i n by ATP i n a l i gh t -dependent k i n a s e - c a t a l y z e d r e a c t i o n (Kuhn and D r y e r , 1972; Bownds e t a l . , 1972). T h i s r e a c t i o n may serve as a c o n t r o l mechanism i n the v i s u a l process (Liebman and Pugh, 1980). F i n a l l y , A l - S a l e h et a l . (1987) have i d e n t i f i e d a d i s u l f i d e b r i d g e between c y s t e i n e s 322 and 323 w h i l e i n d i r e c t evidence suggests t h a t an a d d i t i o n a l d i s u l f i d e l i n k a g e occurs between Cys-110 and Cys-187. The Cys-110 and Cys-187 re s idues are h i g h l y conserved sugges t ing a p o s s i b l e , y e t undetermined, f u n c t i o n a l r o l e f o r these re s idues (Applebury and Hargrave, 1986) . - 1 1 -D. Rhodopsin F u n c t i o n i n V i s u a l T r a n s d u c t i o n The a b s o r p t i o n o f a photon by rhodops in i n i t i a t e s the l i g h t - d e p e n d e n t b i o c h e m i c a l events o f the rod photoreceptor c e l l ( S t r y e r , 1986; Applebury and Hargrave , 1986; Pugh and Cobbs, 1986). L i g h t causes the bound 1 1 - c i s r e t i n a l chromophore to i somer ize to the a l l - t r a n s conformat ion . T h i s leads to a change i n the conformat ion o f rhodops in a l l o w i n g f o r the G - p r o t e i n , t r a n s d u c i n , to b i n d to the cy top la smic sur face o f rhodops in (Kuhn, 1984) ( F i g . 5 ) . The b i n d i n g o f t r a n s d u c i n (composed o f 3 subuni t s G<<=39K, G/=37K and G3r '=8K) to rhodops in promotes the exchange o f bound GDP f o r GTP and the r e l e a s e o f G«c -GTP from i t s rhodops in b i n d i n g s i t e (Fung, 1985). cGMP phosphodies terase i s a c t i v a t e d by G«£-GTP which i n t u r n lowers the cGMP l e v e l s i n the outer segment o f the rod photoreceptor c e l l . cGMP i s r e s p o n s i b l e f o r m a i n t a i n i n g i o n p e r m e a b i l i t y i n the r o d c e l l (Fesenko et a l . , 1985; Yau and N a k a t a n i , 1985). A r e d u c t i o n i n cGMP l e v e l s r e s u l t s i n the c l o s u r e o f the c a t i o n channels and a h y p e r p o l a r i z a t i o n o f the r o d c e l l ( F i g . 5 ) . I n summary, i t i s e v i d e n t t h a t the f u l l under s tand ing o f the s t r u c t u r e and f u n c t i o n of rhodops in i s e s s e n t i a l f o r the complete unders tanding o f the v i s u a l p roce s s . E. Probes o f Rhodopsin S t r u c t u r e and Topography One o f the i n i t i a l approaches to s t u d y i n g rhodops in s t r u c t u r e and topography i n the d i s c membrane was by l i m i t e d p r o t e o l y s i s . E a r l y s t u d i e s i n d i c a t e d t h a t t h e r m o l y s i n c l e a v e d o f f the c a r b o x y l t e r m i n a l 12 amino ac id s o f rhodops in (Hargrave and Fong, 1977) and l a t e r , i t was observed t h a t t h e r m o l y s i n c l e a v e d b leached rhodops in f a s t e r than unbleached rhodops in (Kuhn et a l . , 1982). However, l i m i t e d p r o t e o l y s i s o f r h o d o p s i n i n d i s c membranes u s i n g the enzymes t r y p s i n , chymotryps in and S_^  g r i s e u s protease showed no pre ference f o r b leached vs unbleached rhodops in ( S a a r i , 1974; - 1 2 -F i g u r e 5. Schematic r e p r e s e n t a t i o n o f rod outer segment plasma membrane and d i s c membrane i l l u s t r a t i n g the components i n v o l v e d i n v i s u a l t r a n s d u c t i o n PDE = phosphodies terase ; G = G - p r o t e i n ( t r a n s d u c i n ) ; U n B l - r h o = unbleached r h o d o p s i n ; B l - r h o = b leached r h o d o p s i n . (mod i f i ed from D r . Robert Molday ) . - 1 3 -Molday and Molday, 1979). L i m i t e d p r o t e o l y s i s was used to determine t h a t the carboxy-terminus o f rhodops in was exposed i n d i s c membranes (Hargrave and Fong, 1977). Hence, a m i l d t r y p s i n d i g e s t i o n o f membrane bound rhodops in c l e a v e d o f f the 9 amino a c i d c a r b o x y l t e r m i n a l segment o f rhodops in (Molday and Molday, 1979; Hargrave e t a l . , 1980). A second s i t e o f rhodops in p r o t e o l y s i s y i e l d i n g 2 major p o l y p e p t i d e fragments was found to be i n an exposed sur face loop between h e l i c e s V and V I . Cleavage o f t h i s s i t e by t h e r m o l y s i n , g r i s e u s protease or aureus protease y i e l d e d a carbohydrate c o n t a i n i n g (F^) p o l y p e p t i d e fragment and a d i f f u s e , r e t i n a l c o n t a i n i n g (F^) p o l y p e p t i d e fragment ( S a a r i , 1974; Pober and S t r y e r , 1975; F i n d l a y e t a l . , 1981; Hargrave, 1982). The e x i s t e n c e o f a l i m i t e d number o f p r o t e o l y t i c s i t e s a t s e l e c t i v e reg ions o f the p o l y p e p t i d e c h a i n i n d i c a t e s t h a t rhodops in i s t i g h t l y o rgan ized w i t h i n the l i p i d b i l a y e r o f the d i s c membrane. E a r l y s t u d i e s u t i l i z e d p o l y c l o n a l a n t i r h o d o p s i n a n t i s e r a w i t h immunocytochemical techniques to l o c a l i z e rhodops in i n ROS membranes (Dewey et a l . , 1969; Jan and R e v e l , 1974). These immunologica l reagent s , however, b i n d to m u l t i p l e determinants on rhodops in , and t h e r e f o r e , they have l i m i t e d a p p l i c a t i o n s as s p e c i f i c probes f o r the s t r u c t u r e and f u n c t i o n o f r h o d o p s i n . More r e c e n t l y , s e v e r a l groups have generated monoclonal a n t i b o d i e s aga i n s t bovine rhodops in (MacKenzie and Molday, 1982), f r o g rhodops in ( W i t t e t a l . , 1984) and r a t rhodops in (Fekete and B a r n s t a b l e , 1983) . Monoclona l a n t i b o d i e s s p e c i f i c f o r rhodops in have p r o v i d e d u s e f u l i n f o r m a t i o n on the s t r u c t u r e , o r g a n i z a t i o n and topography o f rhodops in i n the membrane (Molday and MacKenzie , 1983; MacKenzie e t a l . , 1984; Molday and MacKenzie , 1985; DeGr ip , 1985; H i c k s and Molday, 1986; McDowell e t a l . , 1987). S e v e r a l monoclonal a n t i b o d i e s have been r a i s e d a g a i n s t the C-- 1 4 -terminus and N-terminus o f rhodops in i n d i c a t i n g t h a t both o f these reg ions are p r i n c i p a l a n t i g e n i c s i t e s . I n some cases , a n t i r h o d o p s i n a n t i b o d i e s have been p r e c i s e l y l o c a l i z e d by c o m p e t i t i v e i n h i b i t i o n s t u d i e s u s i n g s y n t h e t i c pep t ide analogues (MacKenzie e t a l . , 1984; Hargrave e t a l . , 1986). These a n t i b o d i e s p r o v i d e i n s i g h t i n t o the a c c e s s i b i l i t y o f the C - t e r m i n a l and N-t e r m i n a l rhodops in domains. Moreover, many o f these same a n t i b o d i e s c ros s r e a c t w i t h o ther spec ies i n d i c a t i n g a c o n s e r v a t i o n o f the C - t e r m i n a l and N-t e r m i n a l segments o f rhodops in (MacKenzie e t a l . , 1984; Hargrave e t a l . , 1986) . Monoclonal a n t i b o d i e s aga i n s t a n t i g e n i c s i t e s exposed on the e x t r a c e l l u l a r or i n t r a d i s c a l sur face and the c y t o p l a s m i c s i d e o f the ROS plasma membrane and d i s c membranes shou ld prove p a r t i c u l a r l y v a l u a b l e i n s t u d y i n g the o r g a n i z a t i o n and b i o s y n t h e s i s o f rhodops in by immunocytochemical and b i o c h e m i c a l t echniques . F. A Model o f Rhodopsin S t r u c t u r e i n D i s c Membrane The complete amino a c i d sequence o f bovine rhodops in was f i r s t determined by Ovchinnikov e t a l . (1982) and l a t e r conf i rmed by a n a l y s i s o f CNBr fragments o f rhodops in by Hargrave e t a l . (1983). Subsequent ly , the complementary DNA (cDNA) and genomic DNA c lones encoding bov ine rhodops in were i s o l a t e d and c h a r a c t e r i z e d (Nathans and Hogness, 1983). U s i n g the DNA sequence encoding bovine rhodops in to probe l i b r a r i e s o f human genomic DNA, the genes encoding human cone b l u e , green and red pigments were i s o l a t e d (Nathans e t a l . , 1986). The amino a c i d sequences f o r the v a r i o u s photopigments have shown a h i g h degree o f homology. Homologies or conserved sequences w i t h i n the f a m i l y o f v i s u a l pigments may g ive c l u e s as to the l o c a t i o n o f f u n c t i o n a l domains. Conserved amino a c i d sequences were a l s o observed between the v i s u a l pigments and thefi - adrenerg ic r e c e p t o r (Dixon et a l . , 1986) and the m u s c a r i n i c a c e t y l c h o l i n e r e c e p t o r ( P e r a l t a e t a l . , 1987). - 1 5 -Moreover, not o n l y i s there c o n s i d e r a b l e sequence homology between these two r e c e p t o r p r o t e i n s and r h o d o p s i n , but a l s o they are a c t i v a t e d by G - p r o t e i n , sugges t ing t h a t rhodops in may be long to a f a m i l y o f G - p r o t e i n a c t i v a t e d r e c e p t o r s (Applebury and Hargrave, 1986; H a l l , 1987). A schematic model o f rhodops in was f i r s t proposed by Hargrave e t a l . (1983) ( F i g . 6 ) . W i t h the knowledge o f the t h r e e - d i m e n s i o n a l s t r u c t u r e and f o l d i n g p a t t e r n o f the r e t i n y l - p r o t e i n b a c t e r i o r h o d o p s i n from archaebacter ium Halobacter ium ha lob ium, i t was proposed t h a t rhodops in may e x i s t as a bundle o f seven h e l i c e s (Engelman et a l . , 1980; W a l l a c e , 1982; Hargrave e t a l . , 1983). A 2-d imens iona l model o f rhodops in i n the d i s c membrane i l l u s t r a t e s how the p o l y p e p t i d e c h a i n threads i t s e l f through the membrane w i t h seven predominant ly hydrophobic a l p h a - h e l i c a l r e g i o n s . These h e l i c e s are connected by h y d r o p h i l i c l i n k i n g reg ions ( F i g . 6 ) . T h i s model a l s o p r e d i c t s a h y d r o p h i l i c cy top la smic exposed c a r b o x y l terminus and a h y d r o p h i l i c i n t r a d i s c a l exposed N-terminus (Hargrave e t a l . , 1983). A l l of the v i s u a l pigments have s e r i n e and threon ine r i c h c a r b o x y l - t e r m i n a l segments which can serve as s i t e s o f p h o s p h o r y l a t i o n by rhodops in k i n a s e . Loop I - I I i s the most conserved sequence on the c y t o p l a s m i c sur face o f the v i s u a l pigments (Applebury and Hargrave , 1986). The c y t o p l a s m i c loop V - V I (F^-F2 l o o p ) , as w e l l as b e i n g p a r t i a l l y conserved i n v i s u a l pigments , has been i m p l i c a t e d as the s i t e o f l i gh t -dependent b i n d i n g and a c t i v a t i o n o f t r a n s d u c i n (Kuhn and Hargrave, 1981). The N-terminus o f r h o d o p s i n , o ther than b e i n g a s i t e f o r g l y c o s y l a t i o n , has no o ther conf i rmed f u n c t i o n a l r o l e . The gene f o r bovine rhodops in has been s y n t h e s i z e d w i t h c a r e f u l l y c o n s t r u c t e d s p e c i f i c mutat ions ( F e r r e t t i e t a l . , 1986). A sy s temat i c s t r u c t u r e - f u n c t i o n study o f bovine rhodops in by s p e c i f i c amino a c i d replacements i s c u r r e n t l y b e i n g performed. These s t u d i e s w i l l p r o v i d e -16-F i g u r e 6. A diagrammatic model o f bovine rhodops in i n d i s c membranes, m o d i f i e d from Hargrave e t a l . (1984). The t r y p s i n (T) and aureus V-8 protease (SA) c leavage s i t e s on membrane-bound rhodops in are no ted . - 1 7 -v a l u a b l e i n f o r m a t i o n on the f u n c t i o n a l l y s i g n i f i c a n t amino a c i d domains o f r h o d o p s i n . RETINAL PIGMENT EPITHELIAL CELL STRUCTURE AND FUNCTION The s imple c u b o i d a l r e t i n a l pigment e p i t h e l i u m (RPE) which covers the p o s t e r i o r p a r t o f the e y e b a l l as f a r as the ora s e r r a t a i s predominant ly dark c o l o r e d i n most spec ies owing to the melan in pigment i n i t s c e l l s ( C l a r k , 1986). The c e l l s are p o l a r i z e d w i t h an i n f o l d i n g - r i c h basement membrane and a m i c r o v i l l i - r i c h a p i c a l membrane which i n t e r d i g i t a t e s w i t h the outer segments o f photoreceptor c e l l s ( F i g . 7 ) . The RPE m i c r o v i l l i are separated from d i r e c t contac t s w i t h the photoreceptor c e l l s by a i n t e r p h o t o r e c e p t o r m a t r i x . T i g h t i n t e r c e l l u l a r j u n c t i o n s p l a y an important r o l e i n p r e v e n t i n g the passage o f l a r g e molecu le s , as w e l l as i n e s t a b l i s h i n g a b l o o d - b r a i n b a r r i e r . Aged RPE c e l l s i n c r e a s e i n t h e i r content o f undegradable , p o l y u n s a t u r a t e d f a t t y a c i d s known as a u t o f l u o r e s c e n t age pigment, l i p o f u s c i n ( C l a r k , 1986). The p r i n c i p a l f u n c t i o n s o f the RPE c e l l (as rev iewed by C l a r k , 1986) a re : i ) to s t o r e and t r a n s p o r t v i t a m i n A ( r e t i n o l ) (Goodman and B l a n e r , 1984); i i ) to prevent r e f l e c t i o n by absorb ing s c a t t e r e d l i g h t ; i i i ) to s ec re te components to the i n t e r p h o t o r e c e p t o r m a t r i x and the basement membrane; and i v ) to p a r t i c i p a t e i n the renewal o f photoreceptor c e l l outer segment membrane. Th i s s tudy w i l l focus p r i m a r i l y on the RPE involvement i n ROS renewal . CULTURING OF RPE CELLS RPE s t r u c t u r a l and f u n c t i o n a l s t u d i e s have been g r e a t l y enhanced by the c u l t u r i n g o f RPE c e l l s . RPE c u l t u r e s have been ob ta ined from r a t (Edwards, 1977), human ( A l b e r t e t a l . , 1972), ca t (Stramn e t a l . , 1983), c a t t l e (Basu -18-F i g u r e 7. Schematic r e p r e s e n t a t i o n o f a r e t i n a l pigment e p i t h e l i a l c e l l i n t e r d i g i t a t e d w i t h r o d photoreceptor c e l l outer segments. M o d i f i e d by Dr . D e l y t h R e i d from Rodieck (1973). - 1 9 -et a l . , 1983), and c h i c k (Newsome and Kenyon, 1973). Numerous o ther i n v e s t i g a t o r s have a l s o been s u c c e s s f u l i n e s t a b l i s h i n g pr imary RPE c e l l c u l t u r e s w i t h r e p o r t e d e l abora te m i c r o v i l l i , b a s a l i n f o l d i n g s and pigment g r a n u l e s . I s o l a t i o n o f RPE c e l l s has been performed by bo th d i s s e c t i o n and enzymatic approaches. F i g u r e 8 i l l u s t r a t e s the mechanica l s e p a r a t i o n o f the n e u r a l r e t i n a proper from the pigment e p i t h e l i u m p r i o r to RPE c e l l i s o l a t i o n . Mayerson et a l . (1985) have demonstrated i n the r a t system t h a t h i g h y i e l d s and e x c e l l e n t RPE v i a b i l i t y c o u l d be s t be ach ieved by degrada t ion o f the e x t r a c e l l u l a r mat r i ce s w i t h co l l agenase and h y a l u r o n i d a s e p r i o r to d i s s e c t i o n and d i s s o c i a t i o n i n t r y p s i n . T h i s improved c u l t u r i n g method was p a r t i c u l a r l y u s e f u l i n e s t a b l i s h i n g a c o n t r o l l e d enviroment f o r s t u d y i n g ROS phagocytos i s i n normal and d i sea sed RPE c e l l s . RPE c e l l d i f f e r e n t i a t i o n and genera l morphology can o f t e n be improved by growth on c o l l a g e n mat r i ce s ( H a l l and Quon, 1981). H e l l e r and Jones (1980) have succeeded i n p u r i f y i n g RPE c e l l s from bovine eyes by d i s s o c i a t i o n i n c a l c i u m f ree b u f f e r and c e n t r i f u g a t i o n on a F i c o l l d e n s i t y g r a d i e n t f o l l o w e d by recovery i n t i s s u e c u l t u r e w i t h 90% v i a b i l i t y . More r e c e n t l y bovine RPE c e l l s have been i s o l a t e d by p e r f u s i o n o f the whole bov ine eye which r e s u l t e d i n a h i g h y i e l d o f v i a b l e RPE c e l l s (Trimmers e t a l . , 1984). Serum-free d e f i n e d c u l t u r e medium was a l s o employed f o r c u l t u r i n g bovine and human RPE c e l l s a f t e r i n i t i a l c e l l attachment i n serum supplemented medium (Oka et a l . , 1984). The d o u b l i n g time f o r pr imary c u l t u r e s i n serum supplemented c u l t u r e medium was 50 h o u r s , w h i l e d e f i n e d medium promoted a f a s t e r RPE d o u b l i n g time o f 35 hours (Basu et a l . , 1983; Oka e t a l . , 1984). Both c u l t u r e d melanot i c and amelanot ic bovine RPE c e l l s r e t a i n e d t h e i r i n v i v o c h a r a c t e r i s t i c s , such as, p o l a r i z a t i o n , a p i c a l Figure 8. Schematic diagram showing the anatomica l r e l a t i o n s h i p between the rod outer segments (ROS) o f the n e u r a l r e t i n a (R) and the pigment e p i t h e l i u m (RPE) d u r i n g d i s s e c t i o n . C = C h o r o i d ; S = S c l e r a ; (Feeney-Burns and Berman, 1982) . -21-m i c r o v i l l i and phagocyt i c a b i l i t y (Basu e t a l . , 1983). However, there was a genera l decrease i n melan in pigments i n the me lanot i c RPE c e l l s . PROCESS OF PHAGOCYTOSIS The process o f phagocytos i s occurs i n three f a i r l y d i s t i n c t s t eps : f i r s t , r e c o g n i t i o n and attachment o f the rod outer segments to the RPE c e l l s ; second, engulfment; and f i n a l l y , post engulfment phenomena which i n c l u d e s e q u e n t i a l movement and f u s i o n o f lysosomes w i t h the phagosomes, a drop i n pH, and d i g e s t i o n o f the phagocyt i zed m a t e r i a l . P a r t i c l e s are bound f o r phagocytos i s by s p e c i f i c sur face r e c e p t o r s , r e f e r r e d to as r e c e p t o r -mediated p h a g o c y t o s i s , or by favourab le hydrophobic or charge r e a c t i o n s known as n o n s p e c i f i c phagocy to s i s . RPE c e l l s are capable o f b o t h . Receptor-mediated phagocytos i s by RPE c e l l s was i n i t i a l l y proposed to i n v o l v e sur face Fc r e c e p t o r s s i m i l a r to macrophages ( E l n e r e t a l . , 1981). Monkey RPE c e l l s were shown to p r e f e r e n t i a l l y b i n d and phagocyt i ze RBC coated w i t h IgG and complement ( E l n e r e t a l . , 1981). However, a l a t e r s tudy by Mayerson and H a l l (1986) showed t h a t the c o a t i n g o f RBC and ROS w i t h a n t i s e r a d i d not s i g n i f i c a n t l y a l t e r the b i n d i n g or i n g e s t i o n o f these p a r t i c l e s by RPE c e l l s , thus , e l i m i n a t i n g receptor-media ted ROS or RBC phagocytos i s by RPE Fc r e c e p t o r s . I n the same s tudy , Mayerson and H a l l (1986) demonstrated t h a t RPE c e l l s are capable o f r e c o g n i z i n g RBC, a lgae , b a c t e r i a and y e a s t . However, there i s a s e l e c t i v e pre ference f o r ROS. RPE c e l l s were a l s o capable o f b i n d i n g and i n g e s t i n g p o l y s t y r e n e spheres ( H o l l e y f i e l d and Ward, 1974), as w e l l as r e c o n s t i t u t e d rhodops in i n p h o s p h o l i p i d v e s i c l e s (Shirakawa et a l . , 1987). The s p e c i f i c mechani sm by which ROS b i n d and a t t a c h to RPE c e l l s remains unknown. Once p a r t i c l e b i n d i n g to the RPE c e l l sur face occurs some as y e t undetermined, receptor-media ted or c h a r g e - r e l a t e d transmembrane s i g n a l must i n i t i a t e the i n g e s t i o n proce s s . -22-McLaugh l in e t a l . (1983) developed a method f o r r e p l i c a t i n g the membrane sur face o f r a t r e t i n a l pigment e p i t h e l i u m exp lan t s d u r i n g phagocytos i s o f l a t e x beads f o r v i s u a l i z a t i o n by e l e c t r o n microscopy . Surface r e p l i c a s o f i n i t i a l stages o f phagocytos i s showed the attachment and spread ing o f m i c r o v i l l i over the l a t e x beads. Th i s may be a m o r p h o l o g i c a l movement p r i o r to i n g e s t i o n t h a t a l l o w s a s e q u e n t i a l and c i r c u m f e r e n t i a l i n t e r a c t i o n o f r ecep tor s on the sur face o f the RPE membranes. The beads are engul fed by o v e r l a p p i n g m i c r o v i l l i t h a t resemble "Venus f l y t r a p s " i n sur face r e p l i c a s . F o l l o w i n g s h o r t e n i n g o f the m i c r o v i l l i the beads are engul fed l e a v i n g doughnut l i k e impress ions i n the sur face membrane. These impress ions e v e n t u a l l y become f l a t t e n e d membrane domains (McLaughl in e t a l . , 1983). I n c o n t r a s t C h a i t i n and H a l l (1983b) i d e n t i f i e d saucer-shaped s t r u c t u r e s on the sur face o f r a t RPE c e l l s i n the process o f i n g e s t i o n o f ROS. The d i f f e r e n c e s i n these two observa t ions may be r e l a t e d to the type o f p a r t i c l e b e i n g i n g e s t e d . RPE PHAGOCYTOSIS ASSAYS The f i r s t q u a n t i t a t i v e i n v i t r o s tudy o f phagocytos i s ana lyzed the uptake o f l a t e x microspheres by bovine RPE exp lant s (Feeney and M i x o n , 1976). L a t e r ROS phagocytos i s by r a t RPE c e l l s (Edwards and Szamier , 1977; P h i l i p and B e r n s t e i n , 1981) was demonstrated. I n both in s t ance s phagocytos i s was ana lyzed by c o u n t i n g o f i n d i v i d u a l phagosomes at the l e v e l o f t r a n s m i s s i o n e l e c t r o n microscopy . I n an attempt to de f ine a l e s s t ed ious phagocytos i s assay system, ROS were r a d i o a c t i v e l y l a b e l e d and phagocytos i s was d e f i n e d as the amount o f r a d i o a c t i v i t y a s s o c i a t e d w i t h the RPE c e l l s ( H a l l , 1978; Goldman et a l . , 1979). The phagocytos i s o f r a d i o l a b e l e d ROS by RPE c e l l s was supported by a c t u a l phagosome counts o b t a i n e d from e l e c t r o n - 2 3 -micrographs (Goldman et a l . , 1979). However, no attempt was made i n t h i s system to uncouple ROS b i n d i n g from uptake . The be s t phagocytos i s assay system c h a r a c t e r i z e d to date was developed by C h a i t i n and H a l l (1983a). They used a ROS ant i serum and a double immunofluorescent l a b e l i n g procedure f o r a s say ing the phagocytos i s o f ROS by c u l t u r e d r a t RPE c e l l s . F o l l o w i n g the i n c u b a t i o n o f RPE c e l l s w i t h ROS, the outer segments a t t ached to the sur face o f the c e l l s were l a b e l e d w i t h ROS ant i serum i n c o n j u n c t i o n w i t h tetramethylrhodamine conjugated goat a n t i -r a b b i t IgG. The same c e l l s were then d i s r u p t e d under a graded acetone s e r i e s and the i n t e r n a l ROS were l a b e l e d w i t h the ROS ant i serum f o l l o w e d by f l u o r e s c e i n i s o t h i o c y a n a t e conjugated goat a n t i - r a b b i t IgG. ROS a t t ached to the outer sur face c o u l d be d i s t i n g u i s h e d from the rods t h a t had been i n t e r n a l i z e d by the use o f d i f f e r e n t f l u o r e s c e n t f i l t e r s . A l t h o u g h t h i s assay i s s low and t e d i o u s , i t s u c c e s s f u l l y d i s t i n g u i s h e s ROS b i n d i n g from ROS uptake . U t i l i z i n g t h i s assay system, C h a i t i n and H a l l (1983a) demonstrated t h a t a f t e r a l a g p e r i o d o f 30 min ROS i n g e s t i o n by r a t RPE c e l l s g r e a t l y i n c r e a s e d , w h i l e the number o f sur face bound ROS i n c r e a s e d more s l o w l y . T h i s suggested t h a t the i n g e s t i o n phase o f phagocytos i s occurs r a p i d l y once ROS have a t tached to the c e l l s u r f a c e . R e c e n t l y , H a l l and Abrams (1987) performed e l a b o r a t e s t u d i e s on the k i n e t i c s o f ROS b i n d i n g and i n g e s t i o n by c u l t u r e d r a t RPE c e l l s . They demonstrated t h a t r a t RPE c e l l s were capable o f b i n d i n g , but not i n g e s t i n g ROS a t 1 7 ° C . Furthermore , temperatures l e s s than 10°C r e s u l t e d , not o n l y i n the i n h i b i t i o n o f i n g e s t i o n , but a l s o i n a l o s s i n b i n d i n g o f ROS to the RPE c e l l s . Th i s i s l i k e l y due to the l o s s o f membrane f l u i d i t y . I n t h i s s tudy , H a l l and Abrams (1987) showed t h a t ROS s a t u r a t e d RPE c e l l s were capable o f i n g e s t i n g a d d i t i o n a l ROS a f t e r a recovery p e r i o d o f 2 h , even i n the presence o f p r o t e i n s y n t h e s i s i n h i b i t o r s . T h i s suggests t h a t s p e c i f i c ROS-- 2 4 -b i n d i n g r e c e p t o r s are not degraded i n the RPE c e l l but may be r e c y c l e d back to the c e l l s u r f a c e . PHAGOCYTIC DEFECTS IN THE RPE CELLS There e x i s t s e v e r a l pigment r e t i n o p a t h i e s o f the r e t i n i t i s pigmentosa type which i n v o l v e derangement o f the phagocyt i c p r o c e s s . I n h e r i t e d r e t i n a l degenera t ion i n the Roya l C o l l e g e o f Surgeons (RCS) r a t s has been shown to be a r e s u l t o f a s t r u c t u r a l or f u n c t i o n a l de fec t i n the r e t i n a l pigment e p i t h e l i a l c e l l s (Bok and H a l l , 1971; M u l l e n and L a V a i l , 1976; Edwards and Szamier , 1977). Hence, the RCS r a t c e l l l i n e has been used e x t e n s i v e l y to s tudy the mechanism of p h a g o c y t o s i s . Rat chimera s t u d i e s i d e n t i f i e d the pigment e p i t h e l i a l c e l l as the pr imary s i t e o f gene a c t i o n ( M u l l e n and L a V a i l , 1976). I n i t i a l l y , i n the RCS r a t , normal photoreceptor shedding occurs d u r i n g which there i s a concomitant accumula t ion o f l a m e l l a r bodies i n the e x t r a c e l l u l a r space between the photoreceptor outer segments and the RPE. The accumula t ion o f l a m e l l a r m a t e r i a l i s accompanied by a p r o g r e s s i v e d e t e r i o r a t i o n o f i n d i v i d u a l photoreceptor c e l l s and a consequent d e c l i n e i n r e t i n a l s e n s i t i v i t y . C h a i t i n and H a l l (1983a) have shown t h a t the attachment o f ROS to d y s t r o p h i c pigment e p i t h e l i a l c e l l s occurs a t a normal r a t e , however, o n l y a s m a l l number o f these ROS are i n g e s t e d . The RCS pigment e p i t h e l i u m may l a c k some f a c t o r f o r phagocytos i s or c o n t a i n an abnormal f a c t o r t h a t i n h i b i t s p h a g o c y t i c a c t i v i t y . Both normal and d y s t r o p h i c RPE c e l l s are capable o f p h a g o c y t i z i n g carbon p a r t i c l e s sugges t ing t h a t " n o n - s p e c i f i c " phagocytos i s can s t i l l o c u r r i n d i sea sed RPE c e l l s (Custer and Bok, 1975). ROS packets from normal or d y s t r o p h i c animals are e q u a l l y phagocy t i zed by normal RPE c e l l s c o n f i r m i n g t h a t the pr imary s i t e o f the phagocyt i c de fec t i s the RPE. -25-When two-dimens iona l g e l e l e c t r o p h o r e s i s was performed on membrane-bound p r o t e i n s i n plasma membrane e n r i c h e d f r a c t i o n s from d y s t r o p h i c and normal 125 RPE c e l l s , no d i f f e r e n c e s i n the t o t a l p r o t e i n or I - l a b e l e d sur face p r o t e i n s were observed ( C l a r k and H a l l , 1986). THE ROLE OF THE CYTOSKELETAL SYSTEM IN PHAGOCYTOSIS W i t h the consensus t h a t the i n g e s t i o n phase o f phagocytos i s i s d e f e c t i v e i n the d y s t r o p h i c RPE c e l l s , i t seemed p o s s i b l e t h a t t h i s de fec t might i n v o l v e the c o n t r a c t i l e p r o t e i n a c t i n . A c t i n has been shown to be a major RPE p r o t e i n (Haley e t a l . , 1983) and has been suggested to mediate ROS i n g e s t i o n ( B u m s i d e , 1976). R e c e p t o r - l i g a n d mediated phagocytos i s i n macrophages r e q u i r e s a c t i n - m y o s i n i n t e r a c t i o n s (Bourguignon and S i n g e r , 1977). S tudies w i t h a c t i n a n t i b o d i e s show t h a t a c t i n i s d i s t r i b u t e d n o r m a l l y i n d y s t r o p h i c RPE c e l l s and accumulates beneath each bound ROS i n the i n i t i a l phase o f i n g e s t i o n ( C h a i t i n and H a l l , 1983b). S e v e r a l o ther RPE c y t o s k e l e t a l p r o t e i n s , such as, v i m e n t i n , myosin, s p e c t r i n and a lpha-a c t i n i n , have been i d e n t i f i e d by immunoblots and immunofluorescence ( P h i l p and Nachmias, 1985). I n d i r e c t immunofluorescence l a b e l i n g w i t h a n t i b o d i e s to t u b u l i n were used to s tudy the d i s t r i b u t i o n o f micro tubu le s i n r a t RPE c e l l c u l t u r e s ( I rons and K a l n i n s , 1984). They found no apparent m i c r o t u b u l e de fec t i n both spread ing and f u l l y spread d y s t r o p h i c RPE c e l l s , r e n d e r i n g i t u n l i k e l y t h a t an a l t e r e d d i s t r i b u t i o n o f micro tubu le s i s r e s p o n s i b l e f o r the phagocyt i c de fec t i n these c e l l s . I n essence, both normal and d i sea sed RPE c e l l s appear to c o n t a i n a l l the necessary c y t o s k e l e t a l components f o r ROS p h a g o c y t o s i s . However, one or more o f these components may not be p r o p e r l y a c t i v a t e d upon ROS b i n d i n g , or a l t e r n a t i v e l y , a y e t undetermined c y t o s k e l e t a l p r o t e i n may be l a c k i n g i n d y s t r o p h i c RPE c e l l s . -2 6-THE ROLE OF CARBOHYDRATES IN ROS/RPE INTERACTIONS C e l l sur face carbohydrates on g l y c o p r o t e i n s are thought to be i n v o l v e d i n the r e c o g n i t i o n o f molecules on c e l l s u r f a c e s . On t h i s premise , i n v e s t i g a t i o n s were made i n t o the r o l e o f carbohydrates i n s p e c i f i c l i g a n d -r e c e p t o r r e c o g n i t i o n o f ROS by RPE c e l l s . O ' B r i e n (1976) observed t h a t a s m a l l f r a c t i o n o f rhodops in c o u l d be f u c o s y l a t e d and g a l a c t o s y l a t e d . Consequent ly , he proposed t h a t t h i s a d d i t i o n a l g l y c o s y l a t i o n might s i g n a l shedding o f the ROS t i p and these a d d i t i o n a l sugars may ac t as a l i g a n d f o r subsequent RPE p h a g o c y t o s i s . One o f the most v a l u a b l e b i o c h e m i c a l probes used to s tudy exposed carbohydrates on c e l l sur faces are p l a n t l e c t i n s . H a l l and N i r (1976) found t h a t c o n c a n a v a l i n A (Con A) f e r r i t i n - c o n j u g a t e s , s p e c i f i c f o r mannose and g lucose , l a b e l e d the plasma membrane o f both normal and d y s t r o p h i c RPE c e l l s w i t h s i m i l a r i n t e n s i t i e s . However, McLaughl in and Wood (1980) observed t h a t Lens c u l i n a r i s a g g l u t i n i n (LCA)-peroxidase con jugate , which i s a l s o s p e c i f i c f o r mannose and g lucose , p r e f e r e n t i a l l y s t a i n e d the m i c r o v i l l i o f normal RPE c e l l s more i n t e n s e l y than d i seased RPE c e l l s . The d i s c repancy between these two s t u d i e s may be due to d i f f e r e n c e s i n the m i c r o s p e c i f i c i t i e s o f Con A and LCA. I n c o n t r a s t to t h e i r e a r l i e r work, McLaugh l in e t a l . (1984) found t h a t Lens c u l i n a r i s h a e m a g g l u t i n i n f e r r i t i n -con jugates , s p e c i f i c f o r mannosy l -conta in ing g lycocon juga te s , l a b e l e d the plasma membrane o f d y s t r o p h i c RPE c e l l s more i n t e n s e l y than normal RPE c e l l s . Thus, the proper i n t e r p e t a t i o n o f these l e c t i n b i n d i n g s t u d i e s remains u n c l e a r . However, i t i s p o s s i b l e t h a t the masking o f the LCA b i n d i n g s i t e s may p l a y a r o l e i n ROS r e c o g n i t i o n by RPE c e l l s . L e c t i n b i n d i n g s t u d i e s were a l s o used i n c o n j u n c t i o n w i t h phagocytos i s assays . H a l l and Quon (1980) r e p o r t e d t h a t both ROS and RPE c e l l s must be - 2 7 -coated w i t h l e c t i n s i n order to s i g n i f i c a n t l y i n h i b i t p h a g o c y t o s i s . I n c o n t r a s t , P h i l p and B e r n s t e i n (1980) i n d i c a t e d t h a t Con A coated ROS alone c o u l d i n h i b i t phagocytos i s by as much as 50%. Consequent ly , the d i r e c t i m p l i c a t i o n o f l e c t i n l a b e l i n g on ROS phagocytos i s remains i n c o n c l u s i v e . Another approach to s t u d y i n g the r o l e o f carbohydrates i n c e l l - c e l l a s s o c i a t i o n was to attempt to i n h i b i t the process by h i g h c o n c e n t r a t i o n s o f s imple sugars . I n i t i a l i n h i b i t i o n s t u d i e s have shown t h a t mannose ( P h i l p and B e r n s t e i n , 1980) and L-fucose and D-mannose (Heath and B a s i n g e r , 1983) can i n h i b i t ROS b i n d i n g by RPE c e l l s . Moreover, p o l y s t y r e n e beads coated w i t h mannose were b e l i e v e d to be s p e c i f i c a l l y phagocy t i zed by RPE c e l l s ( S e y f r i e d - W i l l i a m s and M c L a u g h l i n , 1983). Such experiments suggested t h a t sugar s e n s i t i v e , i n p a r t i c u l a r , mannose s e n s i t i v e r e c e p t o r s may e x i s t on the RPE c e l l s u r f a c e . However, recent s t u d i e s by L e n t r i c h i a e t a l . (1987) and Shirakawa et a l . (1987) have c o n v i n c i n g l y shown t h a t h i g h c o n c e n t r a t i o n s o f f r ee g a l a c t o s e , mannose, or N-ace ty l -g lucosamine sugars do not i n h i b i t the b i n d i n g o f r e c o n s t i t u t e d rhodops in v e s i c l e s , d i s c membranes or ROS by RPE c e l l s . A p o s s i b l e e x p l a n a t i o n f o r these c o n f l i c t i n g r e s u l t s maybe t h a t extremely h i g h c o n c e n t r a t i o n s o f sugars may cause n o n - s p e c i f i c i n h i b i t o r y e f f e c t s . A l t e r n a t i v e l y , the stage o f RPE growth at the time o f the phagocytos i s assay may be impor tant . Another i n t e r e s t i n g theory developed from the o b s e r v a t i o n t h a t i n d y s t r o p h i c r a t s , shed ROS t i p s which had accumulated i n the s u b r e t i n a l space f a i l e d to l a b e l w i t h c o l l o d i a l i r o n , w h i l e i n t a c t ROS l a b e l e d i n t e n s e l y w i t h c o l l o d i a l i r o n (Cohen and N i r , 1984). C o l l o d i a l i r o n has p r e v i o u s l y been shown to b i n d s p e c i f i c a l l y to c e l l sur face s i a l i c a c i d r e s idues (Weiss and Sub jec t , 1974). Th i s suggested t h a t changes i n sur face s i a l i c a c i d r e s idues on ROS may be l i n k e d to the l o s s o f s p e c i f i c RPE r e c o g n i t i o n o f shedded ROS fragments . More r e c e n t l y , s i a l a t e d g l y c o p r o t e i n and s i a l i c - 2 8 -a c i d b i n d i n g l e c t i n coated beads were shown not to f a c i l i t a t e the phagocyt i c i n t e r a c t i o n between beads and RPE c e l l s (Tarnowski and M c L a u g h l i n , 1987). Consequent ly , the s p e c i f i c r o l e o f s i a l i c a c i d r e s idues i n receptor-media ted phagocytos i s remains q u e s t i o n a b l e . IMMUNOCYTOCHEMICAL LABELING STUDIES Immunological s t u d i e s have been concern w i t h the s i t e o f ant ibody-a n t i g e n i n t e r a c t i o n . Up u n t i l the mid 70's a l l immunocytochemistry performed i n v o l v e d p o l y c l o n a l a n t i b o d i e s . A s i g n i f i c a n t advancement i n immunologica l r e sea rch was the p r o d u c t i o n o f monoclonal a n t i b o d i e s u s i n g c e l l f u s i o n techniques developed by K o h l e r and M i l s t e i n (1975), as reviewed by M i l s t e i n (1980). A n t i b o d i e s o f d e f i n e d s p e c i f i c i t y produced s e n s i t i v e s e r o l o g i c a l and b i o c h e m i c a l probes . Labe led a n t i b o d i e s have a l s o p r o v i d e d i n s i g h t i n t o the l o c a t i o n and d i s t r i b u t i o n o f s p e c i f i c components i n c e l l s u s i n g l i g h t and e l e c t r o n m i c r o s c o p i c t echniques . S e v e r a l monoclonal a n t i b o d i e s have been r a i s e d aga in s t rod outer segment p r o t e i n s (MacKenzie and Molday, 1982; W i t t e t a l . , 1984; Molday et a l . , 1986; Wong and Molday, 1986) and i n p a r t i c u l a r rhodops in (DeGrip , 1985; Molday and MacKenzie , 1983; MacKenzie e t a l . , 1984). S e v e r a l C - t e r m i n a l s p e c i f i c a n t i - r h o d o p s i n monoclonal a n t i b o d i e s have been p r e c i s e l y l o c a l i z e d u s i n g s y n t h e t i c pept ides (MacKenzie e t a l . , 1984). A n t i r h o d o p s i n s p e c i f i c N-t e r m i n a l a n t i b o d i e s have a l s o been i d e n t i f i e d and p a r t i a l l y c h a r a c t e r i z e d (Molday and MacKenzie , 1983; H i c k s and Molday, 1986; Hargrave e t a l . , 1986). THESIS INVESTIGATION The p r i n c i p a l o b j e c t i v e o f t h i s t h e s i s has been to c h a r a c t e r i z e the ROS l i g a n d ( s ) i n v o l v e d i n s p e c i f i c l i g a n d - r e c e p t o r i n t e r a c t i o n s between ROS and -2 9-RPE c e l l s . I n l i n e w i t h t h i s o b j e c t i v e , the hypothes i s was i n v e s t i g a t e d t h a t rhodops in l o c a l i z e d i n the ROS plasma membrane might ac t as the l i g a n d i n receptor -media ted p h a g o c y t o s i s . The f i r s t s e c t i o n o f t h i s t h e s i s (Chapter 2) i s devoted to f u l l y c h a r a c t e r i z i n g p r e v i o u s l y produced a n t i r h o d o p s i n monoclonal a n t i b o d i e s by c o m p e t i t i v e i n h i b i t i o n assays and immunocytochemical l a b e l i n g s t u d i e s . Moreover, a d d i t i o n a l a n t i r h o d o p s i n monoclonal a n t i b o d i e s were r a i s e d and ana lyzed by SDS-gel e l e c t r o p h o r e s i s and immunoblott ing t e c h n i q u e s . The r e s u l t i n g l i b r a r y o f c h a r a c t e r i z e d a n t i r h o d o p s i n monoclonal a n t i b o d i e s was employed as a b i o c h e m i c a l t o o l to e l u c i d a t e the s t r u c t u r e , f u n c t i o n and topography o f membrane-bound r h o d o p s i n . Chapter 3 i s devoted to s t u d y i n g the p o s s i b l e r o l e o f rhodops in i n the b i n d i n g and phagocytos i s o f rod outer segments by c u l t u r e d bovine RPE c e l l s . For these s t u d i e s , i s o l a t e d and s y n t h e t i c rhodops in pept ide s were employed i n an attempt to i n h i b i t the phagocytos i s o f ROS. Moreover, an N - t e r m i n a l s p e c i f i c a n t i r h o d o p s i n monoclonal ant ibody was s u c c e s s f u l l y used as a b i o c h e m i c a l and immunocytochemical probe i n d e f i n i n g the r o l e o f rhodops in i n receptor -media ted phagocytos i s o f ROS. S ince exper imenta l evidence p r o v i d e d i n Chapter 3 suggests t h a t rhodops in i s not the l i g a n d r e s p o n s i b l e f o r s p e c i f i c ROS b i n d i n g to RPE c e l l s , f u r t h e r b i o c h e m i c a l and immunochemical s t u d i e s are d e s c r i b e d i n Chapter 4 i n an attempt to de f ine the ROS c e l l sur face component(s) i n v o l v e d i n s p e c i f i c ROS/RPE i n t e r a c t i o n s . I n these s t u d i e s , the e f f e c t o f enzyme-treatment on ROS b i n d i n g to RPE c e l l s was ana lyzed by l i m i t e d p r o t e o l y s i s , SDS-gel e l e c t r o p h o r e s i s and western b l o t t i n g t echn iques . L e c t i n - a f f i n i t y and immunoaf f in i ty chromatographic techniques were employed i n an attempt to i s o l a t e the ROS l i g a n d i n v o l v e d i n s p e c i f i c ROS b i n d i n g to RPE c e l l s . These s t u d i e s p r o v i d e d per suas ive evidence t h a t the ROS plasma membrane s p e c i f i c - 3 0 -230kD g l y c o p r o t e i n may ac t as a ROS l i g a n d i n receptor -media ted phagocytos i s o f ROS by RPE c e l l s . CHAPTER 2 A n t i g e n i c Determinants on Rhodopsin as  Def ined by Monoclonal A n t i b o d i e s MATERIALS AND METHODS MATERIALS Chymotryps in , ( T P C K ) - t r y p s i n , soybean t r y p s i n i n h i b i t o r , hypoxanthine , t h y m i d i n e , a m i n o p t e r i n , Tween 20, p h e n y l m e t h y l s u l f o n y l f l u o r i d e and p o l y - L -l y s i n e (M^ = 380,000) were a l l purchased from Sigma Chemical Co. ( S t . L o u i s , MO). Chloramine T, cyanogen bromide, and Carbowax 20 M were o b t a i n e d from F i s h e r S c i e n t i f i c Co. (Ottawa, O n t a r i o ) . P o l y e t h y l e n e g l y c o l 1500, g o l d -c h l o r i d e and t r i f l u o r o a c e t i c a c i d (TFA) were a c q u i r e d from BDH Chemical Co. ( M o n t r e a l , Quebec). Staphlococcus aureus V-8 pro tea se , n o r l e u c i n e and Iodo-Beads were purchased from P i e r c e Chemical Co. (Rockford , I L ) , w h i l e F i c o l l 400, DEAE Sephacel and Sepharose 2B-CL were ob ta ined from Pharmacia F ine Chemicals I n c . (Uppsala , Sweden). I s c o v e ' s m o d i f i e d Dulbecco ' s medium (IMDM), Freund ' s complete and incomplete ad juvant , f e t a l c a l f serum (FCS), p e n i c i l l i n / s t r e p t o m y c i n , and fungizone were a l l purchased from Gibco L a b o r a t o r i e s (Grand I s l a n d , NY) . P r i s t a n e was ob ta ined from A l d r i c h Chemical Co. (Milwaukee, WI) w h i l e AG 1-X10 a n i o n exchange r e s i n was purchased from Bio-Rad L a b o r a t o r i e s (Richmond, CA) . F i n a l l y , r a b b i t anti-mouse I g subc las s s p e c i f i c a n t i b o d i e s were purchased form Boehringer Manneheim B iochemica l s ( I n d i a n a p o l i s , IN) and 125 Na I was ob ta ined from New England Nuc lear (Lach ine , Quebec). A l l o ther chemica l s were o f reagent grade. Phosphate-buf fered s a l i n e (PBS) c o n t a i n e d 0.14 M N a C l , 2.6 mM KC1, 1.4 -32-mM K H 2 P 0 4 and 8.1 nM NaHP0 4 > pH 7 .4 . Pept ides corre sponding to the F ^ - ^ loop (amino a c i d r e s idues 231-252) and to the C - t e r m i n a l segment o f bovine rhodops in were generous g i f t s of D r . P a u l Hargrave; the pept ide corresponding to amino a c i d r e s idues 61-74 o f rhodops in was purchased from I n s t i t u t Armand-Frappier ( L a v a l , Quebec); and the pept ide corre sponding to the 1-16 amino a c i d N - t e r m i n a l segment o f r h o d o p s i n was generous ly s u p p l i e d by Dr . Dan O p r i a n . These pept ide s were s y n t h e s i z e d by the s o l i d - p h a s e M e r r i f i e l d method ( M e r r i f i e l d , 1963). METHODS PROTEIN ASSAYS P r o t e i n c o n c e n t r a t i o n s o f ROS membranes were determined by the method o f Lowry e t a l . (1951) u s i n g BSA as a s t andard . L e c t i n and ant ibody p r o t e i n c o n c e n t r a t i o n s were determined by absorbance at 280 nm u s i n g an e x t i n c t i o n c o e f f i c i e n t (mg/mL) o f 1 .3 . PREPARATION OF ROS MEMBRANES ROS were prepared a c c o r d i n g to the method d e s c r i b e d by Molday et a l . (1987). R o u t i n e l y , ROS were prepared under dim red l i g h t from 50-100 f r e s h bovine eyes ob ta ined from I n t e r c o n t i n e n t a l Packers (Vancouver, B . C . ) The eyes were c a r e f u l l y d i s s e c t e d to remove the r e t i n a from the back o f the o p t i c cup. I s o l a t e d r e t i n a s were g e n t l y s w i r l e d f o r 1 min i n 25 mL of c o l d homogenizat ion s o l u t i o n (20% sucrose , 0.25 mM M g C l ^ , 10 mM g lucose , 5 mM t a u r i n e , 20 mM T r i s - a ce t a te , pH 7.4) and f i l t e r e d twice through a T e f l o n 300 um mesh s c r e e n . A p p r o x i m a t e l y , 5 mL of f i l t r a t e was l a y e r e d on a 22 mL 25-60% sucrose g r a d i e n t c o n t a i n i n g 10 mM t a u r i n e , 10 mM glucose and 20 mM T r i s --33-a c e t a t e , pH 7.4 and c e n t r i f u g e d i n a Beckman SW 27 r o t o r a t 25,000 rpm f o r 50 min at 4 ° C . I n t a c t ROS c o l l e c t e d from a band near the top o f the g r a d i e n t s were d i l u t e d i n 5 volumes o f homogenizat ion s o l u t i o n and washed by c e n t r i f u g a t i o n i n a S o r v a l l SS-34 r o t o r f o r 10 min at 9000 rpm. The p e l l e t was resuspended i n homogenizat ion b u f f e r to a f i n a l c o n c e n t r a t i o n o f 4-6 mg/mL. For CNBr cleavage s t u d i e s , p r o t e o l y s i s and c o m p e t i t i o n assays , ROS d i s c membranes were prepared by hypoton ic l y s i s o f the ROS f o l l o w e d by f l o a t a t i o n on 5% F i c o l l 400 a c c o r d i n g to the method o f Smith e t a l . (1975). The i s o l a t e d d i s c membranes were washed 2 t imes i n 20 mM T r i s - a c e t a t e , pH 7 .4 , by c e n t r i f u g a t i o n at 15,000 rpm f o r 20 min i n a S o r v a l l SS-34 r o t o r . For EM s t u d i e s , ROS d i s c s which m a i n t a i n t h e i r c h a r a c t e r i s t i c f l a t t e n e d d i s c shape were prepared as d e s c r i b e d by Molday e t a l . (1987). B r i e f l y , a f r e s h p r e p a r a t i o n o f rod outer segments was washed i n 20 mM T r i s - a c e t a t e , pH 7 .4 , resuspended i n 2 mL of the same b u f f e r and t r e a t e d w i t h 0 .4 ug/mL of t r y p s i n i n 20 mM T r i s - a c e t a t e f o r 20 min at 23 °C . The r e a c t i o n was stopped by adding 50 uL o f soybean t r y p s i n i n h i b i t o r (8 mg/mL). The ROS d i s c s were then f i x e d w i t h 1.25% g lu ta ra ldehyde i n 0.1 M cacody la te b u f f e r , pH 7 . 2 , c o n t a i n i n g 0.2 M sucrose f o r 15 min a t 4°C and washed 3 t imes i n 20 mM T r i s -a c e t a t e , pH 7 . 2 , by c e n t r i f u g a t i o n at 15,000 rpm f o r 20 min i n an S o r v a l l SS-34 r o t o r . PROTEOLYSIS OF ROS DISC MEMBRANES Rhodopsin i n ROS d i s c membranes was d i g e s t e d w i t h 0.1 mg/mL o f t r y p s i n or S_^  aureus V-8 protease f o r 2 h a t 23°C as p r e v i o u s l y d e s c r i b e d (Molday and MacKenzie , 1983). A f t e r the r e a c t i o n was stopped w i t h soybean t r y p s i n i n h i b i t o r f o r t r y p s i n d i g e s t i o n or p h e n y l m e t h y l s u l f o n y l f l u o r i d e f o r S.  aureus V-8 protease d i g e s t i o n , the d i s c membranes were washed twice i n 10 mM -34-T r i s b u f f e r , pH 7 .4 , by c e n t r i f u g a t i o n at 15,000 rpm f o r 20 min i n a S o r v a l l SS-34 r o t o r . The ROS membrane p e l l e t was resuspended i n T r i s b u f f e r a t a p r o t e i n c o n c e n t r a t i o n o f 3-4 mg/mL. I n the case o f chymotryps in d i g e s t i o n , ROS d i s c s were t r e a t e d w i t h 1 mg/mL chymotryps in f o r 2.5 h a t 2 3 ° C . The r e a c t i o n was stopped w i t h p h e n y l m e t h y l s u l f o n y l f l u o r i d e , washed and resuspended as d e s c r i b e d above. SDS-GEL ELECTROPHORESIS AND GEL TRANSFER ROS and ROS d i s c s were s o l u b i l i z e d i n an equal volume o f d e n a t u r i n g b u f f e r c o n t a i n i n g 4% SDS, 10% 2-mercaptoethanol , 40% sucrose and 10 mM T r i s , pH 7 . 0 . G e n e r a l l y , 5-15 uL o f sample c o n t a i n i n g 7-20 ug p r o t e i n was a p p l i e d to w e l l s o f e i t h e r a 6.5-15% p o l y a c r y l a m i d e g r a d i e n t or 8-10% cont inuous p o l y a c r y l a m i d e s l a b ge l s (0.75 mm x 5 .0 -8 .0 cm) and e l e c t r o p h o r e s i s was c a r r i e d out a t 20-25 mA u s i n g the b u f f e r system of Laemmli (Laemmli, 1970). The c o n c e n t r a t i o n o f b i s - a c r y l a m i d e was 0.2%, 0.4% or 0.8% as i n d i c a t e d . G e l s l i c e s were e i t h e r s t a i n e d w i t h coomassie b lue (Fa i rbanks e t a l . , 1971) or sub j ec ted to e l e c t r o p h o r e t i c t r a n s f e r onto n i t r o c e l l u l o s e paper (Towbin e t a l . , 1979). B r i e f l y , SDS ge l s were washed twice w i t h 100 mL volumes o f t r a n s f e r b u f f e r c o n s i s t i n g o f 20 mM T r i s - a c e t a t e , 2 mM EDTA and 0.01% SDS, pH 7 . 2 . E l e c t r o p h o r e t i c t r a n s f e r was c a r r i e d out i n B io-Rad T r a n s b l o t apparatus or a Hoefer Transphor E l e c t r o p h o r e s i s U n i t i n t r a n s f e r b u f f e r a t 0 .35-0 .50 A f o r 1-3 h a t 4 ° C . F o l l o w i n g t r a n s f e r the n i t r o c e l l u l o s e paper was quenched o f any remaining p r o t e i n b i n d i n g s i t e s by i n c u b a t i o n f o r 1-2 h i n a T r i t o n X-100 immunoblot b u f f e r (0.15 M N a C l , 1 mM EDTA, 1 mM NaN^, 0.2% T r i t o n X-100, 10 mM sodium phosphate, pH 7.5) c o n t a i n i n g 4% BSA or a Tween 20 immunoblot b u f f e r (0.15 M N a C l , 1 mM EDTA, 1 mM NaN^, 0.5% Tween 20 and 20 mM T r i s - a c e t a t e , pH 7 . 4 ) . -35-MONOCLONAL ANTIBODY TECHNIQUES A . Immunization and C e l l F u s i o n Female B a l b / c mice were immunized w i t h i n t r a p e r i t o n e a l i n j e c t i o n s o f 100 ug o f bovine ROS membranes or crude RPE plasma membrane p r e p a r a t i o n s ( L a i r d , 1984) e m u l s i f i e d i n 0.1 mL of Freund ' s adjuvant a t 3 week i n t e r v a l s . Four days a f t e r the t h i r d immunizat ion the sp leen was removed and used f o r c e l l 8 7 f u s i o n . Approx imate ly 2 x 10 sp leen c e l l s were fused w i t h 1 x 10 NS-1 mouse myeloma c e l l s i n 1.0 mL of 50% p o l y e t h y l e n e g l y c o l 1500 as d e s c r i b e d by G a l f r e and M i l s t e i n (1981). A f t e r the f u s i o n the c e l l s were resuspended i n 100 mL of I s c o v e ' s m o d i f i e d Dulbecco ' s medium (IMDM) c o n t a i n i n g 100 uM hypoxanth ine , 0 .4 uM aminopte r in and 16 uM thymidine supplemented w i t h 20% f e t a l c a l f serum, a n t i b i o t i c s (100 U/mL p e n i c i l l i n , 100 U/mL s t r e p t o m y c i n and 0.25 ug/mL f u n g i z o n e ) , and 2 x 10^ B a l b / c feeder thymocytes/mL. A l i q u o t s o f 1 mL were p l a t e d out i n t o 24 w e l l p l a t e s and the fused c e l l s were grown a t 37°C i n an atmosphere o f 5% C O 2 and 95% a i r . P o s i t i v e ant ibody produc ing c e l l s were de tec ted by the s o l i d - p h a s e radioimmune assay method, 10-14 days a f t e r the f u s i o n . B. Hybridoma C l o n i n g , F r e e z i n g and A s c i t e s Format ion P o s i t i v e ant ibody s e c r e t i n g hybridoma c e l l s were c l o n e d by l i m i t i n g s e r i a l d i l u t i o n . Approx imate ly 750-1000 c e l l s were p l a t e d out i n 0.1 mL of IMDM c o n t a i n i n g 20% FCS, 100 uM hypoxanthine and 16 uM thymidine i n 96 w e l l p l a t e s a t a c o n c e n t r a t i o n o f 5 c e l l s / w e l l or 1 c e l l / w e l l w i t h 2 x 10^ mouse thymocyte feeder c e l l s / m L . A f t e r 10-14 days s i n g l e ant ibody s e c r e t i n g c o l o n i e s were assayed by RIA and p o s i t i v e w e l l s were expanded f o r ant ibody p r o d u c t i o n e i t h e r i n c u l t u r e f l u i d or i n a s c i t e s f l u i d . -36-Hybridoma c e l l s were grown to l o g phase i n IMDM supplemented w i t h 10% FCS p r i o r to f r e e z i n g . Approx imate ly 4-6 x 10^ c e l l s i n growth medium supplemented w i t h 10% DMSO i n a t o t a l volume o f 1 mL were s l o w l y f r o z e n i n s m a l l c r y o v i a l s p l a c e d i n a foam i n s u l a t e d c e l l box a t - 7 0 ° C . A f t e r 24 h the c r y o v i a l s were t r a n s f e r r e d to l i q u i d n i t r o g e n and s t o r e d i n d e f i n i t e l y . Monoclonal an t ibody s e c r e t i n g hybridoma c e l l l i n e s were i n j e c t e d i n t o B a l b / c mice f o r the p r o d u c t i o n o f a s c i t e s tumors (MacKenzie and Molday, 1983). B a l b / c mice were i n j e c t e d i n t r a p e r i t o n e a l l y w i t h 0.5 mL o f p r i s t a n e f o l l o w e d 7 days l a t e r by the i n j e c t i o n o f 5-10 x 10^ hybridoma c e l l s . A s c i t e s f l u i d was c o l l e c t e d 10-14 days l a t e r by s a c r i f i c i n g the mice and d r a i n i n g the f l u i d from t h e i r i n t r a p e r i t o n e a l c a v i t y . The a s c i t e s f l u i d was separated from the red b l o o d c e l l s by c e n t r i f u g a t i o n a t 12,000 rpm f o r 10 min i n a S o r v a l l SS-34 r o t o r and immediately f r o z e n at -70°C f o r f u t u r e use . C. Monoclonal Ant ibody P u r i f i c a t i o n Monoclonal a n t i b o d i e s were p u r i f i e d from mouse a s c i t e s f l u i d by ammonium s u l f a t e f r a c t i o n a t i o n and DEAE i o n exchange chromatography (Garvey e t a l . , 1977). B r i e f l y , 1 volume of a s c i t e s f l u i d was d i l u t e d w i t h 3 volumes o f PBS and c e n t r i f u g e d a t 10,000 rpm f o r 10 min i n a S o r v a l l SS-34 r o t o r . The supernatant was p r e c i p i t a t e d w i t h an equal volume of s a t u r a t e d ammonium s u l f a t e s o l u t i o n at 4 °C f o r 1 h . The s o l u t i o n was r e c e n t r i f u g e d as d e s c r i b e d above. The p e l l e t was d i s s o l v e d i n 20 mM T r i s - a c e t a t e , pH 7 .9 , c o n t a i n i n g 40 mM NaCl and d i a l y z e d aga i n s t s e v e r a l changes o f 20 mM T r i s - a c e t a t e , pH 7 .9 , c o n t a i n i n g 20 mM NaCl o v e r n i g h t a t 4 ° C . The d i a l y z a t e was a p p l i e d to a DEAE-Sephacel column (5-6 mg p r o t e i n / m L column) p r e - e q u i l i b r a t e d w i t h 20 mM T r i s -a c e t a t e , pH 7 .9 , c o n t a i n i n g 20 mM N a C l . The ant ibody molecules were e l u t e d w i t h a cont inuous NaCl c o n c e n t r a t i o n g r a d i e n t (20-300 mM) i n 20 mM T r i s -a c e t a t e , pH 7 . 9 . Ant ibody p u r i t y was determined by SDS-polyacrylamide g e l -37-e l e c t r o p h o r e s i s and a c t i v i t y was measured by RIA. Pooled f r a c t i o n s were d i a l y z e d aga in s t 10 mM ammonium b i c a r b o n a t e , pH 7 . 2 , or 10 mM sodium phosphate, pH 7 . 2 , p r i o r to l y o p h i l i z a t i o n and s to rage . D. P r e p a r a t i o n and I o d i n a t i o n o f A n t i b o d i e s and L e c t i n s Goat anti-mouse I g and goat a n t i - r a b b i t Ig were p u r i f i e d by a f f i n i t y chromatography on a mouse Ig or r a b b i t I g Sepharose 4B column, r e s p e c t i v e l y (MacKenzie and Mo1day, 1982). P u r i f i e d a n t i b o d i e s and l e c t i n s were i o d i n a t e d by the chloramine T method (Hunter and Greenwood, 1962) to g ive s p e c i f i c a c t i v i t i e s o f 1-2 x l o ' dpm/ug. Approx imate ly 0 .5 -1 .0 mg o f ant ibody or l e c t i n was d i s s o l v e d i n 1 125 mL o f PBS f o l l o w e d by the a d d i t i o n o f 0.5 mCi-1 .0 mCi o f Na I . The r e a c t i o n was i n i t i a t e d by the a d d i t i o n o f 25 uL o f chloramine T (4 mg/mL i n H^O). A f t e r 10 min the r e a c t i o n was stopped by adding 20 uL o f 4 mg/mL 125 sodium m e t a b i s u l f i t e . The f ree Na I was removed by c e n t r i f u g i n g the r e a c t i o n mix ture f o r 5 min through a Centrex M i c r o f i l t e r U n i t ( S c h l e i c h e r and S c h u e l l ) loaded w i t h 0.5 g o f AG 1-X10 a n i o n exchange r e s i n . The a n t i r h o d o p s i n an t ibody , rho 4D2, was i o d i n a t i o n under more m i l d c o n d i t i o n s u s i n g Iodo-Beads. I n t h i s in s tance no r e d u c i n g reagent was r e q u i r e d to stop the r e a c t i o n . E. Standard Radioimmune Assay A s tandard s o l i d - p h a s e radioimmune assay (RIA) was used to de tec t ant ibody s e c r e t i n g hybridomas accord ing to the procedure developed by MacKenzie and Molday (1982). ROS or ROS d i s c membrane p r e p a r a t i o n s (2 .5 mg/mL) were s o l u b i l i z e d i n 1% T r i t o n X-100 and d i l u t e d to 0.25 mg/mL w i t h d i s t i l l e d H_0. A l i q u o t s o f 25 uL were d r i e d down on f l e x v i n y l U-shaped -38-m i c r o t i t r e w e l l s a t 60°C f o r 2 h . The w e l l s were washed i n d i s t i l l e d H^O p r i o r to b e i n g quenched f o r 30-60 min i n RIA b u f f e r (1% BSA and 0.1% NaN^ i n PBS) . The p l a t e was washed b r i e f l y i n PBS and incubated w i t h 25 uL o f hybridoma c u l t u r e f l u i d f o r 30-60 min at 23 °C . The p l a t e was washed e x t e n s i v e l y w i t h PBS to remove unbound ant ibody and subsequent ly incubated 125 6 w i t h 25 uL o f I - l a b e l e d goat anti-mouse Ig (1-2 x 10 dpm/ug) f o r 1 h a t 2 3 ° C . F i n a l l y the p l a t e was washed e x t e n s i v e l y as be fore and the r a d i o a c t i v i t y was measured i n a Beckman 8000 Gamma Counter . When monoclonal a n t i b o d i e s were c l a s s i f i e d i n t o s p e c i f i c subtypes , i m m o b i l i z e d ROS p r o t e i n s were quenched i n RIA b u f f e r and incubated w i t h hybridoma c u l t u r e f l u i d as d e s c r i b e d above. A f t e r washing i n PBS, the w e l l s were t r e a t e d w i t h subc las s s p e c i f i c r a b b i t anti-mouse immunoglobulin f o r 30-125 60 min f o l l o w e d by washing and ant ibody d e t e c t i o n w i t h I - l a b e l e d goat a n t i - r a b b i t I g . F. So l id -Phase Radioimmune Compet i t ion Assays The b i n d i n g o f ROS, ROS d i s c s , rhodops in pept ides and s y n t h e t i c pept ides to monoclonal a n t i b o d i e s was measured by s o l i d - p h a s e radioimmune c o m p e t i t i o n assays as p r e v i o u s l y d e s c r i b e d (MacKenzie e t a l . , 1984). B r i e f l y , 50 uL o f a n t i g e n was incuba ted w i t h 25 uL o f hybridoma c u l t u r e f l u i d a t a c o n c e n t r a t i o n which gave 80-90% s a t u r a t i o n o f b i n d i n g by the s tandard s o l i d -phase RIA method (MacKenzie and Molday, 1982). A f t e r 1 h , 50 uL o f the mix ture was removed and screened f o r remaining ant ibody a c t i v i t y by the s tandard s o l i d - p h a s e radioimmune assay u s i n g T r i t o n X-100 s o l u b i l i z e d r h o d o p s i n bound to m i c r o t i t r e w e l l s as the i m m o b i l i z e d a n t i g e n . I n some in s t ance s the c o m p e t i t i o n assays were performed under dim red l i g h t . I n o ther c o m p e t i t i v e i n h i b i t i o n assays , rhodops in (0 .3 -0 .5 uM) i n ROS d i s c membranes was incubated i n RIA b u f f e r w i t h p u r i f i e d a n t i r h o d o p s i n -39-monoclonal a n t i b o d i e s ( 0 . 7 -6 .4 uM) i n a t o t a l volume of 200 uL . A f t e r 1 h , 125 6 25 uL o f I - l a b e l e d rho 3A6 (4.0 x 10 dpm/ug) a n t i r h o d o p s i n monoclonal ant ibody was added to each sample and incubated f o r an a d d i t i o n a l 60 min . F i n a l l y 100 uL a l i q u o t s were loaded on top o f 10% BSA i n s m a l l Eppendorf tubes and spun a t 25,000 rpm i n a SW 27 r o t o r f o r 40 min . The r e s u l t i n g membrane p e l l e t s were cu t from the bottom o f the tubes and counted i n a Beckman Gamma 8000 counter . POLYPEPTIDE DETECTION BY MONOCLONAL ANTIBODIES N i t r o c e l l u l o s e t r a n s f e r papers were l a b e l e d f o r 1 h a t 23°C w i t h 10 mL o f 2-10 f o l d d i l u t e d hybridoma c e l l c u l t u r e f l u i d c o n t a i n i n g the monoclonal a n t i b o d y . The t r a n s f e r papers were washed 4-5 times w i t h 50 mL o f T r i t o n X-100 immunoblot b u f f e r (0.15 M N a C l , 1 mM EDTA, 1 mM NaN^, 0.2% T r i t o n X-100, 10 mM sodium phosphate, pH 7.5) f o l l o w e d by a s i n g l e wash w i t h b u f f e r c o n s i s t i n g o f 2 M u r e a , 0 .1 M g l y c i n e , and 1% T r i t o n X-100. The papers were 125 6 subsequent ly t r e a t e d w i t h 10 mL of I - l a b e l e d goat anti-mouse I g (1 x 10 dpm/mL) f o r 1-2 h a t 2 3 ° C . The t r a n s f e r papers were f i n a l l y washed as b e f o r e , a i r - d r i e d and sub jec ted to autoradiography f o r 3-12 h on Kodak Roya l X-Omat f i l m i n a X - r a y i n t e n s i f y i n g s c reen . When the monoclonal ant ibody rho 8A6 was used to l a b e l rhodops in , T r i t o n X-100 was omi t t ed from the immunoblot b u f f e r . A l t e r n a t i v e l y , the n i t r o c e l l u l o s e t r a n s f e r papers , p r e v i o u s l y quenched i n Tween 20 immunoblot b u f f e r ( B a t t e i g e r e t a l . , 1982), were l a b e l e d as above except t h a t the papers were washed i n 20 mM T r i s - a c e t a t e , pH 7 .4 , c o n t a i n i n g 0.15 M N a C l , 1 mM EDTA, 1 mM NaN and 0.05% Tween 20. -40-PREPARATION OF MONOCLONAL ANTIBODY-SEPHAROSE AFFINITY COLUMNS An immunoaf f in i ty column was prepared by the method o f Cuatrecasas (1970) as f o l l o w s : Approx imate ly 10 mL of Sepharose 2B-CL was resuspended i n an equal volume o f H^O and the pH was ad jus ted to 1 0 . 5 . The beads were a c t i v a t e d w i t h 0.5 g o f CNBr f o r 30 min a t 23°C w h i l e m a i n t a i n i n g the pH between 10-11. F o l l o w i n g a c t i v a t i o n the C N B r - a c t i v a t e d Sepharose was washed s e v e r a l times w i t h c o l d 0.01 M sodium borate b u f f e r , pH 8 . 5 . The packed Sepharose was then r e a c t e d w i t h an equal volume of p u r i f i e d monoclonal ant ibody or t r y p s i n (1-2 mg/mL) i n 0.01 M sodium b o r a t e , pH 8 . 5 , a t 4 °C f o r 14 h . The prote in-Sepharose beads were washed s e v e r a l t imes i n 0.01 M ammonium a c e t a t e , pH 7 .0 , by low speed c e n t r i f u g a t i o n and s t o r e d a t 4 °C i n PBS c o n t a i n i n g 0.1% NaN^. R o u t i n e l y , the c o u p l i n g e f f i c i e n c y was found to be 90-95%. CYANOGEN BROMIDE CLEAVAGE OF RHODOPSIN The N - t e r m i n a l 2-39 g l y c o p e p t i d e and the C - t e r m i n a l 318-348 ( l ' - 3 1 ' ) p e p t i d e o f bovine rhodops in were obta ined by cleavage o f rhodops in w i t h cyanogen bromide by m o d i f i c a t i o n of the method o f Hargrave e t a l . (1982). A p p r o x i m a t e l y , 100 mg o f ROS d i s c s were washed once i n 20 mM sodium b o r a t e , pH 8 . 5 , by c e n t r i f u g a t i o n (15,000 rpm X 20 min) and resuspended i n 10 mL of the same b u f f e r . A f t e r the d i s c s were s o l u b i l i z e d i n 1% o c t y l g l u c o s i d e , s o l i d g u a n i d i n e - H C l was added to a f i n a l c o n c e n t r a t i o n o f 6 M. The p r o t e i n s o l u t i o n was purged w i t h n i t r o g e n and 100 mg o f d i t h i o t h r e i t o l was added to the m i x t u r e . A f t e r s t i r r i n g the mix ture a t 37°C f o r 2 h , 100 uL o f e th lyene imine was added and the capped r e a c t i o n was shaken f o r 10 min . Th i s procedure was repeated three times a f t e r which 200 uL o f 2-mercaptoethanol was added to quench the unreacted e t h y l e n e i m i n e . The reduced and aminoethy la ted rhodops in was d i a l y z e d a t 4°C f o r 36 h aga in s t four changes - 4 1 -of d i s t i l l e d water . The p r e c i p i t a t e was c o l l e c t e d by c e n t r i f u g a t i o n (3000 rpm x 15 min) i n a S o r v a l l SS-34 r o t o r f o l l o w e d by two a d d i t i o n a l washes w i t h d i s t i l l e d water . The p e l l e t was resuspended i n 7 mL o f 98% formic a c i d and then ad ju s ted to 70% formic a c i d w i t h d i s t i l l e d water p r i o r to the a d d i t i o n o f 0.5 g o f cyanogen bromide. The r e a c t i o n was a l l o w e d to proceed o v e r n i g h t a t room temperature i n the dark . The mix ture was subsequent ly d r i e d under vacuum and d i s s o l v e d i n d i s t i l l e d water or 1% t r i f l u o r o a c e t i c a c i d (TFA) PURIFICATION OF RHODOPSIN PEPTIDES AND RHODOPSIN The C - t e r m i n a l 318-348 ( l ' - 3 1 ' ) rhodops in pept ide was p u r i f i e d from CNBr-c leaved pept ides by a f f i n i t y chromatography. A 3 mL rho 1D4 monoclonal ant ibody-Sepharose column was washed w i t h four volumes o f 0.05 M ammonium b i c a r b o n a t e b u f f e r , pH 7 .0 . The CNBr-c leaved rhodops in from approx imate ly 20 r e t i n a s was a p p l i e d to the column u s i n g a f l o w r a t e o f 0.2 mL min . A f t e r 14 mL o f 0.05 M ammonium b ica rbona te b u f f e r was passed through the column to remove unbound p e p t i d e , 10 mL of 0.05 M a c e t i c / f o r m i c a c i d , pH 2 .85 , was passed through to e l u t e the bound p e p t i d e . The column was s l o w l y r e -e q u i l i b r a t e d to pH 7.4 and reused as r e q u i r e d . The f r a c t i o n s were d r i e d i n a Savant Speed Vac c o n c e n t r a t o r and each f r a c t i o n was resuspended i n 400 uL of d i s t i l l e d water f o r HPLC or radioimmune assay. The N - t e r m i n a l 2-39 rhodops in pept ide was p u r i f i e d by the same procedure u s i n g a rho 4D2 monoclonal ant ibody-Sepharose a f f i n i t y column. Rhodopsin was p u r i f i e d from s o l u b i l i z e d ROS d i s c s by a f f i n i t y chromatography on a rho 2B2 monoclonal ant ibody-Sepharose column. Approx imate ly 2 mg o f ROS d i s c s were s o l u b i l i z e d i n PBS c o n t a i n i n g 1.5% CHAPS and a p p l i e d to 2 mL of rho 2B2 antibody-Sepharose column. The column -42-was washed i n the CHAPS b u f f e r and bound rhodops in was e l u t e d w i t h the same b u f f e r c o n t a i n i n g 0.1 mg/mL of a s y n t h e t i c 1-16 N - t e r m i n a l rhodops in pept ide analogue. Rhodopsin p u r i t y was determined by SDS-polyacrylamide g e l e l e c t r o p h o r e s i s . ANALYSIS OF RHODOPSIN PEPTIDES BY HIGH PRESSURE LIQUID CHROMATOGRAPHY (HPLC)  AND AMINO ACID ANALYSIS The CNBr^cleaved pept ides were separated by reverse-phase HPLC u s i n g a V a r i a n 5000 L i q u i d Chromatography system w i t h a JU. BONDAPAK C^g column (3.9 x 300 mm). The rhodops in pept ides were e l u t e d w i t h a 5-80% g r a d i e n t o f a c e t o n i t r i l e c o n t a i n i n g 0.1% TFA. The pept ide e l u t i o n was moni tored by the absorbance at 215 nm and f r a c t i o n s were c o l l e c t e d . The 2-39 N - t e r m i n a l CNBr rhodops in pept ide was f u r t h e r c l e a v e d w i t h t r y p s i n by r e a c t i n g 1.5 mL of the 2-39 g l y c o p e p t i d e w i t h approx imate ly 0.5 mL o f t ryps in-Sepharose 2B-CL beads o v e r n i g h t a t 2 3 ° C . The suspens ion was c e n t r i f u g e d to remove the t ryps in-Sepharose i n a c l i n i c a l bench top c e n t r i f u g e (2000 rpm f o r 5 m i n ) . The t r y p t i c c l e a v e d pept ides were then separated by HPLC and the 2-16 t r y p t i c g l y c o p e p t i d e was i d e n t i f i e d by amino a c i d a n a l y s i s . The compos i t ion and c o n c e n t r a t i o n o f pept ides were determined by amino a c i d a n a l y s i s u s i n g a Dionex amino a c i d a n a l y z e r . B r i e f l y , pep t ide samples were added to 1 mL o f 6N HC1 and freezed/thawed 3 times under vacuum. H y d r o l y s i s was c a r r i e d out f o r 24 h at 110°C p r i o r to c o o l i n g and d r y i n g under vacuum i n a Savant Speed Vac c o n c e n t r a t o r . Samples were rehydra ted i n 100 uL o f 0.2 M c i t r a t e , pH 2 . 2 , p r i o r to amino a c i d a n a l y s i s . C o n c e n t r a t i o n de te rmina t ions were based on a n o r l e u c i n e s t andard . - 4 3 -IMMUNOCYTOCHEMICAL LABELING OF ROS MEMBRANES A . P r e p a r a t i o n o f P r o t e i n Gold-Dextran Conjugates Monoclonal a n t i b o d i e s were conjugated to g l u t a r a l d e h y d e - a c t i v a t e d g o l d -d e x t r a n p a r t i c l e s a c c o r d i n g to the method o f H i c k s and Molday (1984) and Molday and L a i r d (1988). B r i e f l y , 8-10 nm p a r t i c l e s were prepared by adding 14 mL of 1% (w/v) sodium c i t r a t e to 200 mL of b o i l i n g d i s t i l l e d water . While the s o l u t i o n was b o i l i n g , 0.2 mL of 10% g o l d c h l o r i d e was added and the r e a c t i o n was cont inued f o r 30 min . A f t e r c o o l i n g , the s i z e o f the g o l d p a r t i c l e s was determined by e l e c t r o n m i c r o s c o p i c a n a l y s i s o f g o l d p a r t i c l e s adsorbed onto p o l y l y s i n e - t r e a t e d formvar-coated g r i d s . The g o l d p a r t i c l e s were s t a b i l i z e d by adding 5 mL o f an aqueous g l u t a r a l d e h y d e - d e x t r a n s o l u t i o n (4 mg/mL) to 200 mL o f the g o l d s o l u t i o n w h i l e s t i r r i n g c o n s t a n t l y (Molday and L a i r d , 1988). A f t e r 30 min the s t a b i l i z e d g o l d p a r t i c l e s were washed f r ee o f excess dext ran by repeated c e n t r i f u g a t i o n i n 10 mM HEPES, pH 7 .0 , i n a Beckman T i 60 r o t o r a t 35,000 rpm f o r 30 min . The f i n a l p e l l e t was resuspended i n 3-5 mL o f HEPES b u f f e r f o r p r o t e i n c o n j u g a t i o n . An a d d i t i o n a l low speed c e n t r i f u g a t i o n (1000 rpm, 5 min) i n a c l i n i c a l c e n t r i f u g e was performed to remove any g o l d aggregates . The concent ra ted c o l l o i d a l g o l d dex t ran s o l u t i o n had an absorbance o f 10-20 at 520 nm. R o u t i n e l y , 1 mg o f ant ibody or l e c t i n i n 10 mM HEPES b u f f e r , pH 7 .0 , was coupled to 3-4 mL o f g l u t a r a l d e h y d e - a c t i v a t e d g o l d - d e x t r a n f o r 1-2 h a t 23°C w i t h cons tant s t i r r i n g . The r e a c t i o n was quenched by the a d d i t i o n o f 2-3 mL of T r i s - b u f f e r e d s a l i n e (0.02 M T r i s , 0.15 M N a C l , pH 7.4) c o n t a i n i n g 1 mg/mL Carbowax 20 M. The r e a c t i o n mix ture was separated from the uncoupled p r o t e i n by l a y e r i n g on top o f 20 mL of 20% w/v sucrose i n T r i s - b u f f e r e d s a l i n e c o n t a i n i n g 1 mg/mL Carbowax 20 M and c e n t r i f u g a t i o n was c a r r i e d out i n a SW 27.0 r o t o r a t 25,000 rpm f o r 2 h . The r e s u l t i n g p e l l e t was -44-resuspended i n 3-4 mL of T r i s - b u f f e r e d s a l i n e c o n t a i n i n g 1 mg/mL BSA and d i a l y z e d a g a i n s t T r i s - b u f f e r e d s a l i n e o v e r n i g h t to remove the sucrose . F i n a l l y the go ld-conjugates were c e n t r i f u g e d at low speed (1000 rpm, 5 min) i n a c l i n i c a l c e n t r i f u g e to remove any g o l d aggrega t ion p r i o r to s torage at 4 °C i n the presence o f 10 mM NaN^. B. Pre-embedding l a b e l i n g o f ROS Membranes I s o l a t e d bov ine r e t i n a s were f i x e d w i t h 1.25% g lu ta ra ldehyde i n cacody la te b u f f e r (0 .1 M sodium c a c o d y l a t e , 6% sucrose , pH 7.2) f o r 1-2 h a t 4 ° C . A f t e r f i x a t i o n the r e t i n a s were washed w i t h s e v e r a l changes o f cacody la te b u f f e r a t 4°C f o l l o w e d by 2 changes o f T r i s - b u f f e r e d s a l i n e (TBS) c o n t a i n i n g 1 mg/mL BSA. P ieces o f f i x e d r e t i n a s were incuba ted w i t h rho 4D2 monoclonal an t ibody d i r e c t l y conjugated to 9 nm g o l d - d e x t r a n p a r t i c l e s f o r 2-4 h a t 23 C f o i l owed by s e v e r a l washes i n TBS c o n t a i n i n g 1 mg/mL BSA. F i n a l l y the samples were r e f i x e d i n g lu ta ra ldehyde as d e s c r i b e d above and prepared f o r t r a n s m i s s i o n e l e c t r o n microscopy. ROS i s o l a t e d from sucrose g r a d i e n t s were washed i n cacody la te b u f f e r by c e n t r i f u g a t i o n i n a S o r v a l l SS-34 r o t o r a t 8000 rpm f o r 8 min . The membranes were f i x e d w i t h 1.25% g lu ta ra ldehyde i n cacody la te b u f f e r f o r 30-60 min at 4 ° C . The samples were washed 3 times i n cacody la te b u f f e r and two times i n TBS c o n t a i n i n g 1 mg/mL BSA by c e n t r i f u g a t i o n i n a S o r v a l l SS 34 r o t o r a t 8000 rpm f o r 8 m i n . The samples were l a b e l e d w i t h a n t i r h o d o p s i n monoclonal a n t i b o d i e s rho 1D4 and rho 4D2 conjugated to 10 nm g o l d - d e x t r a n p a r t i c l e s f o r 1-2 h a t 2 3 ° C . Labe led ROS were washed 3 times i n TBS and 2 times i n cacody la te b u f f e r by c e n t r i f u g a t i o n p r i o r to r e f i x a t i o n w i t h 1.25% g l u t a r a l d e h y d e i n cacody la te b u f f e r f o r 1 h a t 4 ° C . The samples were washed i n cacody la te b u f f e r as d e s c r i b e d before and prepared f o r e l e c t r o n - 4 5 -microscopy . The f i x e d "pancake" shaped d i s c s were adsorbed onto g l a s s c o v e r s l i p s p r e v i o u s l y coated w i t h 0.1 mg/mL p o l y - L - l y s i n e f o r 30 min a t 2 3 ° C . A f t e r washing the samples i n TBS-BSA b u f f e r (0.02 M T r i s , 0.15 M N a C l , pH 7 .4 , c o n t a i n i n g 1 mg/mL BSA), the c o v e r s l i p s were incuba ted w i t h monoclonal ant ibody c u l t u r e f l u i d f o r 1 h a t 2 3 ° C . A f t e r the c o v e r s l i p s were aga in washed i n TBS-BSA b u f f e r , to remove excess an t ibody , the samples were incuba ted w i t h the goat anti-mouse Ig g o l d - d e x t r a n conjugates f o r 1 h . F i n a l l y , the samples were washed i n TBS, r e f i x e d and prepared f o r e l e c t r o n microscopy . C. P r e p a r a t i o n o f Membrane Samples f o r Transmi s s ion E l e c t r o n Microscopy F i x e d membrane samples were pos t f i x e d w i t h 1% osmium t e t r o x i d e i n 50 mM cacody la te b u f f e r c o n t a i n i n g 3% sucrose f o r 30-60 min a t 4 ° C . A f t e r the samples were washed i n cacody la te b u f f e r , they were dehydrated i n an e thano l s e r i e s (50%, 70%, 90%, 95% and two changes o f abso lu te e thano l ) f o r 15 min i n each at 2 3 ° C . Dehydra t ion was cont inued i n a 50:50 mix ture o f abso lute e t h a n o l and propylene oxide f o r 15 min at 23°C f o l l o w e d by a 15 min i n c u b a t i o n i n propylene o x i d e . A r a l d i t e / E p o n r e s i n p e n e t r a t i o n was f a c i l i t a t e d by i n c u b a t i n g the samples i n a 50:50 mix o f propylene ox ide and r e s i n f o r 1-2 h f o l l o w e d by 24 h i n 100% r e s i n . The embedded samples were p l a c e d i n a 60°C oven and a l l o w e d to harden f o r 24 h . The b l o c k s were trimmed and s e c t i o n s (60-80 nm) were cu t on a S o r v a l l 5000 U l t r a Microtome. The u l t r a t h i n s e c t i o n s were c o l l e c t e d on S200 or S300 copper g r i d s . F i n a l l y , the s e c t i o n s were s t a i n e d w i t h s a t u r a t e d u r a n y l ace ta te f o r 5 min and s a t u r a t e d l e a d c i t r a t e f o r 1 min at 23 °C . A l l s e c t i o n s were v iewed under a JEOL 1200EX e l e c t r o n microscope . RESULTS ANTIRHODOPSIN MONOCLONAL ANTIBODIES I n the pas t few years 11 monoclonal a n t i b o d i e s have been r a i s e d aga ins t r a t and bovine rhodops in (reviewed by Molday, 1988). Many o f these a n t i r h o d o p s i n monoclonal a n t i b o d i e s (rho 1D4, rho 3A6, rho 2C1, rho 1C5, rho 3D6 and rho 3C2) were c h a r a c t e r i z e d by l i m i t e d p r o t e o l y s i s and immunoblot t ing s t u d i e s to be s p e c i f i c f o r the C-terminus o f rhodops in (MacKenzie and Molday, 1982; Molday and MacKenzie , 1983). L a t e r , these C-t e r m i n a l s p e c i f i c a n t i r h o d o p s i n a n t i b o d i e s were p r e c i s e l y l o c a l i z e d by s o l i d - p h a s e c o m p e t i t i o n assays u s i n g s y n t h e t i c pep t ide analogues o f rhodops in (MacKenzie e t a l . , 1984; H i c k s and Molday, 1986). I n a d d i t i o n to the C - t e r m i n a l s p e c i f i c a n t i r h o d o p s i n monoclonal a n t i b o d i e s , one monoclonal ant ibody (rho 4B4) was found to b i n d to an i n t e r n a l S_^  g r i s e u s protease s e n s i t i v e s i t e a long the cy top la smic s ide o f rhodops in i n ROS d i s c membranes (MacKenzie and Molday, 1982). A f i n a l c l a s s o f a n t i r h o d o p s i n monoclonal a n t i b o d i e s (rho 4A2, rho 2B2, rho 4D2 and rho 4A3) were determined by l i m i t e d p r o t e o l y s i s and immunoblott ing techniques to be s p e c i f i c f o r the N-t e r m i n a l t w o - t h i r d s o f rhodops in (Molday and MacKenzie , 1983; MacKenzie e t a l . , 1984; H i c k s and Molday, 1986). Two of these a n t i b o d i e s , rho 4A2 and rho 4D2, were shown by c o m p e t i t i v e i n h i b i t i o n assays to be s p e c i f i c f o r the r h o d o p s i n 2-39 N - t e r m i n a l g lycopept ide (Hicks and Molday, 1986). I n t h i s s tudy the b i n d i n g p r o p e r t i e s o f these a n t i r h o d o p s i n monoclonal a n t i b o d i e s were f u r t h e r c h a r a c t e r i z e d and, i n p a r t i c u l a r , the a n t i g e n i c determinants f o r the N - t e r m i n a l s p e c i f i c a n t i r h o d o p s i n a n t i b o d i e s were more p r e c i s e l y l o c a l i z e d . I n a d d i t i o n to these monoclonal a n t i b o d i e s , when mouse myeloma c e l l s were fused w i t h sp leen lymphocytes from immunized mice , two new hybridoma c e l l l i n e s , des ignated rho 5A3 ( L a i r d , 1984) and rho 8A6, were - 4 7 -o b t a i n e d which s e c r e t e d a n t i b o d i e s r e a c t i v e towards T r i t o n X-100 s o l u b i l i z e d ROS. The hybridoma c u l t u r e f l u i d t i t e r s f o r bo th o f these a n t i b o d i e s , as w e l l as the rho 4D2 monoclonal an t ibody , were determined by radioimmune assays ( F i g . 9 ) . Rho 8A6, rho 5A3 and rho 4D2 hybridoma c u l t u r e f l u i d had h a l f maximum b i n d i n g to s o l u b i l i z e d , i m m o b i l i z e d ROS at 14 f o l d , 68 f o l d and 128 f o l d d i l u t i o n s , r e s p e c t i v e l y . IDENTIFICATION OF RHODOPSIN N-TERMINAL MONOCLONAL ANTIBODIES Four a n t i b o v i n e rhodops in monoclonal a n t i b o d i e s , rho 4D2, rho 4A3, rho 4A2 and rho 2B2, p r e v i o u s l y have been shown to b i n d to determinants l o c a t e d a long the N - t e r m i n a l t w o - t h i r d s o f rhodops in (Molday and MacKenzie , 1983; MacKenzie e t a l . , 1984; H i c k s and Molday, 1986; Molday, 1988). A new a n t i b o d y , des ignated as rho 5A3, has been i d e n t i f i e d as an a n t i r h o d o p s i n monoclonal ant ibody by SDS-gel e l e c t r o p h o r e s i s and immunoblot t ing t echn iques . I n order to l o c a l i z e the a n t i g e n i c s i t e f o r t h i s a n t i r h o d o p s i n monoclonal a n t i b o d y , rhodops in i n ROS d i s c s was d i g e s t e d w i t h t r y p s i n and S. aureus V-8 pro tea se . The membrane-bound p r o t e o l y t i c fragments were then separated by SDS-gel e l e c t r o p h o r e s i s and t r a n s f e r r e d to n i t r o c e l l u l o s e paper . The paper s t r i p s were then t r e a t e d w i t h a n t i r h o d o p s i n monoclonal 125 a n t i b o d i e s and d e t e c t i o n was made u s i n g I - l a b e l e d goat anti-mouse Ig as a second a n t i b o d y . R e s u l t s are shown i n F igure 10 f o r rho 5A3 and rho 4D2 monoclonal a n t i b o d i e s . Both monoclonal a n t i b o d i e s bound to the monomeric (M^ = 34,000) and d i m e r i c (M^ = 68,000) spec ies o f rhodops in i n u n t r e a t e d ROS ( lane a) and the p o l y p e p t i d e fragments o f apparent M^ = 32,000 and M^ = 64,000 i n t r y p s i n -t r e a t e d ROS ( lane b ) . I t has been shown t h a t the 32,000 M^ fragment corresponds to rhodops in w i t h a 9 amino a c i d segment removed from the -48-Fi f iure 9. T i t r a t i o n curves o f rho 5A3, rho 4D2 and rho 8A6 monoclonal ant ibody c u l t u r e f l u i d s aga i n s t i m m o b i l i z e d T r i t o n X-100 s o l u b i l i z e d ROS. S o l u b i l i z e d ROS were d r i e d down on m i c r o t i t e r w e l l s and incubated w i t h s e r i a l d i l u t i o n s o f rho. 5A3 ( 0 ) , rho 4D2 ( • ) and rho 8A6 ( A ) monoclonal ant ibody cul ju jre f l u i d . A f t e r washing i n PBS, the m i c r o t i t e r w e l l s were incuba ted w i t h I -l a b e l e d goat anti-mouse Ig a n t i b o d y . - 4 9 -MW X10 J 220-CB 4 D 2 5A3 68-3 4 - | 25 12— a b c a b c is*-. a b c Figure 10. A n a l y s i s o f p o l y p e p t i d e s from unt rea ted and protease d i g e s t e d rod outer segment d i s c s which b i n d rho 4D2 and rho 5A3 monoclonal a n t i b o d i e s . Undiges ted rod outer segment d i s c membranes ( lane a) and r o d outer segment d i s c membranes d i g e s t e d w i t h t r y p s i n ( lane b) or aureus V-8 protease ( lane c) were washed by c e n t r i f u g a t i o n , s o l u b i l i z e d and e l e c t r o p h o r e s i z e d on a cont inuous 10% SDS-polyacrylamide (0.8% b i s ) g e l . The p o l y p e p t i d e s were e i t h e r s t a i n e d by coomassie b lue (CB) or t r a n s f e r r e d to n i t r o c e l l u l o s e paper. T r a n s f e r papers were t r e a t e d w i t h rho 4D2 or rho 5 A ^ c u l t u r e f l u i d . I d e n t i f i c a t i o n o f p r imary ant ibody b i n d i n g was made u s i n g I - l a b e l e d goat anti-mouse I g a n t i b o d y . -50-c a r b o x y l terminus (Molday and MacKenzie , 1983). When ROS d i s c s were t r e a t e d w i t h S_^  aureus V-8 pro tea se , three major rhodops in fragments were observed a t apparent M^= 33,000, 25,000 (F^ f ragment) , and 12,000 (F^ f ragment) . These fragments a r i s e from the removal o f a s m a l l 7 amino a c i d fragment from the C terminus o f rhodops in 239 240 and c leavage a t G l u - Ser w i t h i n the ^^-F^ (231-252) loop ( F i n d l a y et a l . , 1981). The monoclonal a n t i b o d i e s rho 4D2 and rho 5A3 bo th bound to the l a r g e 33,000 M^ fragment and the F^ fragment c o n t a i n i n g the N-terminus o f r h o d o p s i n . These r e s u l t s suggest t h a t rho 4D2 and rho 5A3 a n t i b o d i e s b i n d to determinants l o c a l i z e d a long the N - t e r m i n a l t w o - t h i r d s o f the rhodops in m o l e c u l e . ISOLATION OF N-TERMINAL RHODOPSIN PEPTIDES The N - t e r m i n a l 2-39 rhodops in g l y c o p e p t i d e was i s o l a t e d from CNBr-c l e a v e d bovine rhodops in by a f f i n i t y chromatography and HPLC f r a c t i o n a t i o n . F i g u r e 11A shows a HPLC chromatograph o f w a t e r - s o l u b l e pep t ide s d e r i v e d from a CNBr-d iges t o f r h o d o p s i n . When t h i s p e p t i d e mix ture was passed through a N - t e r m i n a l - s p e c i f i c rho 4D2 antibody-Sepharose column and subsequent ly e l u t e d w i t h a c e t i c a c i d , a major pept ide h a v i n g an e l u t i o n time o f approx imate ly 81 min was observed by HPLC ( F i g . 11B) The amino a c i d c o m p o s i t i o n o f t h i s pept ide corresponded to the 2-39 N - t e r m i n a l g lycopept ide o f rhodops in (Hargrave e t a l . , 1983) (Table I ) . The 2-39 g l y c o p e p t i d e was f u r t h e r d i g e s t e d w i t h t r y p s i n and ana lyzed by HPLC ( F i g . 11C). The peak corresponding to the 2-39 p e p t i d e was absent i n d i c a t i n g t h a t t r y p s i n d i g e s t i o n was complete . However, the t r y p s i n d i g e s t c o n t a i n e d more peaks than expected s o l e l y from the the c leavage o f the 2-39 g l y c o p e p t i d e i n d i c a t i n g t h a t some of the fragments may be due to t r y p s i n a u t o - p r o t e o l y s i s . A broad peak f r a c t i o n a t approx imate ly 62 minutes ( F i g . - 5 1 -20 40 60 80 Time (min) F i g u r e 11. HPLC chromatographs o f pept ides d e r i v e d from CNBr-c leaved bov ine r h o d o p s i n . A) W a t e r - s o l u b l e pept ides from a CNBr-d iges t o f bovine r h o d o p s i n ; B) The CNBr 2-39 N - t e r m i n a l rhodops in g l y c o p e p t i d e i s o l a t e d on a rho 4D2 antibody-Sepharose a f f i n i t y column; C) T r y p s i n d i g e s t o f the 2-39 g l y c o p e p t i d e ; D) HPLC of a 2-16 g l y c o p e p t i d e i s o l a t e d by p r e p a r a t i v e HPLC and i d e n t i f i e d by amino a c i d a n a l y s i s . -52-TABLE I Amino a c i d compos i t ion o f the immunoaf f in i ty p u r i f i e d 2-39 N - t e r m i n a l CNBr fragment o f bovine r h o d o p s i n . * @ Expected Amino a c i d s Amount present i n t e g r a l de tec ted (nmol o f amino a c i d / nmol o f pept ide ) va lue s Asp 3.2 3 Thr 1.9 2 Ser 3.1 3 G l u 5.0 5 Pro 4 .6 5 G l y 4 .3 3 A l a 2.5 2 V a l 1.9 3 Leu 1.4 1 Tyr 2.4 3 Phe 3.4 4 Lys 2.5 1 A r g 1.1 1 _ Values were n o r m a l i z e d w i t h re spec t to G l u . L Hargrave e t a l . , 1983. The amount o f V a l de tec ted was low due to incomplete h y d r o l y s i s o f the two ad jacent r e s i d u e s . G l y was abnormal ly h i g h p o s s i b l y due to some s l i g h t contamina t ion d u r i n g the h y d r o l y s i s . Lys was h i g h e r than expected due to glucosamine re s idues b e i n g p r e s e n t . - 5 3 -11D) was i d e n t i f i e d as the 2-16 N - t e r m i n a l g l y c o p e p t i d e by amino a c i d a n a l y s i s . Heterogene i ty i n the carbohydrate cha ins may account f o r the broad nature o f the 2-39 and 2-16 peaks on the HPLC chromatographs. SPECIFIC LOCALIZATION OF N-TERMINAL ANTIRHODOPSIN MONOCLONAL ANTIBODY  BINDING SITES The b i n d i n g s i t e s f o r rho 4A2 and rho 4D2 have p r e v i o u s l y been l o c a l i z e d to the 2-39 N - t e r m i n a l rhodops in fragment by c o m p e t i t i v e i n h i b i t i o n assays u s i n g a Con A p u r i f i e d rhodops in 2-39 CNBr g l y c o p e p t i d e (Hicks and Molday, 1986). The b i n d i n g s i t e s f o r rho 2B2 and rho 4A2 a n t i b o d i e s were more p r e c i s e l y l o c a l i z e d by radioimmune c o m p e t i t i o n a n a l y s i s u s i n g the CNBr 2-39 and 2-16 N - t e r m i n a l g lycopept ide s o f rhodops in as c o m p e t i t i v e i n h i b i t o r s . As shown i n F i g u r e 12, the 2-39 g l y c o p e p t i d e e f f e c t i v e l y i n h i b i t e d the b i n d i n g o f rho 4A2 and rho 2B2 a n t i b o d i e s to i m m o b i l i z e d r h o d o p s i n . The 2-16 g l y c o p e p t i d e was e s s e n t i a l l y as e f f e c t i v e an i n h i b i t o r as the 2-39 pept ide f o r the rho 2B2 a n t i b o d y . Th i s 2-16 g l y c o p e p t i d e , however, d i d not i n h i b i t the b i n d i n g o f the rho 4A2 ant ibody to rhodops in (Table I I ) . The mix ture o f pept ides r e s u l t i n g from the cleavage o f the 2-39 g l y c o p e p t i d e w i t h t r y p s i n a l s o d i d not i n h i b i t rho 4A2 b i n d i n g ( F i g . 12) , i n d i c a t i n g t h a t t r y p s i n d i g e s t i o n des t royed i t s a n t i g e n i c determinant . As shown i n F i g u r e 13, the p u r i f i e d 2-39 g l y c o p e p t i d e a l s o e f f e c t i v e l y i n h i b i t e d the b i n d i n g o f the rho 4D2 ant ibody to r h o d o p s i n . On the b a s i s o f the va lue s f o r h a l f maximum i n h i b i t i o n , however, the 2-39 g l y c o p e p t i d e was 34 f o l d l e s s r e a c t i v e than rhodops in f o r the rho 4D2 a n t i b o d y . I n an attempt to de f ine more a c c u r a t e l y the a n t i g e n i c determinants o f s e v e r a l N - t e r m i n a l a n t i b o d i e s , the c o n c e n t r a t i o n o f v a r i o u s pep t ide s and s o l u b i l i z e d rhodops in r e q u i r e d to i n h i b i t monoclonal ant ibody b i n d i n g to i m m o b i l i z e d rhodops in by 50% was determined (Table I I ) . Monoclonal - 5 4 -F i g u r e 12. Compet i t ive i n h i b i t i o n o f rho 4A2 and rho 2B2 ant ibody b i n d i n g to rhodops in by CNBr pept ides o f rhodops in and s o l u b i l i z e d r h o d o p s i n . S e r i a l d i l u t i o n s o f T r i t o n X-100 s o l u b i l i z e d ROS d i s c s ( # ) ; immunoaf f in i ty p u r i f i e d 2-39 N-terminus p o l y p e p t i d e ( | ) ; t r y p s i n d i g e s t o f the 2-39 N-terminus p o l y p e p t i d e (O) a n c * HPLC p u r i f i e d 2-16 N-terminus p o l y p e p t i d e (jfk) were p r e i n c u b a t e d w i t h rho 4A2 or rho 2B2 hybridoma c u l t u r e f l u i d . Subsequent ly , the c a p a c i t y o f rho 4A2 and rho 2B2 ant ibody to b i n d s o l u b i l i z e d and i m m o b i l i z e d boy^ne d i s c s was measured by the i n d i r e c t s o l phase radioimmune assay u s i n g I - l a b e l e d goat anti-mouse I g . - 5 5 -I 0.0001 0.001 0.01 0.1 1.0 10 Competing Antigen ( / jM) F i g u r e 13. Compet i t ive i n h i b i t i o n o f rho 4D2 monoclonal ant ibody b i n d i n g to i m m o b i l i z e d rhodops in by the i s o l a t e d 2-39 rhodops in g l y c o p e p t i d e and b leached , s o l u b i l i z e d r h o d o p s i n . S e r i a l d i l u t i o n s o f b l eached , T r i t o n X-100 s o l u b i l i z e d rhodops in ( 0 ) and immunoaf f in i ty p u r i f i e d 2-39 rhodops in g l y c o p e p t i d e ( | | ) were p re incuba ted w i t h rho 4D2 hybridoma c u l t u r e f l u i d . R e s i d u a l rho 4D2 monoclonal ant ibody b i n d i n g to i m m o b i l i z e d rhodops in was de tec ted by a s o l i d - p h a s e radioimmune assay u s i n g I - l a b e l e d goat a n t i -mouse I g as a secondary a n t i b o d y . TABLE I I C o n c e n t r a t i o n (uM) o f Pept ides or Rhodopsin Requi red to I n h i b i t by 50% (I,- n) Monoclonal Ant ibody B i n d i n g to Immobi l i zed Rhodopsin C-Termina l Loop Regions N-Termina l Competing rho rho rho rho rho rho rho rho rho A n t i g e n 1D4 3A6 4B4 8A6 4A2 4D2 2B2 4A3 5A3 Rho ( B l ) @ 0. * ,05 0. * .06 0. ,010 5. 6 0. ,09 0.005 0. ,063 0. .06 0.003 Rho (Unbl)@ 0, * .06 0. * .05 0. .010 1. .41 0.06 0. .63 0.08 2-39 N. . 1 . N. . 1 . N. . 1 . N. , 1 . 0. .14 0.17 0. .70 6. .3 1.12 2-16 N. . 1 . — 1. .12 — 1-16 N. . 1 . 251.0 0. .32 7, .1 63.0 331-348 0. ,16 2. .5 N. . 1 . N. I . N. I . N . I . N. , 1 . 318-348 0. .13 0, .70 N. I . N . I . N. . 1 . 231-252 N. . 1 . 9. .0 N. , 1 . N. . 1 . N . I . 61-74 N. I . N . I . = No I n h i b i t i o n -MacKenzie e t a l . , 1984 Bleached rhodops in (Rho-Bl) or Unbleached rhodops in (Rho-Unbl) s o l u b i l i z e d i n 1% T r i t o n X-100 -57-a n t i b o d i e s rho 5A3 and rho 4A3 i n a d d i t i o n to rho 4D2, rho 4A2 and rho 2B2 were e f f e c t i v e l y i n h i b i t e d by the 2-39 rhodops in g l y c o p e p t i d e . I n the case o f rho 4A2 a n t i b o d y , the 2-39 pept ide was as e f f e c t i v e an i n h i b i t o r as s o l u b i l i z e d r h o d o p s i n . The s y n t h e t i c 1-16 rhodops in p e p t i d e analogue which does not c o n t a i n the carbohydrate cha ins was a l s o an e f f e c t i v e i n h i b i t o r o f the rho 2B2 ant ibody (Table I I ) . Based on va lue s the s y n t h e t i c 1-16 p e p t i d e was a more e f f e c t i v e i n h i b i t o r then the 2-16 p e p t i d e . Thus, i t appears t h a t the Asn l i n k e d carbohydrate cha ins are not r e q u i r e d f o r rho 2B2 b i n d i n g to r h o d o p s i n . The s y n t h e t i c 1-16 pept ide was a l s o as e f f e c t i v e as the 2-39 g l y c o p e p t i d e i n i n h i b i t i n g rho 4A3 b i n d i n g to d e t e r g e n t - s o l u b i l i z e d r h o d o p s i n . High c o n c e n t r a t i o n s o f 1-16 pept ide were a l s o capable o f i n h i b i t i n g rho 4D2 and rho 5A3 b i n d i n g to i m m o b i l i z e d rhodops in (Table I I ) . I n summary, f i v e monoclonal a n t i b o d i e s have been shown to be s p e c i f i c f o r the N - t e r m i n a l 2-39 g l y c o p e p t i d e o f r h o d o p s i n . The a n t i g e n i c determinant f o r a t l e a s t two o f these a n t i b o d i e s (rho 2B2 and rho 4A3) i s l o c a t e d on the 1-16 N - t e r m i n a l g l y c o p e p t i d e , w h i l e the b i n d i n g s i t e f o r two a n t i b o d i e s (rho 4A2 and rho 5A3) encompasses a t l e a s t one o f the t r y p t i c s i t e s a long the 2-39 g l y c o p e p t i d e . EFFECT OF BLEACHING ON MONOCLONAL ANTIBODY BINDING The e f f e c t i v e n e s s o f b leached or unbleached, s o l u b i l i z e d or u n s o l u b i l i z e d , ROS d i s c s to i n h i b i t ant ibody b i n d i n g to T r i t o n X-100 s o l u b i l i z e d , i m m o b i l i z e d rhodops in was e x p l o r e d i n order to determine the r e l a t i v e a c c e s s i b i l i t y o f rhodops in a n t i g e n i c s i t e s on d i s c membranes. I n a p rev ious s tudy , the a c c e s s i b i l i t y o f the a n t i g e n i c s i t e on detergent-s o l u b i l i z e d rhodops in f o r rho 4A2 ant ibody ( s p e c i f i c f o r the N-terminus) b i n d i n g was shown to be s t r o n g l y dependent on the s t a t e o f rhodops in b l e a c h i n g . (Molday and MacKenzie , 1983). -58-Bleached , s o l u b i l i z e d ROS d i s c s were e f f e c t i v e i n i n h i b i t i n g the b i n d i n g o f bo th rho 5A3 and rho 4D2 monoclonal a n t i b o d i e s ( F i g . 14) . However, unbleached, s o l u b i l i z e d d i s c s were 25 times l e s s e f f e c t i v e i n i n h i b i t i n g rho 5A3 b i n d i n g and 12 t imes l e s s e f f e c t i v e i n i n h i b i t i n g rho 4D2 b i n d i n g to i m m o b i l i z e d r h o d o p s i n . B leached , u n s o l u b i l i z e d d i s c membranes were i n e f f e c t i v e i n i n h i b i t i n g the b i n d i n g o f e i t h e r rho 4D2 or rho 5A3 a n t i b o d i e s ( F i g . 14) . Two a d d i t i o n a l N - t e r m i n a l s p e c i f i c a n t i r h o d o p s i n monoclonal a n t i b o d i e s (rho 2B2 and rho 4A2) were a l s o found to more e f f e c t i v e l y b i n d b leached , s o l u b i l i z e d rhodops in than unbleached, s o l u b i l i z e d rhodops in (Table I I ) . These r e s u l t s i n d i c a t e t h a t the N-terminus o f d e t e r g e n t - s o l u b i l i z e d rhodops in must undergo a c o n f o r m a t i o n a l change upon b l e a c h i n g . I n c o n t r a s t , an a n t i r h o d o p s i n C - t e r m i n a l ant ibody (rho 3A6) competed e q u a l l y w e l l w i t h b leached and unbleached rhodops in i n d i s c membranes ( F i g . 14) . I n a p rev ious s tudy , another C - t e r m i n a l s p e c i f i c a n t i r h o d o p s i n ant ibody (rho 1D4) was shown to b i n d unbleached, s o l u b i l i z e d rhodops in as e f f e c t i v e l y as b l eached , s o l u b i l i z e d rhodops in (MacKenzie e t a l . , 1984). Thus, n e i t h e r o f these C - t e r m i n a l s p e c i f i c a n t i b o d i e s was e f f e c t i v e i n d e t e c t i n g a con format iona l change i n rhodops in upon b l e a c h i n g . EFFECT OF FIXATION ON RHO 4D2 BINDING TO ROS MEMBRANES The e f f e c t i v e n e s s o f ROS, s o l u b i l i z e d ROS and g l u t a r a l d e h y d e - f i x e d ROS to i n h i b i t p u r i f i e d r a d i o i o d i n a t e d rho 4D2 ant ibody b i n d i n g to T r i t o n X-100 t r e a t e d , i m m o b i l i z e d ROS d i s c s was i n v e s t i g a t e d i n order to s tudy the r e a c t i v i t y o f the a n t i r h o d o p s i n ant ibody f o r i t s a n t i g e n i c s i t e . As shown i n F i g u r e 15, an e x t r a p o l a t i o n o f the i n h i b i t i o n curve f o r u n f i x e d ROS i n d i c a t e d t h a t g l u t a r a l d e h y d e - f i x e d i n t a c t ROS competed over 30 f o l d more e f f e c t i v e l y than u n f i x e d , i n t a c t ROS f o r rho 4D2 ant ibody b i n d i n g . However, when comparing ! , « v a l u e s , s o l u b i l i z e d ROS were 5000 f o l d more e f f e c t i v e i n -59-1 0 0 » A — A — - A u c 3 O CD E a TJ E 3 E X CO 2 0.001 0.01 0.1 1 Competing Antigen (uM) 10 F i g u r e 14. Compet i t ive i n h i b i t i o n o f rho 5A3, rho 4D2 and rho 3A6 ant ibody b i n d i n g to i m m o b i l i z e d rhodops in by b leached and unbleached r h o d o p s i n . S e r i a l d i l u t i o n s o f T r i t o n X-100 s o l u b i l i z e d , b leached d i s c membranes ( 0 ) ; T r i t o n X-100 s o l u b i l i z e d , unbleached d i s c membranes ( A ) and u n s o l u b i l i z e d , b l eached d i s c membranes ( • ) were p re incuba ted w i t h rho 5A3, rho 4D2 or rho 3A6 hybridoma c u l t u r e f l u i d . Subsequently, the c a p a c i t y o f rho 5A3, rho 4D2 and rho 3A6 ant ibody to b i n d s o l u b i l i z e d and i m m o b i l i z e d r h o ^ n s i n was measured by the i n d i r e c t s o l i d - p h a s e radioimmune assay u s i n g I - l a b e l e d goat anti-mouse I g . -60-•D C 3 O CD E a E a E x CO 65 0.1 1 10 100 Competing Antigen (ug/mL) 1000 F i g u r e 15. 125 Compet i t ive i n h i b i t i o n o f I - l a b e l e d rho 4D2 monoclonal ant ibody b i n d i n g to i m m o b i l i z e d rhodops in by T r i t o n X-100 s o l u b i l i z e d ROS, ROS and g l u t a r a l d e h y d e - f i x e d ROS. S e r i a l d i l u t i o n s o f T r i t o n X-100 s o l u b i l i z e d ROS > R 0 S (O) a n d g l u t a r a l d e h y d e - f i x e d ROS (%) were p r e i n c u b a t e d w i t h I - l a b e l e d rho 4D2 a n t i b o d y . R e s i d u a l ant ibody b i n d i n g was measured by s o l i d - p h a s e radioimmune assay. - 6 1 -i n h i b i t i n g rho 4D2 b i n d i n g to i m m o b i l i z e d rhodops in then f i x e d ROS. These r e s u l t s suggest t h a t the rho 4D2 monoclonal ant ibody b inds to g l u t a r a l d e h y d e - f i x e d rhodops in i n the ROS plasma membrane. Moreover, d e t e r g e n t - s o l u b i l i z a t i o n des t roys the m o r p h o l o g i c a l s t r u c t u r e o f the ROS and exposes the a n t i g e n i c s i t e o f rhodops in i n d i s c membranes f o r rho 4D2 ant ibody b i n d i n g . IMMUNOCYTOCHEMICAL LABELING OF ROS MEMBRANES The o r g a n i z a t i o n and l o c a l i z a t i o n o f rhodops in i n ROS membranes was determined by immunogold-dextran l a b e l i n g s t u d i e s . When a g l u t a r a l d e h y d e -f i x e d r e t i n a was t r e a t e d w i t h rho 4D2 monoclonal ant ibody conjugated to 8 nm g o l d - d e x t r a n p a r t i c l e s , a dense un i form d i s t r i b u t i o n o f g o l d p a r t i c l e s was observed a long the e x t r a c e l l u l a r sur face o f the rod outer segment plasma membrane ( F i g . 16A). Only s c a t t e r e d l a b e l i n g , however, was found a long the sur face o f the rod i n n e r segment plasma membrane ( F i g . 16A). S i m i l a r r e s u l t s were o b t a i n e d when a g l u t a r a l d e h y d e - f i x e d r e t i n a was l a b e l e d w i t h p u r i f i e d rho 4D2 monoclonal ant ibody f o l l o w e d by 9 nm goat anti-mouse Ig g o l d - d e x t r a n p a r t i c l e s ( r e s u l t s not shown). U n f i x e d , i s o l a t e d ROS d i r e c t l y l a b e l e d w i t h 8 nm g o l d - d e x t r a n p a r t i c l e s conjugated to p u r i f i e d rho 4D2 a n t i b o d y , showed no g o l d p a r t i c l e s a long the outer segment plasma membrane ( F i g . 16B) . These r e s u l t s i n c o n j u n c t i o n w i t h the c o m p e t i t i v e i n h i b i t i o n assays demonstrate the i n a c c e s s i b i l i t y o f the rho 4D2 a n t i g e n i c s i t e i n u n f i x e d ROS membranes. Sidedness o f the rhodops in molecule i n ROS d i s c s and ROS plasma membrane was f u r t h e r determined by the immunocytochemical l a b e l i n g o f i s o l a t e d g l u t a r a l d e h y d e - f i x e d ROS w i t h the N - t e r m i n a l s p e c i f i c rho 4D2 monoclonal ant ibody and the C - t e r m i n a l s p e c i f i c rho 1D4 monoclonal ant ibody conjugated to 10 nm g o l d - d e x t r a n p a r t i c l e s . As i l l u s t r a t e d i n F i g u r e 17, rho 1D4 g o l d -Figure 16. Transmi s s ion e l e c t r o n micrographs o f p r e - f i x e d bov ine r o d photoreceptor c e l l s or u n f i x e d i s o l a t e d bovine ROS d i r e c t l y l a b e l e d w i t h rho 4D2 a n t i r h o d o p s i n monoclonal a n t i b o d y - g o l d - d e x t r a n con juga te s . G l u t a r a l d e h y d e - f i x e d bovine rod photoreceptor c e l l s (A) or u n f i x e d i s o l a t e d ROS (B) were t r e a t e d w i t h rho 4D2 monoclonal ant ibody con jugated to 8 nm g o l d - d e x t r a n p a r t i c l e s f o r 4-6 h . A f t e r washing i n cacody la te b u f f e r , the samples were prepared f o r e l e c t r o n microscopy. (RIS) = r o d i n n e r segment (ROS) = rod outer segment. Arrows note l o c a t i o n o f ROS plasma membrane. Bar = 0.5 urn. - 6 3 -d e x t r a n conjugates h e a v i l y l a b e l e d rhodops in on exposed d i s c membranes. No g o l d l a b e l i n g was observed on the ROS plasma membrane. I n c o n t r a s t , rho 4D2 g o l d - d e x t r a n conjugates h e a v i l y l a b e l e d rhodops in on the ROS plasma membrane, but not on exposed d i s c membranes. T h i s l a b e l i n g p a t t e r n i s c o n s i s t e n t w i t h the N - t e r m i n a l r e g i o n o f rhodops in b e i n g exposed on the e x t r a c e l l u l a r sur face o f the ROS plasma membrane and C-terminus b e i n g exposed on the c y t o p l a s m i c s u r f a c e . MONOCLONAL ANTIBODIES AGAINST INTERNAL SEGMENTS OF RHODOPSIN Prev ious s t u d i e s have determined t h a t c leavage o f the rhodops in between Glu-239 and Ser-240 by S_^  aureus V-8 protease e l i m i n a t e s rho 4B4 ant ibody b i n d i n g , sugges t ing t h a t the F^-F^ loop i s the p r i n c i p a l a n t i g e n i c determinant f o r the rho 4B4 ant ibody (MacKenzie and Molday, 1982; L a i r d et a l . , 1987). T h i s was supported by c o m p e t i t i v e i n h i b i t i o n s t u d i e s shown i n 231 F i g u r e 18. S y n t h e t i c F^-F^ 1 ° ° P pept ide o f sequence L y s - G l u - A l a - A l a - A l a -ry i r\ a r a G l n - G l n - G l n - G l u - S e r - A l a - T h r - T h r - G l n - L y s - A l a - G l u - L y s - G l u - V a l - T h r - A r g was found to i n h i b i t rho 4B4 b i n d i n g to r h o d o p s i n . No i n h i b i t o n was observed f o r the C - t e r m i n a l 331-348 ( l ' - 1 8 ' ) p e p t i d e . A 900 f o l d h i g h e r c o n c e n t r a t i o n o f the F ^ - F 2 loop pept ide r e l a t i v e to r h o d o p s i n , however, was r e q u i r e d to o b t a i n half-maximum i n h i b i t i o n . T h i s suggests t h a t the a f f i n i t y o f the ant ibody f o r the pept ide may be a f f e c t e d by the conformat ion o f the p e p t i d e . A l t e r n a t i v e l y , the rho 4B4 ant ibody may r e q u i r e an a d d i t i o n a l s i t e from another r e g i o n o f rhodops in f o r h i g h a f f i n i t y b i n d i n g . Another rhodops in monoclonal a n t i b o d y , des ignated rho 8A6, bound to immunoblots o f p r o t e a s e - d i g e s t e d rhodops in i n a p a t t e r n s i m i l a r to t h a t observed f o r the N - t e r m i n a l ant ibody rho 2B2 ( F i g . 19) . Both rho 2B2 and rho 8A6 a n t i b o d i e s bound to undiges ted rhodops in ( lane a) and the 25,000 M F 1 i r l fragment o f S_^  aureus V-8 protease c l e a v e d rhodops in ( lane b ) . When - 6 4 -Figure 17. Transmi s s ion e l e c t r o n micrographs o f f i x e d i s o l a t e d ROS d i r e c t l y l a b e l e d w i t h a n t i r h o d o p s i n g o l d - d e x t r a n con juga te s . G l u t a r a l d e h y d e - f i x e d ROS were d i r e c t l y l a b e l e d w i t h rho 1D4 (A) or rho 4D2 (B) monoclonal a n t i b o d i e s conjugated to 10 nm go ld-dex t ran p a r t i c l e s . Samples were washed and prepared f o r t r a n s m i s s i o n e l e c t r o n microscopy . The s i n g l e arrows i n d i c a t e the e x t r a c e l l u l a r sur face o f the ROS plasma membrane and the double arrows i n d i c a t e s the cy top la smic sur face o f ROS d i s c membranes. No rho 4D2 monoclonal ant ibody g o l d - d e x t r a n p a r t i c l e s were found a long the ROS d i s c membranes. Bar - 0.2 um. -65-0.0001 0.001 0.01 0.1 1.0 10 Competing Antigen (uM) F i g u r e 18. Compet i t ive i n h i b i t i o n o f rho 4B4 ant ibody b i n d i n g to i m m o b i l i z e d rhodops in by ROS d i s c s ( £ ) , F 1 -F„ s y n t h e t i c rhodops in p e p t i d e (O) o r t n e s y n t h e t i c C - t e r m i n a l 331-348 ( I ' - 1 8 ' ) rhodops in pept ide ( | ) . -66-MWX10 3 68— 34—< 25— 20— dye— a b c C B a b c 2B2 a b c 8 A 6 Figure 19. SDS-gel e l e c t r o p h o r e s i s and immunoblott ing o f ROS d i s c membranes t r e a t e d w i t h p ro tea se s . Undigested bovine d i s c membranes ( l ane a) and d i s c membranes d i g e s t e d w i t h S_^  aureus V-8 protease ( lane b) or chymotryps in ( lane c) were sub jec ted to SDS-gel e l e c t r o p h o r e s i s and e i t h e r s t a i n e d w i t h coomassie b lue (CB) or t r a n s f e r r e d to n i t r o c e l l u l o s e and subsequent ly l a b e l e d w i t h rho 2B2 or rho 8A6 ant ibody and I - l a b e l e d goat anti-mouse Ig f o r autorad iography . -67-rhodops in was c l e a v e d w i t h chymotryps in ( lane c ) , bo th rho 8A6 and rho 2B2 a n t i b o d i e s a l s o bound to a s m a l l e r fragment o f apparent M^ = 20,000. These r e s u l t s i n d i c a t e d t h a t the rho 8A6 ant ibody bound to the N - t e r m i n a l h a l f o f r h o d o p s i n . Immunocytochemical l a b e l i n g s t u d i e s were performed i n order to determine whether the b i n d i n g s i t e f o r rho 8A6 ant ibody was exposed on the cytop la smic s i d e or i n t r a d i s c a l s i d e o f the ROS d i s c membrane. When ROS d i s c s , prepared by a m i l d t r y p s i n treatment i n order to preserve the " p a n c a k e - l i k e " s t r u c t u r e o f the d i s c s (Molday e t a l . , 1987), were l a b e l e d w i t h rho 8A6 or rho 4B4 ant ibody f o l l o w e d by goat anti-mouse Ig g o l d d e x t r a n p a r t i c l e s , heavy l a b e l i n g was observed on the c y t o p l a s m i c s i d e o f the d i s c ( F i g . 20) . When an N - t e r m i n a l a n t i b o d y , rho 4D2, was used as the pr imary a n t i b o d y , no l a b e l i n g o f d i s c s was observed. The l o c a t i o n o f the rho 8A6 a n t i g e n i c determinant on the c y t o p l a s m i c sur face o f d i s c s was f u r t h e r supported by c o m p e t i t i o n assays u s i n g s ea l ed d i s c s . As shown i n F i g u r e 21, s ea l ed d i s c s i n h i b i t the rho 8A6 ant ibody as e f f e c t i v e l y as detergent s o l u b i l i z e d d i s c s . I n an attempt to f u r t h e r de f ine the a n t i g e n i c determinant f o r rho 8A6 a n t i b o d y , c o m p e t i t i v e i n h i b i t i o n assays were performed w i t h s y n t h e t i c pep t ide s ( F i g . 21) . Rho 8A6 ant ibody f a i l e d to compete w i t h the ^-^-^2 1 ° ° P p e p t i d e (Table I I ) or a s y n t h e t i c pept ide analogue o f the segment between rhodops in h e l i x I and I I ( 6 1 V a l - T h r - V a l - G l n - H i s - L y s - L e u - A r g - 7 ° T h r - P r o - L e u -Asn-Tyr ) when pept ide c o n c e n t r a t i o n s o f up to 800 uM were used. PURIFICATION OF THE 1 ' -31 ' C-TERMINAL PEPTIDE OF RHODOPSIN I n an attempt to f u r t h e r i n v e s t i g a t e C - t e r m i n a l s p e c i f i c a n t i b o d i e s , the 318-348 ( l ' - 3 1 ' ) C - t e r m i n a l pep t ide was p u r i f i e d from CNBr-c leaved rhodops in by a f f i n i t y chromatography u s i n g a rho 1D4 antibody-Sepharose column. As shown by HPLC chromatography i n F igure 22, a major peak w i t h a r e t e n t i o n -68-A Figure 20. Transmi s s ion e l e c t r o n micrographs o f i s o l a t e d ROS d i s c membranes l a b e l e d w i t h rho 8A6, rho 4B4 or rho 4D2 immunogold-dextran markers . "Pancake l i k e " d i s c membranes were l a b e l e d w i t h rho 8A6 ( A ) , rho 4B4 (B) or rho 4D2 (C) hybridoma c u l t u r e f l u i d f o l l o w e d by goat anti-mouse I g g o l d -dextran p a r t i c l e s . Diameter o f the g o l d p a r t i c l e s were e i t h e r 9 nm (A) or 12 nm (B and C ) . Bar = 0 . 1 urn. -69-0.0001 0.001 0.01 0.1 1.0 10 Competing Antigen (uM) F i g u r e 21. Compet i t ive i n h i b i t i o n o f rho 8A6 monoclonal ant ibody b i n d i n g to i m m o b i l i z e d rhodops in by T r i t o n X-100 s o l u b i l i z e d d i s c membranes ( H ) > s ea l ed d i s c membranes ( A ) or a 61-74 rhodops in s y n t h e t i c pep t ide ( 0 ) . -7 0-A Time (min) F i g u r e 22. HPLC chromatographs o f CNBr-c leaved bovine rhodops in p e p t i d e s . A) Water s o l u b l e pept ides from the C N B r - d i g e s t i o n o f r h o d o p s i n ; B) HPLC chromatographs o f rho 1D4 immunoaf f in i ty p u r i f i e d p e p t i d e . Amino a c i d a n a l y s i s o f t h i s pep t ide corresponds to the 318-348 C - t e r m i n a l pep t ide o f r h o d o p s i n . The bar represent s the area o f rho 1D4 b i n d i n g as determined by s o l i d - p h a s e radioimmune assay. - 7 1 -time o f 45 min and some s m a l l shoulder peaks w i t h r e t e n t i o n times o f 47 and 49 min were i s o l a t e d from a CNBr rhodops in pept ide mix ture a f t e r chromatography on a rho 1D4 antibody-Sepharose column. When the major pept ide was sub jec ted to amino a c i d a n a l y s i s , i t s c o m p o s i t i o n corresponded to the 318-348 C - t e r m i n a l pept ide o f rhodops in (Table I I I ) . The 318-348 ( l ' - 3 1 ' ) pept ide was approx imate ly 4 f o l d more e f f e c t i v e than the 331-348 ( l ' - 1 8 ' ) pep t ide i n i n h i b i t i n g rho 3A6 ant ibody b i n d i n g to r h o d o p s i n ( F i g . 23) . Moreover, the p u r i f i e d 318-348 p e p t i d e was as e q u a l l y e f f e c t i v e as the 331-348 s y n t h e t i c pept ide i n i n h i b i t i n g rho 1D4 ant ibody b i n d i n g to rhodops in (Table I I ) . 125 INHIBITION OF I-LABELED RHO 3A6 BINDING BY MONOCLONAL ANTIBODIES The c y t o p l a s m i c exposed C - t e r m i n a l segment o f rhodops in i s the s i t e o f rhodops in k i n a s e b i n d i n g (Hargrave e t a l . , 1980) w h i l e the F ^ " F 2 1 ° ° P n a s been proposed as the s i t e o f t r a n s d u c i n b i n d i n g (Applebury and Hargrave , 1986). I t i s p o s s i b l e t h a t monoclonal a n t i b o d i e s s p e c i f i c f o r the c y t o p l a s m i c sur face o f rhodops in c o u l d be used as immunologica l probes i n d e f i n i n g the s p e c i f i c rhodops in s i t e s o f i n t e r a c t i o n w i t h o ther ROS p r o t e i n s i n v o l v e d i n the v i s u a l p roces s . I n an attempt to develop such a f u n c t i o n a l assay system, p r e l i m i n a r y s t u d i e s were performed to determine whether the b i n d i n g o f one monoclonal ant ibody at one rhodops in a n t i g e n i c determinant would i n h i b i t the b i n d i n g o f a second ant ibody at an independent b i n d i n g s i t e . As shown i n F i g u r e 24, l e s s than a t w o - f o l d molar excess o f p u r i f i e d rho 3A6 or rho 1C5 ant ibody 125 p r e i n c u b a t e d w i t h ROS d i s c membranes i n h i b i t e d approx imate ly 90% o f I -l a b e l e d rho 3A6 b i n d i n g , w h i l e a 1 2 - f o l d molar excess o f rho 8A6 or rho 4D2 125 ant ibody had l i t t l e e f f e c t i n i n h i b i t i n g I - l a b e l e d rho 3A6 b i n d i n g . TABLE I I I Amino a c i d compos i t ion o f the immunoaf f in i ty p u r i f i e d 319-348 ( l ' - 3 1 ' ) C - t e r m i n a l CNBr fragment o f bovine r h o d o p s i n . * Expected Amino a c i d Amount present i n t e g r a l d e t e c t e d (nmol o f amino a c i d / va lue s nmol o f pept ide ) Asp 3.2 3 Thr 4 .7 6 Ser 2.5 3 G l u 3.0 3 Pro 2.0 2 G l y 2.3 2 A l a 2.8 3 V a l 2.5 3 Met 0.13 0 Leu 1.8 2 Tyr 0.13 0 Lys 2.1 2 _ Values were n o r m a l i z e d w i t h re spec t to G l u . L Hargrave e t a l . , 1983. The Cys r e s idues were des t royed d u r i n g h y d r o l y s i s . The low va lue s f o r Ser and Thr was p o s s i b l y due to low l e v e l s o f oxygen i n the h y d r o l y s i s tubes . -73-100 TJ C 3 80 O CD, E 60 Q. TJ E E 40 X CO 20 0.001 0.01 0.1 1 10 Competing Antigen (jjM) 100 F i g u r e 23. Compet i t ive i n h i b i t i o n o f rho 3A6 monoclonal ant ibody b i n d i n g to s o l u b i l i z e d rhodops in by T r i t i o n X-100 s o l u b i l i z e d d i s c s ( A ) , ( l ' - 3 1 ' ) 319-348 rhodops in CNBr pept ide (%) or the ( l ' - 1 8 ' ) 330-348 rhodops in s y n t h e t i c pep t ide ( B ) • -74-F i g u r e 24. - I O C I n h i b i t i o n o f I - l a b e l e d rho 3A6 ant ibody b i n d i n g to rhodops in i n d i s c membranes by p u r i f i e d a n t i r h o d o p s i n monoclonal a n t i b o d i e s . ROS d i s c membranes (0 .5 uM rhodopsin) were p re incuba ted i n (a) b u f f e r ; (b) rho 8A6 (6 .4 uM); (c) rho 4D2 (6 .1 uM); (d) rho 4B4 (3 .1 uM); (e) rho 3A6 (0.9 uM); and ( f ) rho 1C5 (0.7 uM) p u r i f i e d a n t i r h o d o p s i n monoclonal, a n t i b o d i e s . A f t e r 1 h , the a b i l i t y o f t r e a t e d ROS d i s c membranes to b i n d I - l a b e l e d rho 3A6 monoclonal an t ibody was measured. Free ant ibody was separated from membrane bound ant ibody by c e n t r i f u g a t i o n o f the t r e a t e d d i s c membranes through 10% BSA. The r e s u l t i n g d i s c membrane p e l l e t s were counted i n a Gamma 8000 c o u n t e r . -7 5-Moreover , 6 - f o l d molar excess o f rho 4B4 ant ibody i n h i b i t e d 45% o f the 125 a b i l i t y o f I - l a b e l e d rho 3A6 to b i n d to ROS d i s c s . A more q u a n t i t a t i v e s tudy ( F i g . 25) showed t h a t even 1 6 - f o l d molar excess rho 8A6 had l i t t l e 125 e f f e c t i n i n h i b i t i n g I - l a b e l e d rho 3A6 b i n d i n g to r h o d o p s i n . However, o n l y 1 uM o f rho 4B4 ant ibody and 0.05 uM rho 1C5 ant ibody were r e q u i r e d to 125 i n h i b i t I - l a b e l e d rho 3A6 b i n d i n g to 0.3 uM of r h o d o p s i n by 50%. These r e s u l t s i n d i c a t e d t h a t monoclonal ant ibody b i n d i n g to the F-^-E^ 1 ° ° P ° f r h o d o p s i n can, i n t u r n , s t e r i c a l l y i n h i b i t the b i n d i n g o f another ant ibody to the C-terminus o f r h o d o p s i n . However, b i n d i n g o f the rho 8A6 ant ibody to the c y t o p l a s m i c sur face ( loop I - I I or I I I - I V ) o f rhodops in had l i t t l e e f f e c t on the b i n d i n g o f an a n t i r h o d o p s i n C - t e r m i n a l ant ibody sugges t ing t h a t these two rhodops in segments are c o n f o r m a t i o n a l l y separated i n d i s c membranes. PURIFICATION OF RHODOPSIN A n t i r h o d o p s i n monoclonal a n t i b o d i e s were used to p u r i f y rhodops in by immunoa f f in i ty chromatography ( F i g . 26) . ROS d i s c membranes ( lane a) were s o l u b i l i z e d i n CHAPS and a p p l i e d to a rho 2B2 a n t i r h o d o p s i n ant ibody-Sepharose immunoaf f in i ty column. The unbound ROS p r o t e i n s were washed through the column ( lane b) u n t i l no f u r t h e r p r o t e i n s were e l u t e d ( lane c ) . The bound r h o d o p s i n , a t apparent = 34,000, was competed from the Sepharose immunoaf f in i ty column w i t h an excess o f a 1-16 s y n t h e t i c pept ide analogue o f rhodops in ( lane d ) . A s i m i l a r p u r i f i c a t i o n o f rhodops in was performed u s i n g a C - t e r m i n a l s p e c i f i c a n t i r h o d o p s i n ant ibody (rho 1D4) immunoaf f in i ty column i n c o n j u n c t i o n w i t h excess l ' - 1 8 ' rhodops in s y n t h e t i c p e p t i d e . P u r i f i e d rhodops in was used i n r e c o n s t i t u t i o n s t u d i e s i n l a t e r exper iments . -7 6-—I 1 I I 0.01 0.1 1 10 Antibody Concentration (JJM) Figure 25. I n h i b i t i o n o f I - l a b e l e d rho 3A6 monoclonal ant ibody b i n d i n g to r h o d o p s i n i n d i s c membranes by p u r i f i e d a n t i r h o d o p s i n monoclonal a n t i b o d i e s . ROS d i s c membranes (0 .3 uM rhodopsin) were p r e i n c u b a t e d i n i n c r e a s i n g c o n c e n t r a t i o n s o f rho 1C5 ( 0 ) , rho 4B4 ( A ) and rho 8A6 monoclonal a n t i b j d ^ e s ( B ) - A f t e r 1 h the a b i l i t y o f the t r e a t e d ROS d i s c membranes to b i n d I - l a b e l e d rho 3A6 monoclonal ant ibody was measured. Free ant ibody was separated from membrane-bound ant ibody by c e n t r i f u g a t i o n of the t r e a t e d d i s c membranes through 10% BSA. The r e s u l t i n g ROS d i s c membrane p e l l e t s were counted i n a gamma counter . -77-MW X10 220 — 9 5 — 34— CB 3 a b e d F i g u r e 26. P u r i f i c a t i o n o f rhodops in by immunoaf f in i ty chromatography and pept ide e l u t i o n as observed by SDS-gel e l e c t r o p h o r e s i s and coomassie b l u e (CB) s t a i n i n g . ROS membranes ( lane a) were s o l u b i l i z e d i n CHAPS and loaded on a N - t e r m i n a l s p e c i f i c a n t i r h o d o p s i n monoclonal ant ibody (rho 2B2) Sepharose 2B-CL column. Unbound p r o t e i n ( lane b) was washed through the column u n t i l no f u r t h e r p r o t e i n was detec ted ( lane c ) . Bound r h o d o p s i n was s p e c i f i c a l l y e l u t e d at an apparent M = 34,000 ( lane d) by a 7 - f o l d molar excess o f a 1-16 s y n t h e t i c rhodops in pept ide analogue. -78-DISCUSSION A p p l i c a t i o n o f monoclonal a n t i b o d i e s as immunologica l probes f o r the s t r u c t u r e and f u n c t i o n o f rhodops in r e q u i r e s d e t a i l e d i n f o r m a t i o n about the l o c a t i o n o f the a n t i g e n i c determinants or ep i topes f o r these a n t i b o d i e s . L i m i t e d p r o t e o l y s i s o f rhodops in i n c o n j u n c t i o n w i t h immunoblott ing techniques have been u s e f u l i n de te rmin ing the genera l l o c a t i o n o f a n t i g e n i c s i t e s on r h o d o p s i n . S y n t h e t i c pept ides and s m a l l pep t ide s d e r i v e d from chemica l or p r o t e o l y t i c cleavage o f rhodops in i n combinat ion w i t h c o m p e t i t i v e i n h i b i t i o n s t u d i e s have proven v a l u a b l e i n o b t a i n i n g more p r e c i s e l o c a l i z a t i o n o f ant ibody b i n d i n g s i t e s . R e s u l t s d e s c r i b e d here i n combinat ion w i t h p r e v i o u s s t u d i e s (MacKenzie e t a l . , 1984; H i c k s and Molday, 1985; Hargrave e t a l . , 1986; Hargrave e t a l . , 1986) i n d i c a t e t h a t the C - t e r m i n a l and N - t e r m i n a l segments o f bovine rhodops in are the most h i g h l y a n t i g e n i c reg ions o f the r h o d o p s i n p o l y p e p t i d e c h a i n . As summarized i n Table IV , s i x IgG monoclonal a n t i b o d i e s had p r e v i o u s l y been generated aga i n s t determinants a long the 331-348 ( l ' - 1 8 ' ) C-t e r m i n a l segment (MacKenzie e t a l . , 1984). Four o f these a n t i b o d i e s (rho 3D6, rho 1D4, rho 2C1, and rho 3C2) r e q u i r e the t e r m i n a l A l a or P r o - A l a group; two a n t i b o d i e s (rho 1C5 and rho 3A6) b i n d f u r t h e r i n and do not r e q u i r e the C - t e r m i n a l seven re s idues as i n d i c a t e d by the b i n d i n g o f these a n t i b o d i e s to aureus V-8 p r o t e a s e - t r e a t e d rhodops in ( F i g . 27) . One ant ibody (rho 1D4) has been s u c c e s s f u l l y used i n the p u r i f i c a t i o n o f the 318-348 pept ide from CNBr-c leaved rhodops in by immunoaf f in i ty chromatography. T h i s ant ibody has a l s o been used w i t h the immunogold-d e x t r a n l a b e l i n g method to l o c a l i z e rhodops in a long the c y t o p l a s m i c sur face o f d i s c s , to map the d i s t r i b u t i o n o f rhodops in i n the r o d photoreceptor c e l l (Molday and L a i r d , 1988) and to study the p r o p e r t i e s o f l i g h t - d e p e n d e n t - 7 9 -TABLE IV P r o p e r t i e s o f A n t i r h o d o p s i n Monoclonal A n t i b o d i e s Ant ibody I g Subtype S p e c i f i c i t y C - t e r m i n a l rho 3D6 rho 1D4 rho 2C1 rho 3C2 rho 3A6 rho 1C5 Loop reg ions rho 4B4 rho 8A6 N - t e r m i n a l rho 4A2 rho 4D2 rho 5A3 rho 2B2 rho 4A3 IgG 3 (L) I g G x (K) I g G 1 (K) IgG]_ (K) IgG 3 (K) IgGj_ (K) I g G 1 (K) I g G 2 b (K) IgG 3 (K) I g G x (K) 345-348 ( l ' - 4 ' ) 341-348 ( l ' - 8 ' ) 341-348 ( l ' - 8 ' ) 341-348 ( l ' - 8 ' ) 337-341 ( 8 ' - 1 2 ' ) 331-340 ( 9 ' - 1 8 ' ) 231-252 Cytopla smic Loop (?) 2-39 2-39 2-39 1-16 1-16 (K) = kappa (L) = lambda Cytoplasmic Surface F i g u r e 27. A diagrammatic model o f bovine rhodops in , m o d i f i e d from Hargrave e t a l . (1983), showing the approximate l o c a t i o n o f the a n t i g e n i c s i t e s f o r v a r i o u s monoclonal a n t i b o d i e s . Cytoplasmic sur face s i t e s o f p r o t e o l y s i s are noted and the seven transmembrane h e l i c a l segments are numbered from l e f t to r i g h t . - 8 1 -p h o s p h o r y l a t i o n o f rhodops in (Molday and MacKenzie , 1985). F i v e monoclonal a n t i b o d i e s have been generated ag a in s t a n t i g e n i c s i t e s a long the N - t e r m i n a l F^ fragment o f aureus V-8 p r o t e a s e - c l e a v e d bovine rhodops in ( F i g . 27) . S tudies d e s c r i b e d here i n d i c a t e t h a t a t l e a s t one o f these a n t i b o d i e s (rho 2B2) b inds to an ep i tope a long the 2-16 N - t e r m i n a l rhodops in g l y c o p e p t i d e . S ince the rho 2B2 ant ibody a l s o b inds to the s y n t h e t i c 1-16 pept ide analogue o f rhodops in which does not c o n t a i n the Asn-l i n k e d carbohydrate c h a i n s , i t can be concluded t h a t the carbohydrate c h a i n i s not p a r t o f the b i n d i n g s i t e . Two a n t i b o d i e s (rho 4A2 and rho 5A3) b i n d to s i t e s encompassing one or two o f the t r y p t i c c leavage s i t e s a long the 2-16 17 21 22 39 g l y c o p e p t i d e , i . e . , Lys -Thr and/or A r g -Ser s i n c e t r y p s i n d i g e s t i o n o f t h i s pept ide r e s u l t s i n a complete l o s s i n b i n d i n g o f these a n t i b o d i e s . Ant ibody rho 4D2, which b inds s t r o n g l y to the 2-39 g l y c o p e p t i d e b inds o n l y weakly to the 2-16 g l y c o p e p t i d e and n e g l i g i b l y to the s y n t h e t i c 1-16 p e p t i d e as measured by radioimmune c o m p e t i t i o n assays . T h i s appears to i n d i c a t e t h a t a pep t ide o ther than the s y n t h e t i c 1-16 N - t e r m i n a l pep t ide i s r e q u i r e d and the carbohydrate re s idues may be e s s e n t i a l f o r rho 4D2 ant ibody b i n d i n g a c t i v i t y . F i n a l l y , rho 4A3 ant ibody b i n d i n g was a l s o i n h i b i t e d by the s y n t h e t i c 1-16 p e p t i d e . A c c o r d i n g l y , i t s b i n d i n g s i t e i s l o c a t e d near the amino te rminus . R e c e n t l y , Hargrave e t a l . (1986) have l o c a l i z e d o ther bovine and r a t rhodops in a n t i b o d i e s to the N-terminus w i t h a s e r i e s o f s y n t h e t i c rhodops in pept ide analogues . I n a d d i t i o n , the a n t i g e n i c determinants o f s e v e r a l a n t i - f r o g rhodops in monoclonal a n t i b o d i e s have been l o c a l i z e d c l o s e to the N-terminus o f rhodops in ( W i t t e t a l . , 1984). A l l N - t e r m i n a l a n t i b o d i e s s t u d i e d here show an i n c r e a s e i n b i n d i n g to b l eached , T r i t o n X-100 s o l u b i l i z e d d i s c s as opposed to unbleached, s o l u b i l i z e d d i s c s . I t i s expected t h a t b l e a c h i n g causes a s t r u c t u r a l change -82-to the rhodops in molecule a l l o w i n g f o r g r e a t e r ant ibody a c c e s s i b i l i t y . The a n t i g e n i c s i t e may be o n l y p a r t i a l l y exposed i n unbleached rhodops in or the N - t e r m i n a l a n t i b o d i e s may be b i n d i n g to s m a l l amounts o f b leached rhodops in i n these p r e p a r a t i o n s . A l though a c o n f o r m a t i o n a l change on the cy top la smic sur face o f rhodops in must occur upon b l e a c h i n g to a l l o w t r a n s d u c i n b i n d i n g , no i n c r e a s e i n C - t e r m i n a l ant ibody b i n d i n g to b leached r h o d o p s i n was observed (MacKenzie e t a l . , 1984). None o f the N - t e r m i n a l a n t i b o d i e s were found to b i n d w e l l to rhodops in i n the membrane. I t appears t h a t the N - t e r m i n a l a n t i g e n i c determinants remain h idden i n the conformat ion o f the rhodops in molecule o n l y to become a v a i l a b l e f o r b i n d i n g upon d e t e r g e n t - s o l u b i l i z a t i o n . However, one ant ibody , rho 4D2, was shown by immunocytochemical l a b e l i n g and c o m p e t i t i v e i n h i b i t i o n assays to have a h i g h degree o f b i n d i n g to rhodops in i n g l u t a r a l d e h y d e - f i x e d ROS. T h i s suggests t h a t the f i x a t i o n process exposes the N - t e r m i n a l rho 4D2 a n t i g e n i c s i t e f o r b i n d i n g . Polans e t a l . (1986) a l s o observed the a b i l i t y o f p o l y c l o n a l and monoclonal a n t i b o d i e s , s p e c i f i c f o r the N - t e r m i n a l domain o f o p s i n , to b i n d the e x t r a c e l l u l a r sur face o f ROS plasma membrane o f a l d e h y d e - f i x e d , but not u n f i x e d f r o g r e t i n a s . I t i s not c l e a r what e f f e c t f i x a t i o n has on t h i s r e g i o n o f r h o d o p s i n . The a b i l i t y o f rho 4D2 ant ibody to b i n d to rhodops in i n the plasma membrane i n f i x e d ROS was used i n l a t e r s t u d i e s i n v o l v i n g ROS/RPE i n t e r a c t i o n s . Immunocytochemical l a b e l i n g s t u d i e s o f i s o l a t e d ROS u s i n g rho 1D4 and rho 4D2 monoclonal a n t i b o d i e s conjugated to g o l d - d e x t r a n p a r t i c l e s v e r i f i e d t h a t the C-terminus o f rhodops in was exposed to the cytop la sm, w h i l e the N-terminus o f plasma membrane l o c a l i z e d rhodops in was exposed to the e x t r a c e l l u l a r s u r f a c e . Two monoclonal a n t i b o d i e s rho 4B4 and rho 8A6 s p e c i f i c f o r i n t e r n a l segments o f rhodops in exposed on the cy top la smic sur face o f d i s c s have been -Re-generated and p a r t i a l l y c h a r a c t e r i z e d . The b i n d i n g o f rho 4B4 to rhodops in 239 240 was e l i m i n a t e d by S_^  aureus V-8 protease d i g e s t i o n a t the G l u - Ser s i t e (MacKenzie and Molday, 1982). Furthermore, a s y n t h e t i c p e p t i d e o f the F ^ - F 2 loop i n h i b i t e d rho 4B4 ant ibody b i n d i n g to r h o d o p s i n . Immunocytochemical l a b e l i n g s t u d i e s and c o m p e t i t i v e i n h i b i t i o n s t u d i e s u s i n g s ea l ed d i s c v e s i c l e s i n d i c a t e d t h a t the rho 4B4 b i n d i n g s i t e i s l o c a l i z e d on the c y t o p l a s m i c sur face o f d i s c membranes (MacKenzie and Molday, 1982). These r e s u l t s support the v iew t h a t the a n t i g e n i c determinant f o r t h i s ant ibody i s l o c a l i z e d a long the F^-F^ loop between h e l i x V and V I o f r h o d o p s i n . T h i s segment o f r h o d o p s i n , l i k e the N-terminus and C-terminus o f r h o d o p s i n , i s h i g h l y conserved i n o ther mammalian spec ies (MacKenzie e t a l . , 1984), as w e l l as i n f r o g rhodops in from red rod photoreceptor c e l l s (Hicks and Molday, 1986). The ant ibody rho 8A6 appears to b i n d to another i n t e r n a l loop r e g i o n o f rhodops in exposed on the cy top la smic sur face o f d i s c membranes. T h i s i s based on the f i n d i n g t h a t t h i s ant ibody b inds to the N - t e r m i n a l F^ fragment o f S_^  aureus p r o t e a s e - d i g e s t e d rhodops in and to a s m a l l e r fragment from chymotryps in c leavage . I n c o n t r a s t to the N - t e r m i n a l a n t i b o d i e s , however, the rho 8A6 ant ibody has been shown to b i n d to the c y t o p l a s m i c sur face o f d i s c s . On the b a s i s o f the rhodops in model (Hargarve e t a l . , 1984; Ovchinnikov e t a l . , 1982) and p r o t e o l y t i c s t u d i e s u s i n g chymotryps in , i t would appear t h a t the rho 8A6 ant ibody b inds to c y t o p l a s m i c loops between h e l i x I and I I or I I I and IV ( F i g . 27) . The i n a b i l i t y o f a s y n t h e t i c pept ide o f the segment between h e l i x I and I I ( V a l . . . . Tyr) to i n h i b i t b i n d i n g to rhodops in may i n d i c a t e t h a t t h i s ant ibody does not b i n d to t h i s segment, or a l t e r n a t i v e l y , b i n d i n g i s dependent on a s p e c i f i c " l o o p " conformat ion as may be found i n longer rhodops in fragments. Such a requirement has been - 8 4 -demonstrated i n immunochemical s t u d i e s o f lysozyme (Arnon et a l . , 1971). F u r t h e r s t u d i e s u s i n g s m a l l e r CNBr-fragments or p r o t e a s e - d e r i v e d rhodops in pept ides or s y n t h e t i c analogues may h e l p to more c l e a r l y de f ine the a n t i g e n determinant f o r t h i s a n t i b o d y . The reason f o r the h i g h degree o f a n t i g e n c i t y found f o r the N - t e r m i n a l and C - t e r m i n a l reg ions o f rhodops in i s not known. Many p r o t e i n s have been shown to e x h i b i t h i g h a n t i g e n i c r e a c t i v i t y a long N - t e r m i n a l and C - t e r m i n a l r eg ions (Anderer and Schlumberger, 1965; A l t s c h u h and Regenmortel , 1982; Quesniaux et a l . , 1983). Severa l recent s t u d i e s i n d i c a t e t h a t there i s a h i g h degree o f c o r r e l a t i o n between the a n t i g e n i c i t y o f p e p t i d e reg ions and segmental m o b i l i t y (Ta iner e t a l . , 1984). The C-terminus o f rhodops in i s h i g h l y a c c e s s i b l e to a n t i b o d i e s as w e l l as p r o t e o l y t i c enzymes i n both membrane and s o l u b i l i z e d s t a t e s and may e x h i b i t such segmental m o b i l i t y . The N-terminus , on the o ther hand, i s l e s s a c c e s s i b l e f o r e i t h e r ant ibody b i n d i n g or p r o t e o l y s i s when rhodops in i s embedded i n the d i s c membrane. A n t i g e n i c de terminant s , however, become a c c e s s i b l e upon s o l u b i l i z a t i o n (Molday and MacKenzie , 1983). Such treatment i s known to a l t e r the conformat ion o f rhodops in and impart a l e s s - o r d e r e d s t r u c t u r e . T h i s may r e s u l t i n an i n c r e a s e i n segmental m o b i l i t y and a cor re spond ing increa se i n ant ibody b i n d i n g . The c y t o p l a s m i c loop and i n t r a d i s c a l i n t e r n a l loop reg ions appear to be l e s s a n t i g e n i c p o s s i b l y due to reduced segmental m o b i l i t y . N e v e r t h e l e s s , two a n t i b o d i e s rho 4B4 and rho 8A6 have been shown to be d i r e c t e d a g a i n s t a n t i g e n i c determinants o f rhodops in exposed on cy top la smic sur face o f d i s c membranes. These a n t i b o d i e s , however, show s i g n i f i c a n t l y reduced b i n d i n g a c t i v i t y to s y n t h e t i c pept ides and may depend to a g rea te r degree on the conformat ion o f the pept ide as i t appears i n the i n t a c t r h o d o p s i n m o l e c u l e . A computer model ing program was used to p r e d i c t a n t i g e n i c reg ions o f -85-rhodops in based on the a c c e s s i b i l i t y , h y d r o p h i l i c i t y and f l e x i b i l i t y o f the v a r i o u s pept ide segments (Parker e t a l . , 1986; J a n i n , 1979). A composite graph i d e n t i f i e d the N - t e r m i n a l 1-39 segment, c y t o p l a s m i c loops I - I I and V-VI ( F 1 - F 2 ) , i n t r a d i s c a l loops IV-V and V I - V I I , and the C - t e r m i n a l segment as h i g h l y a n t i g e n i c reg ions ( F i g . 28) . Monoclonal a n t i b o d i e s d e s c r i b e d i n t h i s r e p o r t would g e n e r a l l y support t h i s p r e d i c t i o n w i t h known a n t i b o d i e s aga ins t the C - t e r m i n a l and N - t e r m i n a l domains o f rhodops in , as w e l l as the F ^ - F 2 loop and an a d d i t i o n a l cy top la smic loop ( I - I I loop or I I I - I V loop) segment. T h i s , however, does not e l i m i n a t e the p o s s i b i l i t y t h a t monoclonal a n t i b o d i e s can be r a i s e d aga i n s t " l e s s a n t i g e n i c " reg ions o f the rhodops in molecu le . whereas a n t i r h o d o p s i n a n t i b o d i e s are p r o v i n g to be h i g h l y u s e f u l t o o l s f o r s t r u c t u r a l and f u n c t i o n a l s t u d i e s , they are a l s o v a l u a b l e i n immunocytochemical s t u d i e s on the d i s t r i b u t i o n o f rhodops in and r e l a t e d p r o t e i n s i n c e l l membranes (Hicks and Molday, 1986; Molday e t a l . , 1986). The a b i l i t y o f a n t i r h o d o p s i n a n t i b o d i e s to be used i n immunoa f f in i ty chromatography techniques f o r the p u r i f i c a t i o n o f pep t ide e l u t e d rhodops in i s an added advantage f o r f u n c t i o n a l s t u d i e s i n v o l v i n g r e c o n s t i t u t e d r h o d o p s i n . F u n c t i o n a l rhodops in expressed i n COS-1 c e l l s was p u r i f i e d e s s e n t i a l l y to homogenity u s i n g rho 1D4 immunoaf f in i ty chromatography f o l l o w e d by p e p t i d e e l u t i o n (Oprian e t a l . , 1987). T h i s a b i l i t y to p u r i f y expressed , f u l l y f u n c t i o n a l rhodops in from COS-1 c e l l s a l l o w s f o r f u r t h e r experiments i n t o the f u n c t i o n a l reg ions o f rhodops in u s i n g s i t e - d i r e c t e d mutagenesis t echnology . Moreover , p r e l i m i n a r y s t u d i e s u s i n g rho 2B2 a n t i r h o d o p s i n immunoaf f in i ty chromatography to separate b leached from unbleached rhodops in have been encouraging (unpubl i shed r e s u l t s ) . These s t u d i e s u t i l i z e d the g r e a t e r a f f i n i t y o f the a n t i r h o d o p s i n N - t e r m i n a l s p e c i f i c ant ibody f o r -86-RHODOPSIN 100 80 120 160 200 240 Residue Number 280 320 F i g u r e 28. Computer generated rhodops in composite graph i l l u s t r a t i n g the p r e d i c t e d pept ide segments o f g rea te s t a n t i g e n i c i t y . The seven transmembrane h e l i c a l segments are numbered from l e f t to r i g h t . T h i s composite sur face p r o f i l e o f rhodops in was generated u s i n g a program a v a i l a b l e from the A l b e r t a Pept ide I n s t i t u t e (B iochemis t ry Department, U n i v e r s i t y o f A l b e r t a ) . - 8 7 -bleached rhodops in over unbleached rhodops in and s p e c i f i c pep t ide e l u t i o n . The use o f a n t i r h o d o p s i n monoclonal a n t i b o d i e s to d i r e c t l y study t r a n s d u c i n and rhodops in k inase b i n d i n g may prove q u i t e u s e f u l . However, the l a r g e s i z e o f an IgG molecule bound to one rhodops in s i t e can s t e r i c a l l y i n h i b i t another independent ant ibody b i n d i n g s i t e on rhodops in as shown by rho 4B4 ant ibody ( s p e c i f i c f o r the F ^ " F 2 1 ° ° P ) a b i l i t y to i n h i b i t the b i n d i n g o f a C - t e r m i n a l s p e c i f i c (rho 3A6) a n t i b o d y . T h i s problem may p o s s i b l y be overcome by u s i n g monoclonal ant ibody Fab fragments i n such b i n d i n g i n h i b i t i o n s t u d i e s . I n g e n e r a l , monoclonal a n t i b o d i e s ag a in s t a n t i g e n i c s i t e s exposed on the e x t r a c e l l u l a r or i n t r a d i s c a l sur face and the c y t o p l a s m i c s i d e o f ROS plasma and d i s c membranes shou ld prove p a r t i c u l a r l y v a l u a b l e i n s t u d y i n g the o r g a n i z a t i o n and b i o s y n t h e s i s o f rhodops in by immunocytochemical and b i o c h e m i c a l techniques and i n t e r a c t i o n s o f rhodops in w i t h o ther ROS p r o t e i n s . F i n a l l y , one a n t i r h o d o p s i n monoclonal ant ibody (rho 4D2) was s u c c e s s f u l l y used as a b i o c h e m i c a l and immunocytochemical probe f o r s t u d y i n g the b i n d i n g and i n g e s t i o n o f ROS by bovine r e t i n a l pigment e p i t h e l i a l c e l l s as d e s c r i b e d i n the next two Chapters . -88-CHAPTER 3 Evidence A g a i n s t the Role o f Rhodopsin i n Rod Outer Segment B i n d i n g to RPE C e l l s MATERIALS AND METHODS MATERIALS Hanks' ba l anced s a l t s o l u t i o n (HBSS), Du lbecco ' s phosphate-buf fered s a l i n e (dPBS) and the enzymes co l l agenase Type I V , t e s t i c u l a r h y a l u r o n i d a s e , g lucose oxidase Type V , l a c t o p e r o x i d a s e and p e p s i n were a l l o b t a i n e d from Sigma Chemical Co. ( S t . L o u i s , MO). RPMI 1640 medium was purchased from Gibco (Grand I s l a n d , NY) w h i l e t r y p s i n (1:250) was purchased from D i f c o L a b o r a t o r i e s ( D e t r o i t , M I ) . U l t r a g e l was purchased from LKB Instruments I n c . (Bromma, Sweden). Neuraminidase from A r t h r o b a c t e r u r e a f a c i e n s was ob ta ined from Boer inger Mannheim Chemical Co. (Penzburg, W. Germany). R i c i n u s  communis a g g l u t i n i n (RCA) I and I I were prepared from c a s t o r beans by the method o f N i c o l s o n and B l a u s t e i n (1972). T r i s - b u f f e r e d s a l i n e (TBS) c o n s i s t e d o f 0.02 M T r i s - H C l , pH 7 .4 , c o n t a i n i n g 0.15 M N a C l . METHODS RPE ISOLATION AND TISSUE CULTURE Bovine RPE c e l l s were i s o l a t e d and c u l t u r e d by a m o d i f i c a t i o n o f the method o f Basu e t a l . (1983). B r i e f l y , f r e s h l y enuc lea ted eyes were d i s s e c t e d under s t e r i l e c o n d i t i o n s by i n c i s i n g the eye b a l l c i r c u m f e r e n t i a l l y a t the l e v e l o f the ora s e r r a t a , and the cornea , l ens and -89-v i t r e o u s humour were removed. The r e t i n a was g e n t l y pee led from the u n d e r l y i n g RPE c e l l s u s i n g f i n e f o r c e p s . The o p t i c cup was r i n s e d twice w i t h 2 mL o f HBSS to remove r e s i d u a l broken ROS and c e l l u l a r d e b r i s . The RPE c e l l s were e n z y m a t i c a l l y removed from the B r u c h ' s membrane by adding 1-2 mL o f d i g e s t i o n s o l u t i o n c o n t a i n i n g 0.25% t r y p s i n , 105 u n i t s / m L co l l agenase Type IV and 50 u n i t s / m L t e s t i c u l a r h y a l u r o n i d a s e i n PBS, pH 7 .4 . The o p t i c cups were g e n t l y rocked at 37°C f o r 60-90 min d u r i n g t h i s procedure . The RPE c e l l s were r e l e a s e d from the B r u c h ' s membrane by a s p i r a t i o n and washed by low speed c e n t r i f u g a t i o n i n RPMI 1640 medium c o n t a i n i n g 10% FCS and a n t i b i o t i c s (100 U/mL p e n i c i l l i n , 100 U/mL s t r e p t o m y c i n and 0.25 ug/mL f u n g i z o n e ) . An a d d i t i o n a l treatment w i t h 0.1% t r y p s i n i n PBS was o c c a s i o n a l l y c a r r i e d out f o r 3-5 min a t room temperature i n order to separate the sheets o f RPE c e l l s . The RPE c e l l s were r o u t i n e l y p l a t e d out i n RPMI 1640 medium w i t h 10% FCS a t approx imate ly 20,000-30,000 c e l l s per w e l l i n 24 w e l l assay p l a t e s c o n t a i n i n g 13 mm g la s s c o v e r s l i p s or formvar-coated g o l d g r i d s . F i n a l l y , the RPE c e l l s were incubated a t 37°C i n an environment o f 5% CX>2 and 95% a i r . The c u l t u r e medium was changed d a i l y f o r the f i r s t 4 days to remove any c e l l u l a r d e b r i s from the c u l t u r e s . PREPARATION OF PLASMA MEMBRANE FREE DISC MEMBRANES ROS d i s c membranes, f r ee o f contaminat ing plasma membrane, were prepared from r i c i n - g o l d - d e x t r a n l a b e l e d ROS as d e s c r i b e d by Molday and Molday (1987a, 1987b). B r i e f l y , neuramin ida se - t rea ted (0.025 u n i t s / m L f o r 2-4 h) i n t a c t ROS were l a b e l e d w i t h 8-10 nm g o l d - d e x t r a n p a r t i c l e s conjugated to R i c i n u s communis a g g l u t i n i n (RCA I or RCA I I ) . Labe led ROS were o s m o t i c a l l y d i s r u p t e d by i n c u b a t i o n o v e r n i g h t i n 10 mM T r i s - a c e t a t e , pH 7 . 2 . D i s c membranes were then separated from the r i c i n - g o l d - d e x t r a n l a b e l e d plasma - 9 0 -membrane by c e n t r i f u g a t i o n on a 20-50% (w/w) sucrose g r a d i e n t . ROS plasma membrane-free d i s c membranes were c o l l e c t e d a t 30% (w/w) sucrose and washed once i n 10 mM T r i s - a c e t a t e , pH 7 . 2 , by c e n t r i f u g a t i o n a t 15,000 rpm f o r 20 min i n a S o r v a l l SS-34 r o t o r . The d i s c p e l l e t was resuspended i n 10 mM T r i s -a c e t a t e , pH 7 . 2 , and s t o r e d a t - 7 0 ° C . RADIOIODINATION OF ROS O s m o t i c a l l y i n t a c t ROS from 15 bovine r e t i n a s were c o l l e c t e d from a cont inuous sucrose g r a d i e n t and washed i n approx imate ly 20 mL of i s o l a t i o n medium (20% sucrose , 2 mM M g C l 2 > 0.13 M NaCl and 20 mM T r i s - a c e t a t e , pH 7.2) by c e n t r i f u g a t i o n i n a S o r v a l l SS-34 r o t o r a t 9000 rpm f o r 10 min at 4 ° C . The ROS p e l l e t was g e n t l y resuspended i n 2 mL o f i s o l a t i o n medium and l a c t o p e r o x i d a s e c a t a l y z e d i o d i n a t i o n was performed i n the dark a c c o r d i n g to a m o d i f i e d method o f C l a r k and H a l l (1982). B r i e f l y , 20 uL o f 0.5 M @ -D-g lucose , 75 ug o f l a c t o p e r o x i d a s e , 40 mU o f g lucose oxidase and 500 u C i o f 125 Na I were added per mL o f r e a c t i o n m i x t u r e . A f t e r 45 min a t 4 C the r e a c t i o n was stopped by the a d d i t i o n o f 4 mM N a l . The i o d i n a t e d ROS were washed 4 t imes i n i s o l a t i o n medium by c e n t r i f u g a t i o n as d e s c r i b e d above. The f i n a l p e l l e t was resuspended i n 2 mL o f i s o l a t i o n medium and a 10 uL a l i q u o t was counted i n a Beckman Gamma 8000 c o u n t e r . The number o f ROS/mL was determined by counts i n a hemacytometer chamber. The s p e c i f i c a c t i v i t y 2 o f the i o d i n a t e d ROS was approx imate ly 0 .5 -3 .0 x 10 dpm/1000 ROS. ANTIRHODOPSIN RHO 4D2 AND ITS F ( A B ' ) ^ FRAGMENT The a n t i r h o d o p s i n monoclonal an t ibody , rho 4D2, was p u r i f i e d from mouse a s c i t e s f l u i d as p r e v i o u s l y d e s c r i b e d i n Chapter 2. P e p s i n d i g e s t i o n o f the p u r i f i e d rho 4D2 monoclonal ant ibody was performed a c c o r d i n g to the method o f Lamoyi (1986). Approx imate ly 2 mg/mL of rho 4D2 ant ibody was - 9 1 -d i a l y z e d o v e r n i g h t a t 4 C aga i n s t 0 .1 M sodium a c e t a t e , pH 7 . 0 . Immediately be fore d i g e s t i o n the pH o f the p r o t e i n s o l u t i o n was a d j u s t e d to pH 4.2 w i t h 2 M a c e t i c a c i d . A f t e r warming the p r o t e i n to 3 7 ° C , 60 uL o f p e p s i n (2 mg/mL i n 0 .1 M sodium aceta te pH 4.2) was added. The r e a c t i o n was stopped by adding 25 uL o f 2 M T r i s - H C l , pH 8.0 and a d j u s t i n g the pH to n e u t r a l i t y w i t h 1 M NaOH. The F ( a b ' ) 2 fragment was i s o l a t e d on an U l t r a g e l AcA-34 column. I t s p u r i t y was conf i rmed by SDS-gel e l e c t r o p h o r e s i s , and the absence o f the 125 Fc fragment was demonstrated by Western b l o t t i n g u s i n g I - l a b e l e d P r o t e i n A . Both rho 4D2 monoclonal ant ibody and i t s F ( a b ' ) 2 fragment were coupled to g o l d - d e x t r a n p a r t i c l e s as d e s c r i b e d i n Chapter 2. MONOCLONAL ANTIBODY LABELING OF ROS I n t a c t ROS c o l l e c t e d from 6 r e t i n a s on a cont inuous sucrose g r a d i e n t were d i l u t e d w i t h 15 mL of cacody la te b u f f e r (0 .1 M sodium c a c o d y l a t e , 6% sucrose) and c e n t r i f u g e d i n a S o r v a l l SS-34 r o t o r f o r 8 min a t 8000 rpm. The ROS p e l l e t was g e n t l y resuspended i n the same b u f f e r and r e c e n t r i f u g e d as b e f o r e . The ROS p e l l e t was resuspended and f i x e d w i t h 1.25% g l u t a r a l d e h y d e i n cacody la te b u f f e r f o r 1-2 h a t 4 ° C . The f i x e d ROS were washed three times i n c acody la te b u f f e r and twice i n TBS by c e n t r i f u g a t i o n i n a S o r v a l l SS 34 r o t o r f o r 8 min a t 5000 rpm. The f i n a l p e l l e t was resuspended i n 250 uL o f u n d i l u t e d a n t i r h o d o p s i n immunogold-dextran reagent and g e n t l y r o t a t e d f o r 4-10 h a t 2 3 ° C . The immunogold l a b e l e d ROS were washed three t imes i n cacody la te b u f f e r by c e n t r i f u g a t i o n i n an Eppendorf c e n t r i f u g e and r e f i x e d as be fore w i t h 1.25% g lu ta ra ldehyde i n cacody la te b u f f e r f o r 1 h a t 4 ° C . F i n a l l y , the l a b e l e d ROS were prepared f o r the phagocytos i s assay by 3 washes w i t h cacody la te b u f f e r f o l l o w e d by 2 a d d i t i o n a l washes w i t h HBSS i n an Eppendorf c e n t r i f u g e . - 9 2 -I n o ther exper iments , g l u t a r a l d e h y d e - f i x e d , I - l a b e l e d ROS (1 mg/mL) were incuba ted w i t h a n t i r h o d o p s i n monoclonal a n t i b o d i e s (rho 1D4 or rho 4D2) (1 mg/mL) f o r 2 h a t 2 3 ° C . The l a b e l e d ROS were washed 3 t imes i n HBSS by c e n t r i f u g a t i o n f o r 10 min at 8000 rpm i n a S o r v a l l SS-34 r o t o r . F i n a l l y , the ROS p e l l e t s were resuspended i n 1 mL of HBSS and prepared f o r phagocytos i s a s says . PHAGOCYTOSIS ASSAY I s o l a t e d bovine RPE c e l l s grown to conf luency (80,000-100,000 c e l l s / w e l l ) i n 24 w e l l p l a t e s c o n t a i n i n g one 13 mm g la s s c o v e r s l i p per w e l l were washed i n RPMI 1640 medium c o n t a i n i n g 10% FCS p r i o r to per forming the phagocytos i s assay. When p e p t i d e s , d i s c s or ROS were used i n i n h i b i t i o n s t u d i e s , the RPE c e l l s were p re incuba ted w i t h the a p p r o p r i a t e i n h i b i t o r i n 20 mM NaN^ f o r 1.5 h a t 37 °C . S ince NaN^ was present i n the pept ide s used as i n h i b i t o r s , i t was i n c l u d e d i n a l l phagocytos i s i n h i b i t i o n experiments i n v o l v i n g the p e p t i d e s . Under these c o n d i t i o n s , the presence o f 20 mM NaN^ was observed to have no e f f e c t on the morphology o f RPE c e l l s , nor d i d i t prevent the phagocytos i s o f ROS. The RPE c e l l s were washed thoroughly i n dPBS and each c o v e r s l i p was p l a c e d i n 0.5 mL o f RPMI 1640 medium c o n t a i n i n g 125 7 10% FCS. The I - l a b e l e d ROS were resuspended i n HBSS and 0.5 mL (1 x 10 125 I - l a b e l e d ROS) was added to each w e l l . The phagocytos i s assay was c a r r i e d out a t 37°C f o r 2-3 h f o l l o w e d by washing i n dPBS. The g l a s s c o v e r s l i p s were d r i e d and counted i n a Beckman Gamma 8000 c o u n t e r . I n many assays , RPE c e l l s were p re incuba ted w i t h ROS i n the absence o f NaN^, w h i l e i n s t i l l o ther assays , no p r e i n c u b a t i o n step was r e q u i r e d . 125 R o u t i n e l y , I - l a b e l e d ROS-treated RPE c e l l s y i e l d e d a maximum number 4 o f counts o f 1 .5-10.0 x 10 d p m / c o v e r s l i p . I n c o n t r o l samples, when 13 mm 125 g l a s s c o v e r s l i p s w i t h no RPE c e l l s were t r e a t e d w i t h I - l a b e l e d ROS, the -93-maximum number o f counts was 0 .5 -2 .5 x 10 d p m / c o v e r s l i p . When phagocytos i s assays were performed f o r e l e c t r o n microscopy, each g l a s s c o v e r s l i p or formvar-coated g r i d c o n t a i n i n g the RPE c e l l s was incuba ted w i t h approx imate ly 5 x 10^. ROS/mL at 37°C f o r the des igna ted p e r i o d o f t i m e . The c e l l s were washed i n cacody la te b u f f e r and prepared f o r e l e c t r o n microscopy. ELECTRON MICROSCOPY Samples prepared f o r e l e c t r o n microscopy were f i x e d w i t h 1.25% g l u t a r a l d e h y d e i n cacody la te b u f f e r f o r 1 h a t 4 ° C . For t r a n s m i s s i o n e l e c t r o n microscopy the c e l l s were p o s t - f i x e d i n 1% osmium t e t r o x i d e , dehydrated i n e t h a n o l and embedded i n A r a l d i t e / E p o n . Cut s e c t i o n s were s t a i n e d w i t h u r a n y l ace ta te and l e a d c i t r a t e as d e s c r i b e d i n Chapter 2. RPE c e l l s prepared f o r scanning e l e c t r o n microscopy were dehydrated i n a graded e t h a n o l s e r i e s (50%, 70%, 90% and 2 changes o f abso lu te f o r 15 min each) and c r i t i c a l p o i n t d r i e d from CO^ i n a P o l a r o n apparatus (Lewis e t a l . , 1975). The c e l l s were coated w i t h e i t h e r g o l d - p a l l a d i u m i n a Technics Hummer V s p u t t e r - c o a t e r f o r secondary e l e c t r o n imaging or w i t h carbon i n a B a l z e r MED 010 carbon evaporator f o r b a c k s c a t t e r imaging . A l l e l e c t r o n m i c r o s c o p i c samples were viewed under a JEOL 1200EX e l e c t r o n microscope . -94-RESULTS MORPHOLOGY OF CULTURED BOVINE RPE CELLS Bovine RPE c e l l s were s u c c e s s f u l l y i s o l a t e d from f r e s h bov ine eyes by an enzymatic d i g e s t i o n procedure . V i a b l e RPE c e l l s were recovered i n t i s s u e c u l t u r e on a g l a s s subs t r a te and a l l o w e d to grow to conf luency w i t h a d o u b l i n g time o f approx imate ly 52 h . The genera l m o r p h o l o g i c a l f ea tures o f bov ine RPE c e l l s i n c u l t u r e are i l l u s t r a t e d i n F i g u r e 29. A t the l e v e l o f scanning e l e c t r o n microscopy (SEM), the RPE c e l l s appeared f l a t t e n e d w i t h an abundance o f m i c r o v i l l i ( F i g . 29A). The RPE c e l l s a l s o had a few 1-2 um i n diameter membrane v e s i c l e s (presumably bound ROS) a t t ached to t h e i r c e l l s u r f a c e . A t the l e v e l o f scanning t r a n s m i s s i o n e l e c t r o n microscopy (STEM), l a r g e melanosomes were observed i n pigmented bovine RPE c e l l s ( F i g . 29B). C l e a r vacuo le s were a l s o present a long w i t h numerous r i b b o n - l i k e m i t o c h o n d r i a . PHAGOCYTOSIS OF ROS BY CULTURED RPE CELLS Bovine RPE c e l l s ma inta ined i n c u l t u r e f o r one week were capable o f b i n d i n g and p h a g o c y t i z i n g e i t h e r u n f i x e d or g l u t a r a l d e h y d e - f i x e d , i s o l a t e d bovine ROS. A f t e r i n c u b a t i n g RPE c e l l s w i t h ROS f o r 2 hours a t 3 7 ° C , many ROS were observed by SEM to be bound to the m i c r o v i l l i - r i c h sur face o f the RPE c e l l ( F i g . 30A). A n a l y s i s by t r a n s m i s s i o n e l e c t r o n microscopy (TEM) i n d i c a t e d t h a t many ROS fragments had been i n g e s t e d or were i n the process o f b e i n g engul fed by the RPE c e l l s ( F i g . 30B). D a r k - s t a i n i n g melan in granules d i s p e r s e d between the ROS-conta in ing phagosomes were a l s o prominent i n these c e l l s . RPE c e l l s were capable o f i n g e s t i n g many g l u t a r a l d e h y d e -f i x e d ROS o f d i f f e r e n t s i z e s and shapes as shown i n F i g u r e 31A. Many o f the ROS fragments have r e t a i n e d t h e i r m o r p h o l o g i c a l c h a r a c t e r i s t i c s and, i n Figure 29. Scanning and scanning t r a n s m i s s i o n e l e c t r o n micrographs o f c u l t u r e d bovine RPE c e l l s . E n z y m a t i c a l l y i s o l a t e d bovine RPE c e l l s were c u l t u r e d on a g la s s subs t r a te and prepared f o r scanning e l e c t r o n microscopy (A) or grown on formvar coated g o l d g r i d s and prepared f o r scanning t r a n s m i s s i o n e l e c t r o n microscopy ( B ) . The arrows i n (A) denote v e s i c l e s , p o s s i b l y r o d outer segments, a t t ached to the c e l l s u r f a c e . Ribbon-shaped m i t o c h o n d r i a (M) w i t h i d e n t i f i a b l e c r i s t a e are shown amongst the pigment ( P ) . Bar = 2 urn. - 9 6 -Figure 30. B i n d i n g and phagocytos i s o f f i x e d ROS by c u l t u r e d bov ine RPE c e l l s . A) Scanning e l e c t r o n micrograph o f the m i c r o v i l l i - r i c h sur f ace o f a RPE c e l l c o n t a i n i n g s e v e r a l bound ROS. Arrows denote the p o s i t i o n o f the ROS. B) Transmis s ion e l e c t r o n micrograph o f a RPE c e l l i n the process o f e n g u l f i n g a ROS. The RPE c e l l a l s o conta ins a ROS phagosome (Ph) and numerous dark-s t a i n i n g melan in granules ( P ) . Bar — 0.5 um. - 9 7 -F i g u r e 31. Transmi s s ion e l e c t r o n micrographs o f r e t i n a l pigment e p i t h e l i a l c e l l s which had been incubated w i t h e i t h e r g l u t a r a l d e h y d e - f i x e d ROS (A) or u n f i x e d ROS (B) . Ph = phagosome; N - n u c l e u s . Bar = 1 urn. -98-p a r t i c u l a r , s t acks o f d i s c s surrounded by a plasma membrane. I n c o n t r a s t , the ROS morphology o f i n g e s t e d , u n f i x e d ROS was c o n s i d e r a b l y d i s r u p t e d . ( F i g . 31B). EFFECT OF PEPTIDES ON BINDING AND PHAGOCYTOSIS 125 A q u a n t i t a t i v e assay u s i n g I - l a b e l e d ROS was developed to measure the i n t e r a c t i o n o f ROS w i t h c u l t u r e d RPE c e l l s . As shown i n F i g u r e 32, p r e i n c u b a t i o n o f RPE c e l l s w i t h i n c r e a s i n g c o n c e n t r a t i o n s o f u n l a b e l e d ROS 125 r e s u l t e d i n a 75% maximum i n h i b i t i o n o f b i n d i n g and phagocytos i s o f I -l a b e l e d ROS by c u l t u r e d RPE c e l l s . The i n a b i l i t y to comple te ly i n h i b i t 125 b i n d i n g o f I - l a b e l e d ROS to RPE c e l l s may be due to r e c e p t o r turnover i n the c e l l a l l o w i n g f o r a d d i t i o n a l ROS attachment and i n g e s t i o n d u r i n g the chase p e r i o d . Whi le a p r e i n c u b a t i o n w i t h 2 - f o l d excess o f u n l a b e l e d ROS i n 125 the presence o f NaN^ r e s u l t e d i n a 40% i n h i b i t i o n o f b i n d i n g o f I -l a b e l e d ROS to c u l t u r e d RPE c e l l s , l i t t l e i n h i b i t i o n o f b i n d i n g was observed when RPE c e l l s were p r e i n c u b a t e d w i t h excess f rozen-thawed, plasma membrane-f ree d i s c membranes ( F i g . 33) . RPE c e l l s were v i s i b l y u n a f f e c t e d by p r e i n c u b a t i o n i n the presence o f NaN^, and a f t e r i t s removal , were capable 125 o f b i n d i n g and i n t e r n a l i z i n g I - l a b e l e d ROS. These r e s u l t s i n d i c a t e t h a t the s p e c i f i c l i g a n d r e s p o n s i b l e f o r receptor-media ted phagocytos i s o f ROS appears to be present o n l y on the ROS plasma membrane and not on the ROS d i s c membrane. T h i s i n h i b i t i o n assay was a l s o used to assess the p o s s i b l e r o l e o f the N - t e r m i n a l segment o f rhodops in on the i n t e r a c t i o n o f ROS w i t h RPE c e l l s . As 125 shown i n F i g u r e 33, no s i g n i f i c a n t i n h i b i t i o n o f I - l a b e l e d ROS b i n d i n g and phagocytos i s was observed w i t h a 12-100- fo ld excess o f CNBr-der ived N-t e r m i n a l 2-39 rhodops in pept ide or a s y n t h e t i c 1-16 N - t e r m i n a l pep t ide -99-F i g u r e 32. 125 I n h i b i t i o n o f I - l a b e l e d ROS b i n d i n g and phagocytos i s by p r e i n c u b a t i o n s w i t h u n l a b e l e d ROS. I n c r e a s i n g c o n c e n t r a t i o n s o f u n l a b e l e d ROS were p r e i n c u b a t e d f o r 3 h a t 37 C w i t h 1 week o l d bov ine RPE c e l l s grown to conf luency on 13 nun g la s s c o v e r s l i p s i n 24 well^ajssay p l a t e s . A f t e r washing, the bovine RPE c e l l s were t r e a t e d w i t h 1 x 10 I - l a b e l e d ROS/wel l f o r 3 h . The c o v e r s l i p s were aga in washed, d r i e d and counted i n a Beckman Gamma 8000 c o u n t e r . Va lues represent mean + S .D. n = 3 g la s s c o v e r s l i p s . -100-120 T5 C 100 D O CD E 80 d ~o E D 60 E X O 40 6^ I . I J .X. 20 -...X.-. F i g u r e 33. 7 125 I n h i b i t i o n o f b i n d i n g and phagocytos i s o f 1.0 x 10 I - l a b e l e d bovine ROS by bovine ROS, ROS d i s c membranes and pept ide s o f r h o d o p s i n . Conf luent c u l t u r e s o f bovine RPE c e l l s were p r e i n c u b a t e d w i t h a) RPE c e l l c u l t u r e f l u i d ; b) 2.0 x 10 ROS/mL; c) 12.6 uM 1-16 N - t e r m i n a l s y n t h e t i c rhodops in p e p t i d e ; d) 11.2 uM 330-348 C - t e r m i n a l s y n t h e t i c rhodops in p e p t i d e ; e) 1.5 uM o f immunoaf f in i ty p u r i f i e d 2-39 N - t e r m i n a l rhodogs in g l y c o p e p t i d e ; or f ) 75 ug o f frozen-thawed ROS d i s c s f o r 1.5 h a t 37 C i n 24 w e l l assay p l a t e s c o n t a i n i n g 13 mm g l a s s ^ c j v g r s l i p s . A f t e r washing i n dPBS the RPE c e l l s were incubated w i t h 1 x 10 I - l a b e l e d ROS/wel l f o r 2-3 h at 3 7 ° C . The c o v e r s l i p s were washed, d r i e d and counted; g) c o n t r o l c o v e r s l i p s c o n t a i n i n g no RPE c e l l s . Values represent mean + S . D . n = 3 - 6 g l a s s c o v e r s l i p s . - 1 0 1 -analogue o f r h o d o p s i n . Molar excess was c a l c u l a t e d based on the e s t imated amount o f rhodops in i n the ROS plasma membrane a v a i l a b l e f o r b i n d i n g to RPE r e c e p t o r s i n each w e l l . A s y n t h e t i c 330-348 pept ide analogue o f the C-terminus o f rhodops in a l s o showed no i n h i b i t o r y e f f e c t ( F i g . 33) . BINDING AND PHAGOCYTOSIS OF ANTIBODY LABELED ROS The p o s s i b l e r o l e o f rhodops in i n b i n d i n g and phagocytos i s o f ROS by RPE c e l l s was a l s o s t u d i e d u s i n g immunocytochemical l a b e l i n g methods. I n i t i a l l y 125 g l u t a r a l d e h y d e - f i x e d , i n t a c t , I - l a b e l e d ROS were t r e a t e d w i t h 1 0 - f o l d molar excess o f e i t h e r a n t i r h o d o p s i n rho 4D2 or rho 1D4 monoclonal a n t i b o d i e s i n an attempt to i n h i b i t ROS b i n d i n g and phagocytos i s by RPE c e l l s ( F i g . 34) . A g a i n molar excess was based on the e s t imated amount o f r h o d o p s i n a v a i l a b l e i n the plasma membrane f o r ant ibody b i n d i n g . The rho 4D2 monoclonal ant ibody was i n e f f e c t i v e i n i n h i b i t i n g ROS b i n d i n g and phagocytos i s by c u l t u r e d RPE c e l l s . L i k e w i s e rho 1D4 a n t i b o d y - t r e a t e d , 125 I - l a b e l e d ROS s e r v i n g as a c o n t r o l , d i d not i n h i b i t b i n d i n g o f the ROS to RPE c e l l s ( F i g . 34) . To ensure t h a t f i x e d ROS were b e i n g adequate ly coated w i t h rho 4D2 monoclonal ant ibody p r i o r to per forming the assay, f i x e d ROS fragments were l a b e l e d w i t h rho 4D2 ant ibody conjugated to 10 nm g o l d p a r t i c l e s . As shown by TEM i n F i g u r e 35A, g o l d p a r t i c l e s were found evenly d i s t r i b u t e d a long the a c c e s s i b l e plasma membrane o f the ROS. Scanning e l e c t r o n microscopy conf i rmed t h a t the ROS were evenly coated w i t h rho 4D2 immunogold p a r t i c l e s ( F i g . 35B). Larger g o l d p a r t i c l e s coupled to ant ibody molecules e x e r t a s t e r i c h indrance e f f e c t r e s u l t i n g i n a reduced d e n s i t y o f g o l d l a b e l i n g ( r e s u l t s not shown). The e f f e c t o f l a b e l i n g f i x e d ROS w i t h rho 4D2 a n t i b o d y - g o l d - d e x t r a n p a r t i c l e s on b i n d i n g and phagocytos i s by bovine RPE c e l l s was a l s o s t u d i e d - 1 0 2 -Figure 34. The e f f e c t o f l a b e l i n g f i x e d ROS w i t h a n t i r h o d o p s i n monoclonal a n t i b o d i e s on t h e i r a b i l i t y to be bound and phagocyt i zed by bovine RPE c e l l s . G l u t a r a l d e h y d e - f i x e d I - l a b e l e d ROS (1 x 10 ROS/wel l ) were t r e a t e d (a) w i t h b u f f e r ; (b) w i t h p u r i f i e d C - t e r m i n a l rho 1D4 monoclonal a n t i b o d y ; or (c) w i t h p u r i f i e d N - t e r m i n a l rho 4D2 monoclonal ant ibody p r i o r to be ing incuba ted w i t h 1 week o l d bovine RPE c e l l s grown to conf luency on 13 mm g la s s c o v e r s l i p s i n 24 w e l l assay p l a t e s . A f t e r 3 h the g l a s s c o v e r s l i p s were washed and counted. Values repre sent the average o f 2-4 g l a s s c o v e r s l i p s . - 1 0 3 -Figure 35. A t r a n s m i s s i o n e l e c t r o n micrograph (A) and a scanning e l e c t r o n micrograph (B) o f g l u t a r a l d e h y d e - f i x e d ROS l a b e l e d d i r e c t l y w i t h rho 4D2 a n t i r h o d o p s i n monoclonal ant ibody conjugated to 9 nm g o l d - d e x t r a n p a r t i c l e s . The arrows denote the in tense d e n s i t y o f g o l d p a r t i c l e s a l o n g the ROS plasma membrane. Bar = 0.2 urn. -104-by e l e c t r o n microscopy . As i l l u s t r a t e d i n F igure 36A,B, rho 4D2 ant ibody-g o l d - d e x t r a n l a b e l e d ROS were seen both on the sur face and w i t h i n phagosomes o f the RPE c e l l s by TEM. B i n d i n g and phagocytos i s was a l s o observed f o r ROS dense ly l a b e l e d w i t h g o l d - d e x t r a n p a r t i c l e s conjugated to the F(ab')^ fragment o f the rho 4D2 rhodops in ant ibody ( F i g . 36C). T h i s i n d i c a t e d t h a t the phagocytos i s o f the ant ibody g o l d - d e x t r a n l a b e l e d ROS d i d not occur by way o f b i n d i n g to p u t a t i v e F £ r ecep tor s on the RPE c e l l s . When RPE c e l l s c o n t a i n i n g a n t i b o d y - g o l d - d e x t r a n l a b e l e d ROS were m a i n t a i n e d a t 37°C f o r 4-20 h a r e d i s t r i b u t i o n and c l u s t e r i n g o f g o l d p a r t i c l e s i n s i d e the ROS c o n t a i n i n g phagosome was observed ( F i g . 36D). A n t i b o d y - g o l d - l a b e l e d ROS bound to the sur face o f RPE c e l l s c o u l d a l s o be d i s t i n g u i s h e d from those which had been p h a g o c y t i z e d by scanning e l e c t r o n microscopy and b a c k s c a t t e r imaging. As shown i n F i g u r e 37A, secondary e l e c t r o n imaging o f a carbon-coated sample enabled one to v i s u a l i z e the sur face morphology o f the RPE c e l l and de tec t sur face bound ROS. Wi th b a c k s c a t t e r e l e c t r o n imaging g o l d p a r t i c l e s were observed i n c l u s t e r s on the sur face o f the ROS ( F i g . 37B). The comparison o f the secondary and b a c k s c a t t e r e l e c t r o n images o f the same f i e l d a l l o w s f o r the d i s t i n c t i o n between sur face bound ROS and ROS t h a t had been i n t e r n a l i z e d by the RPE c e l l . Phagocytos i s o f immunogold-dextran l a b e l e d ROS was f u r t h e r viewed by TEM ( F i g . 37C). These r e s u l t s c o n f i r m the f i n d i n g t h a t RPE c e l l s e f f e c t i v e l y b i n d and i n t e r n a l i z e ant ibody g o l d - d e x t r a n l a b e l e d ROS. -105-Figure 36. E l e c t r o n micrographs i l l u s t r a t i n g the b i n d i n g and phagocytos i s o f rho 4D2 immunogold-dextran l a b e l e d ROS by c u l t u r e d bov ine RPE c e l l s . G l u t a r a l d e h y d e - f i x e d bovine ROS l a b e l e d w i t h e i t h e r the rho 4D2 monoclonal ant ibody (A,B) or i t s F ( a b ' ) . fragment (C) conjugated to 9 nm g o l d - d e x t r a n p a r t i c l e s were incubated w i t h c u l t u r e d bovine RPE c e l l s f o r 2-3 h . Note t h a t both i n t e n s e l y and s p a r s e l y l a b e l e d ROS have been p h a g o c y t i z e d by the RPE c e l l s . Rho 4D2 immunogold l a b e l e d f i x e d ROS incubated w i t h bov ine RPE c e l l s f o r 20 h (D) . Arrows denote the r e d i s t r i b u t i o n and c l u s t e r i n g o f g o l d p a r t i c l e s i n s i d e the RPE c e l l . Bar — 0.5 urn. -106-Figure 37. E l e c t r o n micrographs i l l u s t r a t i n g the b i n d i n g and phagocytos i s o f immunogold-dextran l a b e l e d ROS. G l u t a r a l d e h y d e - f i x e d ROS were d i r e c t l y l a b e l e d w i t h rho 4D2 monoclonal ant ibody conjugated to 12 nm g o l d - d e x t r a n p a r t i c l e s p r i o r to f e e d i n g to bovine RPE c e l l s . A) Scanning e l e c t r o n micrograph u s i n g secondary e l e c t r o n imaging o f an RPE c e l l w i t h a sur face bound ROS as noted by the arrow. B) B a c k s c a t t e r e l e c t r o n image o f the same r e g i o n o f the c e l l . Go ld p a r t i c l e s were de tec ted not o n l y from the sur face bound ROS but a l s o from a ROS which had been p h a g o c y t i z e d . C) Transmi s s ion e l e c t r o n micrograph o f another RPE c e l l showing g o l d - l a b e l e d ROS w i t h i n the RPE c e l l . Bar = 0 . 5 urn. -107-DISCUSSION The s e l e c t i v i t y o f RPE c e l l s f o r the phagocytos i s o f ROS prov ide s s t rong evidence t h a t the sur face o f the ROS plasma membrane c o n t a i n s s p e c i f i c membrane components or l i g a n d s which i n t e r a c t w i t h r e c e p t o r s on RPE c e l l s (Mayerson and H a l l , 1986). O ' B r i e n (1976) has suggested t h a t rhodops in may serve as the r e c o g n i t i o n component. Rhodopsin, the major membrane p r o t e i n o f ROS d i s c s , i s a l s o present i n s i g n i f i c a n t q u a n t i t i e s i n the ROS plasma membrane (Dewey e t a l . , 1969; H i c k s and Molday, 1986). Two mannose and N-a c e t y l g l u c o s a m i n e - c o n t a i n i n g o l i g o s a c c h a r i d e cha ins l i n k e d to asparagine re s idues a long the N - t e r m i n a l segment o f rhodops in may be i n v o l v e d i n t h i s r e c o g n i t i o n proce s s . The l e c t i n s , c o n c a n a v a l i n A and wheat germ a g g l u t i n i n , which b i n d to mannose and N - a c e t y l glucosamine re s idues on rhodops in (Molday and Molday, 1979), r e s p e c t i v e l y , have been shown to b i n d to the e x t r a c e l l u l a r sur face o f ROS (Molday and Molday, 1979; H a l l and N i r , 1976; N i r and H a l l , 1979; McLaughl in and Wood, 1980; B r i d g e s , 1981). Excess mannose had e a r l i e r been r e p o r t e d to i n h i b i t the b i n d i n g o f ROS to RPE c e l l s ( P h i l i p and B e r n s t e i n , 1980) and a l s o i n h i b i t F c - m e d i a t e d phagocytos i s by macrophages. (Sung e t a l . , 1983). R e c e n t l y , however, the r o l e o f mannose as an i n h i b i t o r o f ROS b i n d i n g to RPE c e l l s has been d i s p u t e d s i n c e h i g h c o n c e n t r a t i o n s o f mannose f a i l e d to i n h i b i t r e c o n s t i t u t e d r h o d o p s i n v e s i c l e s , d i s c membranes or ROS b i n d i n g to RPE c e l l s ( L e n t r i c h i a e t a l . , 1987; Shirakawa et a l . , 1987). I n the present s tudy , both q u a n t i t a t i v e assays and e l e c t r o n microscopy were used to f u r t h e r examine the p o s s i b l e r o l e o f r h o d o p s i n , and i n p a r t i c u l a r , i t s N - t e r m i n a l segment i n the b i n d i n g and phagocytos i s o f ROS by c u l t u r e d bov ine RPE c e l l s . Bovine RPE c e l l s were s u c c e s s f u l l y i s o l a t e d and c u l t u r e d i n v i t r o on -108-g l a s s c o v e r s l i p s . These c e l l s g e n e r a l l y e x h i b i t e d normal e p i t h e l i a l c e l l c h a r a c t e r i s t i c s w i t h an abundance o f rough endoplasmic r e t i c u l u m , m i t o c h o n d r i a and a p i c a l m i c r o v i l l i , as w e l l as a predominance o f pigment i n the m e l a n o t i c RPE c e l l s . However, i n some areas o f the bov ine RPE c e l l c u l t u r e s , the c e l l s l a c k e d the t y p i c a l e p i t h e l o i d morphology. Oka e t a l . (1984) a l s o observed t y p i c a l e p i t h e l o i d growth i n pr imary c u l t u r e s o f human and bov ine RPE c e l l s w i t h s e v e r a l areas o f the c u l t u r e s e x h i b i t i n g i r r e g u l a r c e l l growth. I n agreement w i t h our s tudy , Basu e t a l . (1983) r e p o r t e d t h a t bo th me lanot i c and amelanot ic bovine RPE c e l l s became more e p i t h e l i a l - l i k e a f t e r 4-6 days i n c u l t u r e and were o c c a s i o n a l l y b i n u c l e a t e . C u l t u r e d bovine RPE c e l l s were f u n c t i o n a l l y capable o f not o n l y b i n d i n g , but a l s o , i n t e r n a l i z i n g ROS. Both g l u t a r a l d e h y d e - f i x e d and u n f i x e d ROS were e f f e c t i v e l y and abundantly phagocyt i zed by bovine RPE c e l l s . G lu ta ra ldehyde c r o s s - l i n k i n g o f ROS membrane p r o t e i n s f a c i l i t a t e d the p r e s e r v a t i o n and genera l m o r p h o l o g i c a l appearance o f RPE-phagocyt ized ROS. I n c o n t r a s t , u n f i x e d RPE phagocy t i zed ROS were q u i c k l y d i s t o r t e d and degraded as the ROS-c o n t a i n i n g phagosome moved toward the b a s a l r e g i o n o f the RPE c e l l . Pep t ide i n h i b i t i o n s t u d i e s u s i n g a s y n t h e t i c 1-16 p e p t i d e analogue and an immunoa f f in i ty p u r i f i e d 2-39 N - t e r m i n a l g l y c o p e p t i d e o f rhodops in f a i l e d 125 to i n h i b i t the b i n d i n g and phagocytos i s o f I - l a b e l e d ROS. These r e s u l t s i n d i c a t e d t h a t N - t e r m i n a l rhodops in pept ides d i d not b i n d to r e c e p t o r s on RPE c e l l s and i n h i b i t phagocytos i s even a t a 1 2 - 1 0 0 - f o l d g r e a t e r c o n c e n t r a t i o n than t h a t expected f o r rhodops in i n ROS plasma membranes. The a b i l i t y o f rhodops in i n d i s c membranes to i n h i b i t b i n d i n g and phagocytos i s was a l s o i n v e s t i g a t e d . For these s t u d i e s , d i s c membranes separated from plasma membranes on a sucrose g r a d i e n t were f r o z e n and thawed to expose the i n t r a d i s c a l sur face ( C l a r k and Molday, 1979). These unsea led d i s c membranes -109-a l s o f a i l e d to s i g n i f i c a n t l y i n h i b i t I - l a b e l e d ROS b i n d i n g to RPE c e l l s , sugges t ing t h a t the l i g a n d r e s p o n s i b l e f o r s p e c i f i c r eceptor -media ted phagocytos i s i s found o n l y i n the ROS plasma membrane. I n c o n t r a s t , excess u n l a b e l e d ROS were found to e f f e c t i v e l y i n h i b i t up to 75% o f the b i n d i n g and 125 phagocytos i s o f I - l a b e l e d ROS by RPE c e l l s . The i n a b i l i t y to i n h i b i t RPE c e l l s from f u r t h e r b i n d i n g and i n g e s t i o n o f r a d i o i o d i n a t e d ROS may be due to a d d i t i o n a l ROS s p e c i f i c - r e c e p t o r s y n t h e s i s or s p e c i f i c - r e c e p t o r r e c y c l i n g to the c e l l sur face d u r i n g the chase p e r i o d . H a l l and Abrams (1987) have suggested t h a t i n t e r n a l i z e d ROS s p e c i f i c - r e c e p t o r s i n the r a t RPE c e l l s are not degraded and are p o s s i b l y s o r t e d back to the c e l l sur face a f t e r a r ecovery p e r i o d o f 1-2 h . Thus, d u r i n g the chase p e r i o d , f u r t h e r ROS b i n d i n g to these r e c y c l e d recep tor s may o c c u r . These s t u d i e s , however, do not exclude the p o s s i b i l i t y t h a t rhodops in i n the ROS plasma membrane may c o n t a i n o l i g o s a c c h a r i d e cha ins w i t h a d i f f e r e n t sugar c o m p o s i t i o n . These a l t e r e d o l i g o s a c c h a r i d e cha ins may, i n t u r n , serve as l i g a n d s f o r the b i n d i n g o f ROS to RPE c e l l s as proposed by O ' B r i e n (1976). To i n v e s t i g a t e t h i s p o s s i b i l i t y , f i x e d ROS were dense ly l a b e l e d w i t h s m a l l g o l d - d e x t r a n p a r t i c l e s conjugated to an N - t e r m i n a l s p e c i f i c a n t i r h o d o p s i n monoclonal ant ibody i n an e f f o r t to i n h i b i t b i n d i n g and phagocytos i s o f ROS. The i n t e n s i t y o f g o l d l a b e l i n g o f ROS was dependent on the s i z e o f the g o l d p a r t i c l e conjugated to the rho 4D2 a n t i b o d y . Larger g o l d p a r t i c l e s (14-25 nm diameter) o n l y s p a r s e l y l a b e l e d f i x e d ROS ( r e s u l t s not shown), whereas s m a l l e r g o l d p a r t i c l e s (8-10 nm diameter) were dense ly d i s t r i b u t e d over the c e l l sur face ( L a i r d and Molday, 1988; Molday and L a i r d , 1988). As v i s u a l i z e d by both scanning and t r a n s m i s s i o n e l e c t r o n microscopy , RPE c e l l s e f f e c t i v e l y bound and phagocyt i zed ant ibody g o l d - d e x t r a n l a b e l e d ROS. These ant ibody g o l d - d e x t r a n l a b e l e d ROS d i d not b i n d to F c r ecep tor s on RPE c e l l s s i n c e ROS l a b e l e d w i t h g o l d - d e x t r a n p a r t i c l e s conjugated to -110-F ( a b ' ) 2 fragments o f t h i s a n t i r h o d o p s i n ant ibody were a l s o r e a d i l y p h a g o c y t i z e d by RPE c e l l s . Mayerson and H a l l (1986) p r e v i o u s l y r e p o r t e d the absence o f F c r eceptor-media ted ROS i n g e s t i o n . Whereas o ther segments o f rhodops in c o u l d , i n p r i n c i p l e , serve as the r e c o g n i t i o n l i g a n d , t h i s would be u n l i k e l y s i n c e the l a r g e s i z e o f the a n t i b o d y - g o l d - d e x t r a n p a r t i c l e s would be expected to s t e r i c a l l y i n h i b i t the b i n d i n g o f ROS to the RPE c e l l s , and a r e d u c t i o n or e l i m i n a t i o n o f b i n d i n g and phagocytos i s o f ROS shou ld 125 have been observed. A l s o , when f i x e d I - l a b e l e d ROS were incuba ted w i t h excess rho 4D2 a n t i b o d y , no i n h i b i t i o n o f phagocytos i s was observed, e l i m i n a t i n g the p o s s i b i l i t y t h a t n o n s p e c i f i c phagocytos i s had occur red due to d e x t r a n - g o l d p a r t i c l e s bound to the ROS c e l l s u r f a c e . These r e s u l t s taken together i n d i c a t e t h a t rhodops in i s not the l i g a n d on the ROS plasma membrane r e s p o n s i b l e f o r b i n d i n g o f ROS to RPE c e l l s . More l i k e l y , one or more membrane components s p e c i f i c f o r the ROS plasma membrane serves as t h i s r e c o g n i t i o n l i g a n d . Recent s t u d i e s have conf i rmed the e x i s t e n c e o f g l y c o p r o t e i n s s p e c i f i c f o r the ROS plasma membrane (Molday and Molday, 1987a). The p o s s i b l e r o l e o f these g l y c o p r o t e i n s and o ther ROS plasma membrane s p e c i f i c p r o t e i n s as r e c o g n i t i o n l i g a n d s i n the b i n d i n g and phagocytos i s o f ROS by RPE c e l l s i s i n v e s t i g a t e d i n the next Chapter . -111 -CHAPTER 4 I d e n t i f i c a t i o n and I s o l a t i o n o f a Plasma Membrane S p e c i f i c 230kD  G l y c o p r o t e i n and i t s P o s s i b l e Role i n ROS b i n d i n g to RPE C e l l s MATERIALS AND METHODS MATERIALS The p h o s p h o l i p i d s L-«V - d i o l e o y l l e c i t h i n and L - « C - d i o l e o y l phosphat idy le thano lamine were purchased from A v a n t i P o l a r L i p i d s I n c . (Birmingham, A L ) . Concanava l in A (Con A) was purchased from Sigma Chemical Co. (St L o u i s , MO). METHODS MONOCLONAL ANTIBODIES D r . D e l y t h R e i d generated a monoclonal ant ibody s e c r e t i n g hybridoma c e l l l i n e , des ignated ROS 1B3, by the f u s i o n o f NS-1 mouse myeloma c e l l s w i t h lymphocytes from a mouse immunized w i t h an e n r i c h e d p r e p a r a t i o n o f ROS plasma membrane (Molday and Molday, 1987b). C u l t u r e supernatants from c loned ROS 1B3 hybridoma c e l l s , as w e l l as from a n t i r h o d o p s i n (rho 1D4 and rho 4D2) ant ibody s e c r e t i n g hybridoma c e l l s (Hicks and Molday, 1986) and a n t i s p e c t r i n - l i k e (ROS 4B2) ant ibody s e c r e t i n g hybridoma c e l l s (Wong and Molday, 1986) were used i n many exper iments . The monoclonal ant ibody rho 4D2 was p u r i f i e d from mouse a s c i t e s f l u i d by ammonium s u l f a t e f r a c t i o n a t i o n and DEAE i o n exchange chromatography as d e s c r i b e d i n Chapter 2. However, the ROS 1B3 ant ibody was p u r i f i e d from mouse a s c i t e s f l u i d by a m o d i f i c a t i o n o f t h i s procedure . B r i e f l y , 1 volume o f a s c i t e s f l u i d was d i l u t e d w i t h 3 volumes o f PBS and c e n t r i f u g e d at -112-10,000 rpm f o r 10 min i n a S o r v a l l SS 34 r o t o r . The supernatant was p r e c i p i t a t e d w i t h an equal volume o f s a t u r a t e d ammonium s u l f a t e s o l u t i o n at 4°C f o r 1 h . The s o l u t i o n was r e c e n t r i f u g e d as d e s c r i b e d above. The p e l l e t was d i s s o l v e d i n 20 mM T r i s - a c e t a t e , pH 7 .9 , c o n t a i n i n g 40 mM NaCl and d i a l y z e d a g a i n s t s e v e r a l changes o f 20 mM T r i s - a c e t a t e , pH 7 . 9 , c o n t a i n i n g 20 mM NaCl o v e r n i g h t a t 4 ° C . The d i a l y z a t e was c e n t r i f u g e d a t 10,000 rpm f o r 10 min i n a S o r v a l l SS 34 r o t o r . The r e s u l t i n g p e l l e t was r e d i s s o l v e d i n 20 mM T r i s - a c e t a t e , pH 7 .9 , c o n t a i n i n g 100 mM N a C l . F i n a l l y , the p u r i f i e d an t ibody was d i a l y z e d aga i n s t 10 mM sodium b o r a t e , pH 8 . 5 , c o n t a i n i n g 100 mM NaCl p r i o r to c o u p l i n g to Sepharose 2B-CL beads as d e s c r i b e d i n Chapter 2. SDS-gel e l e c t r o p h o r e s i s conf i rmed t h a t the ant ibody p r e p a r a t i o n was over 95% p u r e . NEURAMINIDASE AND TRYPSIN TREATMENTS OF ROS MEMBRANES I n t a c t ROS and ROS d i s c membranes were prepared as d e s c r i b e d i n Chapter 2. I n t a c t ROS membranes from 25-75 bovine r e t i n a s , suspended i n 2-5 mL o f homogenizat ion b u f f e r (20% sucrose , 0.25 mM M g C ^ , 10 mM g l u c o s e , 5 mM t a u r i n e , 20 mM T r i s - a c e t a t e , pH 6 .8-7 .2 ) or ROS d i s c membranes (2-4 mg/mL) suspended i n 10 mM T r i s - a c e t a t e , pH 6 .8 , were t r e a t e d w i t h 0.025 u n i t s / m L o f neuraminidase f o r 2-4 h a t 4 ° C . The d i g e s t e d ROS were washed 3 t imes i n homogenizat ion b u f f e r by c e n t r i f u g a t i o n at 8000 rpm f o r 10 min , w h i l e the d i g e s t e d ROS d i s c membranes were washed 3 t imes i n 10 mM T r i s - a c e t a t e , pH 6 .8 , by c e n t r i f u g a t i o n a t 15,000 f o r 15 min i n a S o r v a l l SS-34 r o t o r . The r e s u l t i n g ROS membrane p e l l e t was resuspended i n homogenizat ion b u f f e r and the ROS d i s c membrane p e l l e t was resuspended i n 10 mM T r i s - a c e t a t e , pH 6 . 8 . F i n a l l y , the membrane samples were prepared f o r phagocytos i s assays , column chromatography, e l e c t r o n microscopy or SDS-gel e l e c t r o p h o r e s i s . -113-I n t a c t ROS and I - l a b e l e d ROS membranes (4-6 mg/mL) were d i g e s t e d w i t h t r y p s i n (1-5 ug/mL) f o r 20 min a t 4 °C i n homogenizat ion b u f f e r , w h i l e ROS d i s c membranes (4-6 mg/mL) were d i g e s t e d w i t h t r y p s i n (1 ug/mL) f o r 20 min a t 4 °C i n 20 mM T r i s - a c e t a t e , pH 7 . 2 , c o n t a i n i n g 20% sucrose . A f t e r 20-60 uL o f soybean t r y p s i n i n h i b i t o r (8 mg/mL) was added to s top the r e a c t i o n , the d i g e s t e d ROS membranes were washed 3 t imes i n homogenizat ion b u f f e r by c e n t r i f u g a t i o n a t 8,000-15,000 rpm f o r 10-15 min i n a S o r v a l l SS-34 r o t o r . Supernatants from t r y p s i n - t r e a t e d ROS, a long w i t h t r y p s i n - t r e a t e d ROS and t r y p s i n - t r e a t e d ROS d i s c membranes were prepared f o r SDS-gel e l e c t r o p h o r e s i s , column chromatography and phagocytos i s s t u d i e s . T r y p s i n t r e a t e d ROS were a l s o prepared f o r e l e c t r o n microscopy as d e s c r i b e d i n Chapter 2. A p p r o x i m a t e l y , 0 . 5 -1 .0 mL o f a r e c o n s t i t u t e d p r o t e i n v e s i c l e p r e p a r a t i o n (see page 118), suspended i n 10 mM T r i s - a c e t a t e , pH 7 .2 , c o n t a i n i n g 10% sucrose was d i g e s t e d w i t h t r y p s i n (1 ug/mL) f o r 20 min a t 4 ° C . A f t e r the r e a c t i o n was stopped by the a d d i t i o n o f 10-20 uL o f soybean t r y p s i n i n h i b i t o r , the v e s i c l e s were used i n phagocytos i s s t u d i e s . SDS-GEL ELECTROPHORESIS AND IMMUNOBLOTTING ROS membranes, ROS d i s c s membranes and enzyme-treated ROS membranes were s o l u b i l i z e d i n SDS and sub jec ted to e l e c t r o p h o r e s i s as d e s c r i b e d i n Chapter 2. G e l s l i c e s were e i t h e r s t a i n e d w i t h coomassie b l u e or e l e c t r o p h o r e t i c a l l y t r a n s f e r r e d to n i t r o c e l l u l o s e paper f o r l a b e l i n g w i t h monoclonal a n t i b o d i e s 125 or I - l a b e l e d l e c t i n s . B r i e f l y , n i t r o c e l l u l o s e papers were quenched i n 20 mM T r i s - a c e t a t e , pH 7 .4 , c o n t a i n i n g 0.15 M N a C l , 1 mM EDTA, 1 mM NaN^ and 0.5% Tween 20 f o r 1 h a t 2 3 ° C . The papers were r i n s e d i n Tween 20 immunoblot b u f f e r (20 mM T r i s -a c e t a t e , pH 7 .4 , 0.15 M N a C l , 1 mM EDTA, 1 mM NaN and 0.05% Tween 20) p r i o r -114-to i n c u b a t i n g w i t h e i t h e r 5-10 f o l d d i l u t e d hybridoma c u l t u r e f l u i d or I -l a b e l e d Con A or RCA I I l e c t i n s (1-2 x 10 6 dpm/mL) f o r 30-60 min a t 2 3 ° C . A f t e r washing e x t e n s i v e l y i n Tween 20 immunoblot b u f f e r , n i t r o c e l l u l o s e 125 papers , p r e v i o u s l y l a b e l e d w i t h pr imary a n t i b o d i e s were incuba ted w i t h I -l a b e l e d goat anti-mouse I g (1 x 10^ dpm/mg) f o r 1-2 h a t 2 3 ° C . A f t e r washing as b e f o r e , the papers were a i r - d r i e d and sub jec ted to au torad iography . I n some cases , n i t r o c e l l u l o s e t r a n s f e r papers were t r e a t e d w i t h neuraminidase (0.025 un i t s /mL) f o r 1 h a t 23°C p r i o r to quenching and 125 l a b e l i n g w i t h I - l a b e l e d RCA I I . DIRECT ANTIBODY BINDING STUDIES 125 The b i n d i n g o f I - l a b e l e d rho 4D2 monoclonal ant ibody to f i x e d ROS membranes was measured by i n c u b a t i n g 50 ug o f g l u t a r a l d e h y d e - f i x e d ROS 125 5 membranes w i t h 15 ug o f I - l a b e l e d rho 4D2 (3 .4 x 10 dpm/ug) i n a t o t a l volume o f 125 uL . A f t e r 1 h , 100 uL o f the assay mix ture was removed and a p p l i e d to the top o f s m a l l p o l y e t h y l e n e tubes c o n t a i n i n g 500 uL o f 10% BSA. The membrane bound ant ibody was separated from the unbound ant ibody by c e n t r i f u g i n g the tubes i n a SW 27 r o t o r a t 25,000 rpm f o r 30 min . The r e s u l t i n g membrane p e l l e t s were cut from the bottom o f the tubes and counted i n a Beckman Gamma 8000 counter . I n some cases , g l u t a r a l d e h y d e - f i x e d ROS were p r e i n c u b a t e d w i t h 15 ug o f u n l a b e l e d rho 4D2 ant ibody f o r 1 h p r i o r to per forming the assay as d e s c r i b e d above. AFFINITY CHROMATOGRAPHY a) RCA I a f f i n i t y chromatography P u r i f i e d RCA I l e c t i n (10-15 mg) was coupled to C N B r - a c t i v a t e d 2B-CL -115 -beads (10 mL) as d e s c r i b e d i n Chapter 2. Approx imate ly 7-10 mg o f n e u r a m i n i d a s e - t r e a t e d ROS were washed 2 t imes i n 20 mM T r i s - a c e t a t e , pH 7 .4 , by c e n t r i f u g a t i o n at 12,000 rpm f o r 15 min i n a S o r v a l l SS 34 r o t o r . The ROS p e l l e t was resuspended i n 1 mL o f 20 mM T r i s - a c e t a t e , pH 7 .4 , c o n t a i n i n g 200 mM NaCl and s o l u b i l i z e d i n 2.5% T r i t o n X-100. The ROS p r o t e i n s were a p p l i e d to a 2 mL RCA I a f f i n i t y column (1 .5 mg RCA I/mL packed Sepharose 2B-CL beads) e q u i l i b r a t e d w i t h c o l d 20 mM T r i s - a c e t a t e , pH 7 .4 , c o n t a i n i n g 200 mM NaCl and 0.2% T r i t o n X-100. The column was incubated a t 4 °C f o r 30-60 min p r i o r to washing w i t h 10 volumes o f 20 mM T r i s - a c e t a t e , pH 7 .4 , c o n t a i n i n g 200 mM NaCl and 0.2% T r i t o n X-100 at a f l o w r a t e o f 1-2 mL/min. Subsequently , the b u f f e r was exchanged f o r 20 mM T r i s - a c e t a t e , pH 7 .4 , c o n t a i n i n g 200 mM NaCl and 0.5% CHAPS. A f t e r an a d d i t i o n a l 10 volumes o f b u f f e r was passed through the column, the bound p r o t e i n s were e l u t e d w i t h 10 mL o f 0.2 M ga lac tose i n 20 mM T r i s - a c e t a t e , pH 7 .4 , c o n t a i n i n g 0.5% CHAPS a t 2 3 ° C . A f t e r measuring the absorbances o f the ga l ac to se e l u t e d f r a c t i o n s a t 280 nm, these 2 mL f r a c t i o n s were poo led and concent ra ted i n a Savant Speed Vac c o n c e n t r a t o r . F i n a l l y , the column f r a c t i o n s were sub jec ted to g e l e l e c t r o p h o r e s i s and g e l t r a n s f e r or prepared f o r r e c o n s t i t u t i o n s t u d i e s by d i a l y s i s aga in s t s e v e r a l changes of 20 mM T r i s -a c e t a t e , pH 7 .4 , c o n t a i n i n g 0.125% c h o l a t e . The ga l ac to se e l u t e d p r o t e i n s were s t o r e d a t 4 °C or -20°C u n t i l r e q u i r e d . I n some i n s t a n c e s , supernatants from t r y p s i n d i g e s t e d , neuraminidase-125 t r e a t e d I - l a b e l e d ROS or u n l a b e l e d ROS (1 mL) were a p p l i e d to 1-2 mL RCA-I a f f i n i t y column p r e v i o u s l y e q u i l i b r a t e d w i t h 20 mM T r i s - a c e t a t e , pH 7 . 2 , c o n t a i n i n g 200 mM N a C l . The t r y p s i n - t r e a t e d ROS supernatants were incuba ted w i t h the RCA I Sepharose beads f o r 30-45 min at 4 ° C , f o l l o w e d by washing w i t h 27 mL of 20 mM T r i s - a c e t a t e , pH 7 . 2 , c o n t a i n i n g 200 mM NaCl a t a f l o w r a t e o f 1-2 mL/min. The a f f i n i t y column was e l u t e d w i t h 8 mL o f 20 mM -116-T r i s - a c e t a t e , pH 7 . 2 , c o n t a i n i n g 200 mM NaCl and 0.2 M g a l a c t o s e . R a d i o a c t i v e l y l a b e l e d samples (100 uL) from each 3 mL column f r a c t i o n were counted on a Beckman Gamma 8000 counter . F i n a l l y , s e l e c t e d column f r a c t i o n s o b t a i n e d from a f f i n i t y chromatography o f u n l a b e l e d t r y p s i n - t r e a t e d ROS supernatants were prepared f o r phagocytos i s s t u d i e s by d i a l y s i s a g a i n s t s e v e r a l changes o f 10 mM T r i s - a c e t a t e , pH 7 . 2 , c o n t a i n i n g 20 mM N a C l . b) Immunoaf f in i ty Chromatography P u r i f i e d ROS 1B3 monoclonal ant ibody (6-8 mg) was coupled to CNBr-a c t i v a t e d Sepharose 2B-CL beads (5 mL) as d e s c r i b e d i n Chapter 2. Approx imate ly 15 mg o f ROS were washed 2 t imes i n PBS by c e n t r i f u g a t i o n at 15,000 rpm f o r 15 min i n a S o r v a l l SS 34 r o t o r . The p e l l e t was resuspended i n 4 mL o f PBS and s o l u b i l i z e d i n 3.5-4% CHAPS. The ROS p r o t e i n s were a p p l i e d to the ROS 1B3 immunoaf f in i ty column (2-3 mL packed beads ) , p re -e q u i l i b r a t e d w i t h PBS c o n t a i n i n g 0.5% CHAPS, and incubated w i t h the Sepharose beads f o r 1 h a t 2 3 ° C . Approx imate ly 20 mL o f CHAPS-conta in ing PBS was passed through the column at a f l o w r a t e o f 1-2 mL/min and c o l l e c t e d i n 1-2 mL f r a c t i o n s p r i o r to the e l u t i o n o f bound p r o t e i n molecules w i t h 10 mL o f 50 mM g l y c i n e , pH 2 . 8 , c o n t a i n i n g 300 mM NaCl and 0.5% CHAPS. A 25 uL sample from each f r a c t i o n was d r i e d down i n m i c r o t i t e r p l a t e w e l l s and probed f o r ROS 1B3 b i n d i n g by the s o l i d - p h a s e radioimmune assay. Column samples were a l s o probed f o r ROS 1B3 b i n d i n g a c t i v i t y by SDS-gel e l e c t r o p h o r e s i s and immunoblott ing t echniques . F r a c t i o n s c o n t a i n i n g ROS 1B3 b i n d i n g a c t i v i t y were n e u t r a l i z e d by adding a 50 uL a l i q u o t o f 1 M T r i s -a c e t a t e , pH 8 . 0 , to each f r a c t i o n , and f i n a l l y , prepared f o r r e c o n s t i t u t i o n s t u d i e s by d i a l y s i s aga i n s t s e v e r a l changes o f 20 mM T r i s - a c e t a t e , pH 7 .2 , c o n t a i n i n g 0.125% c h o l a t e . - 1 1 7 -RECONSTITUTION STUDIES For r e c o n s t i t u t i o n s t u d i e s , p h o s p h o l i p i d v e s i c l e s were made by combining 50 mg o f L- ° < - d i o l e o y l l e c i t h i n w i t h 50 mg o f ~L-°i-dioleoyl phosphat idy le thanolamine i n a mixture o f benzene/methanol (70:30 r a t i o ) f o l l o w e d by l y o p h i l i z a t i o n (Madden et a l . , 1983). E x a c t l y 25 mg o f t h i s l i p i d mix ture was d i s s o l v e d i n 5 mL o f 20 mM T r i s - a c e t a t e , pH 7 . 2 , 2 c o n t a i n i n g 0.125% c h o l a t e . N i t r o g e n pres sure (100-200 l b / i n ) was used to f o r c e t h i s m u l t i l a m e l l a r v e s i c l e p r e p a r a t i o n through a l i p i d v e s i c l e e x t r u d e r ( L i p e x Biomembranes I n c . , Vancouver, B . C . ) c o n t a i n i n g 2 po lycarbonate f i l t e r s (0 .1 um pore s i z e ) a t a f l o w r a t e o f 20 mL/min. The v e s i c l e s were r e a p p l i e d to the v e s i c l e ex t ruder and passed through the membrane 9 more t imes i n order to o b t a i n u n i l a m e l l a r v e s i c l e s w i t h an approximate diameter o f 100 nm (Hope et a l . , 1985). S i z e d l i p i d v e s i c l e s were combined w i t h equal volumes o f 0 .05-0 .5 mg/mL p u r i f i e d r h o d o p s i n , RCA I e l u t e d p r o t e i n s , immunoaf f in i ty i s o l a t e d p r o t e i n s or s o l u b i l i z e d ROS p r o t e i n s i n 20 mM T r i s - a c e t a t e , pH 7 . 2 , c o n t a i n i n g 0.125% c h o l a t e . The p r o t e i n v e s i c l e s were immediately d i a l y z e d a g a i n s t 4 changes o f 300 volumes o f 20 mM T r i s - a c e t a t e , pH 7 . 2 , a t 4 °C f o r 24-48 h . The r e c o n s t i t u t e d p r o t e i n v e s i c l e s were used i n phagocytos i s s t u d i e s 36-48 hours a f t e r d i a l y s i s . PHAGOCYTOSIS ASSAYS I n t h i s s tudy , phagocytos i s assays were performed i n v i t r o i n an attempt to d e f i n e the components i n v o l v e d i n s p e c i f i c ROS b i n d i n g to c u l t u r e d bovine RPE c e l l s . ROS phagocytos i s s t u d i e s were measured: a) by d i r e c t l y r a d i o i o d i n a t i n g ROS p r i o r to i n c u b a t i n g w i t h RPE c e l l s or b) by immunochemically p r o b i n g ROS-treated RPE c e l l s w i t h a r a d i o i o d i n a t e d a n t i r h o d o p s i n monoclonal a n t i b o d y . -118-a) D i r e c t R a d i o i o d i n a t i o n o f ROS Method I n t h i s assay, ROS were r a d i o i o d i n a t e d by the l a c t o p e r o x i d a s e method (as d e s c r i b e i n Chapter 3) p r i o r to b e i n g incubated w i t h bovine RPE c u l t u r e s 125 ( F i g . 38a) . The r a d i o l a b e l a s s o c i a t e d w i t h the I - l a b e l e d ROS-treated RPE c e l l s was c o n s i d e r e d a measurement o f the t o t a l phagocyt i c p roce s s , where no d i s t i n c t i o n was made between sur face bound ROS and ROS which had been i n g e s t e d . I n these s t u d i e s , c o n f l u e n t c u l t u r e s o f bovine RPE c e l l s were p r e i n c u b a t e d w i t h ROS membranes, ROS d i s c membranes, supernatants from p r o t e a s e - t r e a t e d ROS or r e c o n s t i t u t e d p r o t e i n v e s i c l e s f o r 1.5-3 h a t 37 °C , i n 0.5 mL of RPMI 1640 medium c o n t a i n i n g 10% f e t a l c a l f serum. C o v e r s l i p s p r e i n c u b a t e d w i t h ROS or ROS d i s c membranes were washed v i g o r o u s l y i n 3 (200 mL) beakers o f dPBS and 1 (100 mL) beaker o f RPMI 1640 medium to remove the unbound membranes. The washed c o v e r s l i p s were p l a c e d i n 24 w e l l assay p l a t e s c o n t a i n i n g 0.5 mL RPMI 1640 medium supplemented w i t h 10% FCS. F i n a l l y , the a b i l i t y o f the RPE c e l l s to phagocyt ize ROS was measured by 7 125 adding 1-1.5 x 10 I - l a b e l e d ROS i n 0.5 mL HBSS to each w e l l f o r 3 h a t 3 7 ° C . The g la s s c o v e r s l i p s were washed as b e f o r e , d r i e d and counted i n a Beckman Gamma 8000 counter . The maximum number o f counts f o r sample and c o n t r o l c o v e r s l i p s were as d e s c r i b e d i n Chapter 3. b) A n t i b o d y L a b e l i n g Method The second assay procedure used i n s t u d y i n g phagocytos i s was developed to d i s t i n g u i s h sur face bound ROS from ROS t h a t had been i n g e s t e d ( F i g 38b). I n t h i s assay, the advantage o f h a v i n g an a n t i r h o d o p s i n monoclonal ant ibody (rho 4D2) which bound to rhodops in i n g l u t a r a l d e h y d e - f i x e d ROS plasma membrane (Chapter 2) was e x p l o i t e d . I n these s t u d i e s , v a r i o u s c o n c e n t r a t i o n s o f u n t r e a t e d and enzyme--119-PHAGOCYTOSIS ASSAYS a) Direct Radioiodinatlon Method b) Antibody Labeling Method ROS kit. RPE , « » 125 125 i i r M i I 1 " ^ " ^ p . 1 2 » ROS + RPE Triton X - 1 W W ; I-rho 402 125 A 11 © J 1 2 5 l - r ho4D2 ,_>12i ' 7 A i V v V v v \ / v V y W u V \ © Figure 38. A schematic r e p r e s e n t a t i o n o f the assays used f o r the d e t e c t i o n of ROS phagocytos i s by c u l t u r e d bovine RPE c e l l s , a) I n the f i r s t assay, t o t a l ROS phagocytos i s ( b i n d i n g + i n g e s t i o n ) was measured by t r e a t i n g bovine RPE c e l l s w i t h r a d i o i o d i n a t e d ROS. b) I n a second assay procedure , ROS-t r e a t e d RPE c jg l l s were g l u t a r a l d e h y d e - f i x e d and s u r f ace-bound ROS were de tec ted by I - l a b e l e d rho 4D2 ant ibody ( I ) . T o t a l ROS phagocytos i s was measured by f i r s t p e r m e a b i l i z i n g the ROS-treated RPE c e l l s w i t h T r i t o n X-100 p r i o r to ROS d e t e c t i o n w i t h I - l a b e l e d rho 4D2 ant ibody ( I I ) . ROS i n g e s t i o n was d e f i n e d as the t o t a l ROS phagocytos i s minus surface-bound ROS ( I I - D . -120-t r e a t e d ROS were incubated w i t h c u l t u r e d bovine RPE c e l l s f o r s p e c i f i e d p e r i o d s o f t ime . Samples were washed i n dPBS and f i x e d w i t h 1.25% g l u t a r a l d e h y d e i n 0.1 M cacody la te b u f f e r c o n t a i n i n g 6% sucrose f o r 60 min a t 4 ° C . A f t e r washing w i t h s e v e r a l changes o f PBS over a p e r i o d o f 1 h , some samples were f u r t h e r t r e a t e d w i t h 2% T r i t o n X-100 f o r 30-45 min a t 4 °C i n order to p e r m e a b i l i z e the c e l l s . F i n a l l y , the f i x e d and f i x e d - p e r m e a b i l i z e d samples were quenched i n RIA b u f f e r (1% BSA and 0.1% NaN^ i n PBS) f o r 30 min 125 and incuba ted w i t h 250 uL o f I - l a b e l e d rho 4D2 monoclonal ant ibody ( 0 . 5 - 1 . 5 x 10^ dpm/ug) f o r 1 h a t 2 3 ° C . The samples were washed v i g o r o u s l y i n 3 separate beakers o f PBS, a i r - d r i e d , and f i n a l l y , counted i n a Beckman Gamma 8000 c o u n t e r . I n some cases , c u l t u r e d RPE c e l l s were p r e i n c u b a t e d w i t h supernatants from p r o t e a s e - t r e a t e d ROS f o r 1.5-3 h p r i o r to per forming the phagocytos i s assay as d e s c r i b e d above. C u l t u r e d bov ine RPE c e l l s which were not t r e a t e d w i t h ROS were f i x e d or f i x e d and T r i t o n X-100 p e r m e a b i l i z e d 125 p r i o r to p r o b i n g w i t h I - l a b e l e d rho 4D2 monoclonal a n t i b o d y . R o u t i n e l y , the maximum number o f counts f o r c o n t r o l s used i n b i n d i n g s t u d i e s was 2 .0-3 5.5 x 10 d p m / c o v e r s l i p , w h i l e the maximum counts f o r p e r m e a b i l i z e d c o n t r o l 4 samples was 2 . 0 - 4 . 0 x 10 d p m / c o v e r s l i p . Counts from these background c o n t r o l s were s u b t r a c t e d from the a p p r o p r i a t e t e s t samples. I n some s t u d i e s , a s tudent t - t e s t was employed to determine the p r o b a b i l i t y o f s i g n i f i c a n t d i f f e r e n c e between the mean v a l u e s o f c o n t r o l and t e s t samples. Based on a 95% conf idence l i m i t (P< 0.05) when the mean va lues d i f f e r e d by g r e a t e r than 15% the d i f f e r e n c e was s t a t i s t i c a l l y s i g n i f i c a n t u n l e s s o therwise no ted . - 1 2 1 -RESULTS PHAGOCYTOSIS ASSAYS I n e a r l y s t u d i e s w i t h c u l t u r e d r a t RPE c e l l s , phagocytos i s was assayed e i t h e r by t r e a t i n g RPE c e l l s w i t h r a d i o a c t i v e l y l a b e l e d ROS ( H a l l , 1978) or by e l e c t r o n microscopy (Edwards and Szamier , 1977). I n l a t e r s t u d i e s , C h a i t i n and H a l l (1983) used a ROS ant i serum and i n d i r e c t immunofluorescence to s tudy the phagocytos i s o f r a t ROS by r a t RPE c e l l s . A l t h o u g h t h i s assay system s u c c e s s f u l l y d i s t i n g u i s h e d c e l l sur face a t tached ROS from ROS which had been i n g e s t e d , the assay was s low and l a b o r i o u s . N e v e r t h e l e s s , C h a i t i n and H a l l (1983) demonstrated the importance o f e s t a b l i s h i n g an assay system which would q u a n t i t a t e b i n d i n g as a separate phagocyt i c process from i n g e s t i o n , as t h e i r study showed t h a t o n l y i n g e s t i o n was d e f e c t i v e i n d i sea sed RCS r a t s . I n our s tudy , an attempt was made to develop an a l t e r n a t e , q u a n t i t a t i v e assay system f o r d i s t i n g u i s h i n g sur face bound ROS from i n g e s t e d ROS. T h i s assay system was, i n t u r n , s u c c e s s f u l l y used to s tudy the k i n e t i c s o f ROS phagocytos i s by bov ine RPE c e l l s . I n t h i s assay system (as shown i n F i g . 38b) , ROS-treated bovine RPE c e l l s were f i r s t f i x e d i n g l u t a r a l d e h y d e . I n order to determine the ex tent o f ROS b i n d i n g , the f i x e d ROS-treated RPE c e l l s were d i r e c t l y t r e a t e d w i t h a r a d i o i o d i n a t e d a n t i r h o d o p s i n monoclonal ant ibody (rho 4D2). T h i s monoclonal ant ibody was p r e v i o u s l y shown to be capable o f b i n d i n g to rhodops in i n g l u t a r a l d e h y d e - f i x e d ROS plasma membrane (Chapter 2 ) . I n a d d i t i o n to ROS b i n d i n g to RPE c e l l s , t o t a l phagocytos i s ( b i n d i n g p l u s i n g e s t i o n ) was measured by p e r m e a b i l i z i n g g l u t a r a l d e h y d e - f i x e d , ROS-treated RPE c e l l s w i t h 125 T r i t o n X-100 p r i o r to p r o b i n g w i t h I - l a b e l e d rho 4D2 ant ibody ( F i g . 38b). Thus, ROS i n g e s t i o n was d e f i n e d as the t o t a l amount o f phagocytos i s minus ROS b i n d i n g to the c e l l s u r f a c e . -122-The e f f e c t o f T r i t o n X-100 treatment o f g l u t a r a l d e h y d e - f i x e d ROS on rho 4D2 ant ibody b i n d i n g was s t u d i e d i n order to determine the ex tent o f ROS p e r m e a b i l i z a t i o n . As shown i n Table V , g l u t a r a l d e h y d e - f i x e d ROS adsorbed to 125 p o l y l y s i n e coated g l a s s c o v e r s l i p s were capable o f b i n d i n g I - l a b e l e d rho 4D2 monclonal a n t i b o d y . When f i x e d samples were f u r t h e r t r e a t e d w i t h T r i t o n 125 X-100 p r i o r to l a b e l i n g w i t h I - l a b e l e d rho 4D2, o n l y a 20% i n c r e a s e i n b i n d i n g was observed. T h i s r e s u l t i n d i c a t e d t h a t the treatment o f g l u t a r a l d e h y d e - f i x e d , i n t a c t ROS w i t h T r i t o n X-100 f a i l e d to s i g n i f i c a n t l y p e r m e a b i l i z e the m o r p h o l o g i c a l s t r u c t u r e o f the ROS. Hence, a c o r r e c t i o n f a c t o r o f o n l y 20% was i n t r o d u c e d i n a l l subsequent phagocytos i s assays to a l l o w f o r the increa se i n ant ibody b i n d i n g to T r i t o n X-100 t r e a t e d ROS. KINETIC STUDIES ON ROS PHAGOCYTOSIS BY BOVINE RPE CELLS The ant ibody l a b e l i n g method f o r d e t e c t i n g ROS phagocytos i s was used to s tudy the k i n e t i c s o f bovine ROS b i n d i n g and i n g e s t i o n by bov ine RPE c e l l s . As shown i n F i g u r e 39, i n g e s t i o n of ROS by bov ine RPE c e l l s appeared to be a s a t u r a b l e p r o c e s s . ROS b i n d i n g s t e a d i l y i n c r e a s e d over a p e r i o d o f 5 h ( F i g . 39a) , w h i l e i n g e s t i o n reached a maximum a f t e r 2 h ( F i g . 39b) . Other ROS phagocytos i s k i n e t i c s t u d i e s u s i n g r a t RPE c e l l s and a double immunofluorescence l a b e l i n g technique demonstrated t h a t ROS b i n d i n g reached s a t u r a t i o n a f t e r approx imate ly 3-4 h , w h i l e i n g e s t i o n reached a maximum a f t e r approx imate ly 2-4 h ( C h a i t i n and H a l l , 1983; H a l l and Abrams, 1987). I n subsequent s t u d i e s , RPE c e l l s were t r e a t e d w i t h ROS f o r 3.0 h . I n o ther k i n e t i c s t u d i e s , ROS b i n d i n g to c u l t u r e d bov ine RPE c e l l s was shown to be c o n c e n t r a t i o n dependent r e a c h i n g a maximum v a l u e a t 1.0 x 10^ ROS/we l l as shown i n F i g u r e 40. H a l l and Abrams (1987) showed t h a t ROS - 1 2 3 -Table V 125 The e f f e c t o f T r i t o n X-100 treatment on I - l a b e l e d rho 4D2 ant ibody  b i n d i n g to g l u t a r a l d e h y d e - f i x e d rod outer segments. 125 I - l a b e l e d rho 4D2 dpm Bound F i x e d ROS 17,981 + 3573 F i x e d ROS ( T r i t o n X-100) 21,814 + 4825 C o n t r o l 984 I n t a c t ROS were f i x e d w i t h 1.25% g lu ta ra ldehyde i n 0 .1 M cacody la te b u f f e r c o n t a i n i n g 6% sucrose f o r 1 h , washed and adsorbed onto 13 mm g la s s c o v e r s l i p s p r e v i o u s l y coated w i t h 0.1 mg/mL p o l y - L - l y s i n e . Samples were washed, quenched i n RIA b u f f e r (1% BSA and 0.1% NaN^ i n PBS) and immediately t r e a t e d w i t h 1251- labe led rho 4D2 antibc-djr or incuba ted w i t h 2% T r i t o n X-100 f o r 45 min p r i o r to b e i n g t r e a t e d w i t h I - l a b e l e d rho 4D2 a n t i b o d y . The c o v e r s l i p s were washed, d r i e d and counted i n a Gamma 8000 c o u n t e r . P o l y l y s i n e - c o a t e d , quenched, T r i t o n X-100 t r e a t e d c o v e r s l i p s w i t h no ROS adsorbed to the s u r f a c e , served as the c o n t r o l . Values repre sent mean + S .D. n = 3-4 g l a s s c o v e r s l i p s . -124-Time (hours) Figure 39. The e f f e c t o f i n c u b a t i o n time on the b i n d i n g (A) and i n g e s t i o n (B) o f ROS by bovine RPE c e l l s . One week o l d bovine RPE c e l l s grown to conf luency on 13 mm g la s s c o v e r s l i p s i n 24 w e l l p l a t e s were t r e a t e d w i t h 1 x 10 ROS/wel l f o r s p e c i f i e d p e r i o d s o f t ime . The c o v e r s l i p s were washed, g l u t a r a l d e h y d e - f i x e d and ROS b i n d i n g was measured by t r e a t i n g the c e l l s w i t h I - l a b e l e d rho 4D2 monoclonal a n t i b o d y . T o t a l ROS phagocytos i s was determined by the p j j m e a b i l i z a t i o n o f the RPE c e l l s i n T r i t o n X-100 p r i o r to ROS d e t e c t i o n w i t h I - l a b e l e d rho 4D2 ant ibody^ ROS i n g e s t i o n was d e f i n e d as the t o t a l amount o f ant ibody b i n d i n g minus I - l a b e l e d rho 4D2 ant ibody b i n d i n g to s u r f a c e - a t t a c h e d ROS. I n a d d i t i o n , a c o r r e c t i o n f a c t o r o f 20% was i n t r o d u c e d to a l l o w f o r the s l i g h t increa se i n ant ibody b i n d i n g to T r i t o n X-100 t r e a t e d ROS. Values represent mean + S .D. n = 3 g la s s c o v e r s l i p s . -125-F i g u r e 40. The e f f e c t o f c o n c e n t r a t i o n on ROS b i n d i n g to bovine RPE c e l l s . I n c r e a s i n g c o n c e n t r a t i o n o f ROS were incubated w i t h one week o l d c u l t u r e d bovine RPE c e l l s f o r 3 h a t 37 C. The c o v e r s l i p s were washed, ^ 5 g l u t a r a l d e h y d e - f i x e d and ROS b i n d i n g was measured by t r e a t i n g w i t h I -l a b e l e d rho 4D2 monoclonal a n t i b o d y . Values represent mean + S .D. n = 3-4 g l a s s c o v e r s l i p s . -126-b i n d i n g to r a t RPE c e l l s was a l s o a s a t u r a b l e process a t 17 C. I n subsequent exper iments , 1-1.5 x 10 7 ROS/wel l were incuba ted w i t h the bovine RPE c e l l s f o r s p e c i f i e d p e r i o d s o f t ime . EFFECT OF BLEACHING AND TEMPERATURE ON ROS BINDING The e f f e c t o f ROS b l e a c h i n g on the b i n d i n g o f ROS to RPE c e l l s was e x p l o r e d . As i l l u s t r a t e d i n F i g u r e 41 , b leached and unbleached ROS were found to b i n d e q u a l l y w e l l to bovine RPE c e l l s sugges t ing t h a t the ROS l i g a n d ( s ) i n v o l v e d i n s p e c i f i c receptor-media ted b i n d i n g to RPE c e l l s i s exposed i n both l i g h t and dark-adapted ROS. C o l l e y and H a l l (1986) have a l so shown t h a t bo th l i g h t and dark-adapted ROS were phagocy t i zed by r a t RPE c e l l s to the same e x t e n t . When ROS were f ed to RPE c e l l s a t 4 ° C , a complete e l i m i n a t i o n o f b i n d i n g to the RPE c e l l s was observed ( F i g . 4 1 ) . T h i s was i n g e n e r a l agreement w i t h the s t u d i e s o f H a l l and Abram (1987). They demonstrated i n the r a t system t h a t temperatures o f 17°C or above a l l o w e d f o r maximum ROS b i n d i n g w h i l e temperatures below 10°C e l i m i n a t e d ROS b i n d i n g . H a l l and Abrams (1987) a t t r i b u t e d t h i s l a c k o f ROS b i n d i n g a t low temperatures to the l o s s o f membrane f l u i d i t y . EFFECT OF TRYPSIN AND NEURAMINIDASE ON ROS PHAGOCYTOSIS The e f f e c t o f t r e a t i n g i s o l a t e d i n t a c t ROS w i t h t r y p s i n , t r y p s i n i n c o n j u n c t i o n w i t h neuraminidase , and neuraminidase a lone on ROS b i n d i n g to RPE c e l l s was i n v e s t i g a t e d i n an attempt to de f ine the ROS components r e q u i r e d f o r s p e c i f i c ROS b i n d i n g ( F i g 4 2 ) . Treatment o f ROS w i t h t r y p s i n (5 ug/mL) or neuraminidase f o l l o w e d by t r y p s i n r e s u l t e d i n a 70% i n h i b i t i o n o f ROS b i n d i n g to bovine RPE c e l l s . However, neuraminidase treatment a lone r e s u l t e d i n a 40% increa se i n ROS b i n d i n g to RPE c e l l s . When a lower t r y p s i n -127-Figure 41 . The e f f e c t o f b l e a c h i n g and temperature on ROS b i n d i n g to RPE c e l l s . Unbleached (a) and b leached (b) ROS (1 x 10 ROS/wel l ) were incubated w i t h c o n f l u e n t c u l t u r e s o f bovine RPE c e l l s f o r 3 h a t 37 C. A l t e r n a t i v e l y , b leached ROS (1 x 10 ROS/wel l ) were incuba ted w i t h bovine RPE c e l l s f o r 3 h a t 4 °C ( c ) . The RPE cu l tures^were washed, g l u t a r a l d e h y d e -f i x e d and sur face bound ROS were de tec ted by I - l a b e l e d rho 4D2 monoclonal a n t i b o d y . Va lues represent mean + S .D. n = 3-4 g la s s c o v e r s l i p s . -128-FlRure 42. The e f f e c t o f t r y p s i n and neuraminidase on ROS b i n d i n g to RPE c e l l s . U n t r e a t e d ROS ( a ) ; t r y p s i n - t r e a t e d (5 ug/mL) ROS ( b ) ; neuraminidase (0.025 un i t s /mL) f o l l o w e d by t r y p s i n treatment (5 ug/mL) o f ROS (c) or n e u r a m i n i d a s e - t r e a t e d (0.025 un i t s /mL) ROS (d) were incuba ted w i t h c o n f l u e n t c u l t u r e s o f bovine RPE c e l l s f o r 3 h a t 3 7 ° C . The RPE c e l l s if^re washed, g l u t a r a l d e h y d e - f i x e d and sur face bound ROS were de tec ted by I - l a b e l e d rho 4D2 a n t i b o d y . Va lues repre sent mean + S .D. n = 3-4 g l a s s c o v e r s l i p s . -129-c o n c e n t r a t i o n (1 ug/mL) was used to t r e a t i n t a c t ROS, a decrease o f 60% i n the a b i l i t y o f ROS to b i n d to RPE c e l l s was observed ( F i g . 43A) . T h i s treatment o f ROS w i t h t r y p s i n a l s o r e s u l t e d i n an approximate 40% decrease i n ROS i n g e s t i o n ( F i g . 43B) . These r e s u l t s i n d i c a t e d t h a t t r y p s i n treatment o f ROS was a l t e r i n g , not o n l y the b i n d i n g phase o f the phagocytos i s proces s , but a l s o the i n g e s t i o n phase. I n order to c o n f i r m t h a t m i l d enzymatic d i g e s t i o n o f ROS membranes was 125 not a r t i f i c i a l l y a f f e c t i n g I - l a b e l e d rho 4D2 b i n d i n g to rhodops in i n ROS membranes, equal c o n c e n t r a t i o n s o f u n t r e a t e d and enzyme-treated ROS were g l u t a r a l d e h y d e - f i x e d and incubated w i t h approx imate ly 3 - f o l d molar excess 125 I - l a b e l e d rho 4D2 ant ibody (Table V I ) . I n a l l cases , no s i g n i f i c a n t 125 decrease i n I - l a b e l e d rho 4D2 b i n d i n g was observed. The s p e c i f i c i t y o f rho 4D2 b i n d i n g to ROS was conf i rmed i n c o n t r o l exper iments . U n t r e a t e d , g l u t a r a l d e h y d e - f i x e d ROS incubated w i t h 3 - f o l d molar excess o f u n l a b e l e d 125 rho 4D2 ant ibody i n h i b i t e d the b i n d i n g o f I - l a b e l e d rho 4D2 ant ibody by 95%. ANALYSIS OF TRYPSIN AND NEURAMINIDASE TREATMENTS OF ROS BY SDS-GEL  ELECTROPHORESIS AND WESTERN BLOTTING I n an attempt to de f ine the ROS p r o t e i n s a f f e c t e d by t r y p s i n and neuraminidase , enzyme-treated ROS were sub jec ted to SDS-gel e l e c t r o p h o r e s i s and western b l o t t i n g ( F i g . 4 4 ) . The coomassie b l u e s t a i n i n g p a t t e r n o f u n t r e a t e d ROS ( lane a) was s i m i l a r to t h a t o b t a i n e d by o ther re searcher s (Kuhn, 1980; Fung and S t r y e r , 1980; Molday and Molday, 1979) w i t h a major r h o d o p s i n band a t apparent H^<= 34,000, t r a n s d u c i n s u b u n i t bands a t apparent M^= 39,000 and M^= 37,000 and a h i g h molecu la r weight band o f apparent M^= 220,000. Numerous o ther coomassie b lue s t a i n i n g bands c o u l d a l s o be -130-Figure 43 . The e f f e c t o f t r y p s i n on ROS b i n d i n g (A) and i n g e s t i o n (B) by bovine RPE c e l l s . U n t r e a t e d (a) or t r y p s i n - t r e a t e d (1 ug/mL) (b) ROS (1 x 10 ROS/wel l ) were incubated w i t h c o n f l u e n t c u l t u r e s o f bovine RPE c e l l s f o r 3 h a t 37 °C . A f t e r washing, the RPE c | l ] . s were f i x e d i n g lu t a ra ldehyde and sur face bound ROS were de tec ted w i t h I - l a b e l e d rho 4D2 a n t i b o d y . T o t a l ROS phagocytos i s was measured by f i r s t p e r m g a b i l i z i n g the f i x e d RPE c e l l s w i t h T r i t o n X-100 p r i o r to d e t e c t i o n w i t h I - l a b e l e d rho 4D2 a n t i b o d y . ROS i n g e s t i o n was d e f i n e d as t o t a l ROS phagocytos i s minus sur face-bound ROS w i t h c a c o r r e c t i o n f a c t o r o f 20% i n t r o d u c e d to a l l o w f o r a s l i g h t i n c r e a s e i n I - l a b e l e d rho 4D2 ant ibody b i n d i n g to T r i t o n X-100 t r e a t e d ROS. Values represent mean + S .D. n = 3 g l a s s c o v e r s l i p s . - 1 3 1 -Table VI The e f f e c t o f neuraminidase and t r y p s i n on I - l a b e l e d rho 4D2 ant ibody  b i n d i n g to g l u t a r a l d e h y d e - f i x e d ROS. ROS I - l a b e l e d rho 4D2 Treatment dpm Bound a) u n t r e a t e d 26,096 + 1,960 b) neuraminidase (0.025 un i t s /mL) 27,491 + 2,155 c) neuraminidase (0.025 un i t s /mL) f o l l o w e d by t r y p s i n (1 ug/mL) 28,433 + 280 d) t r y p s i n (1 ug/mL) 27,190 + 1,666 e) t r y p s i n (5 ug/mL) 29,201 + 148 f ) rho 4D2 ant ibody (15 ug) 1,332 + 280 U n t r e a t e d , enzyme-treated and rho 4 D 2 ^ a r i t . i l , 0 d y - t r e a t e d ROS were g l u t a r a l d e h y d e - f i x e d and incubated w i t h I - l a b e l e d rho 4D2 ant ibody (15 ug) f o r 1 h . S e p a r a t i o n o f membrane-bound ant ibody from unbound ant ibody was performed by c e n t r i f u g a t i o n through 10% BSA i n a SW 27 r o t o r a t 25,000 rpm f o r 30 m i n . The r e s u l t i n g membrane p e l l e t s were counted i n a Gamma 8000 c o u n t e r . Values represent mean + S .D. n = 3. - 1 3 2 -Figure 44. SDS-gel e l e c t r o p h o r e s i s and western b l o t a n a l y s i s o f neuraminidase and t r y p s i n t r e a t e d ROS. Po lypept ide s from u n t r e a t e d ROS ( a ) , neuraminidase-t r e a t e d ROS (b) and neuraminidase f o l l o w e d by t r y p s i n (1 ug/mL) t r e a t e d ROS (c) were separated by SDS-gel e l e c t r o p h o r e s i s on a 6-15% g r a d i e n t p o l y a c r y l a m i d e (0.8% b i s ) g e l and e i t h e r s ta ined^w^th coomassie b l u e (CB) or t r a n s f e r r e d to n i t r o c e l l u l o s e and t r e a t e d w i t h I - l a b e l e d RCA I I . - 1 3 3 -d e t e c t e d . No d i f f e r e n c e i n coomassie b lue s t a i n i n g p a t t e r n was observed f o r n e u r a m i n i d a s e - t r e a t e d or n e u r a m i n i d a s e / t r y p s i n - t r e a t e d ROS ( lanes b , c ) . 125 I - l a b e l e d RCA I I o n l y f a i n t l y l a b e l e d g l y c o p r o t e i n s a t apparent M^= 34,000 and 110,000 i n western b l o t s o f u n t r e a t e d ROS ( lane a ) . However, when 125 ROS were t r e a t e d w i t h neuraminidase , I - l a b e l e d RCA I I was observed to h e a v i l y l a b e l bands a t apparent M r= 230,000 and 110,000 ( lane b ) . L a b e l i n g 125 o f the 230kD g l y c o p r o t e i n by I - l a b e l e d RCA I I was e l i m i n a t e d when i n t a c t n e u r a m i n i d a s e - t r e a t e d ROS were d i g e s t e d w i t h 1 ug/mL t r y p s i n ( l ane c ) . 125 However, t h i s low c o n c e n t r a t i o n o f t r y p s i n o n l y reduced the I - l a b e l e d RCA I I b i n d i n g to the band at H O k D . Due to the d i f f i c u l t y i n s e p a r a t i n g h i g h molecu la r weight p r o t e i n s on SDS-polyacrylamide g e l s , the p o s s i b i l i t y e x i s t e d t h a t r i c i n was l a b e l i n g the major 220kD g l y c o p r o t e i n i n neuramin ida se - t rea ted ROS and not a d i s t i n c t 230kD g l y c o p r o t e i n . I n order to i n v e s t i g a t e t h i s p o s s i b i l i t y , p r o t e i n s from und ige s ted ROS, neuramin ida se - t rea ted ROS and n e u r a m i n i d a s e / t r y p s i n - t r e a t e d ROS were separated by SDS-gel e l e c t r o p h o r e s i s p r i o r to t r a n s f e r r i n g to 125 n i t r o c e l l u l o s e paper and l a b e l i n g w i t h I - l a b e l e d Con A ( F i g . 4 5 ) . I n t h i s 125 s tudy , I - l a b e l e d Con A bound i n t e n s e l y to rhodops in at apparent M = 34,000 and to the ROS 1.2 p r o t e i n a t apparent M r= 220,000. The m o b i l i t y o f the Con A l a b e l e d band at 220kD d i d not change upon neuraminidase t rea tment , 125 nor d i d t r y p s i n e l i m i n a t e I - l a b e l e d Con A b i n d i n g to the 220kD band. These r e s u l t s c o n f i r m t h a t the r i c i n b i n d i n g g l y c o p r o t e i n a t 230kD i s d i s t i n c t from the Con A b i n d i n g 220kD g l y c o p r o t e i n . ANALYSIS OF TRYPSIN AND NEURAMINIDASE TREATMENTS OF ROS BY TRANSMISSION  ELECTRON MICROSCOPY ROS were ana lyzed by t r a n s m i s s i o n e l e c t r o n microscopy i n order to determine i f the morphology o f the enzyme-treated ROS was s i g n i f i c a n t l y - 1 3 4 -F i g u r e 45. A n a l y s i s o f neuraminidase and t r y p s i n t r e a t e d ROS p o l y p e p t i d e s by Con A b i n d i n g . P o l y p e p t i d e s from untrea ted ROS ( a ) , n e u r a m i n i d a s e - t r e a t e d ROS (b) and neuraminidase f o l l o w e d by t r y p s i n (1 ug/mL) t r e a t e d ROS (c) were separated by SDS-gel e l e c t r o p h o r e s i s on a 6-15% g r a d i e n t p o l y a c r y l a m i d e (0.8% b i s ) g e l and e i t h e r s t a i n e d w i t h coomassie b l u e (CB) or^ J i r ans fe r red t o n i t r o c e l l u l o s e paper . The t r a n s f e r paper was incuba ted w i t h I - l a b e l e d Con A and b i n d i n g was d e t e c t e d by autoradiography. -135-a l t e r e d . As shown i n F i g u r e 46, no d i s t i n c t m o r p h o l o g i c a l d i f f e r e n c e s were observed among u n t r e a t e d , t r y p s i n - t r e a t e d and n e u r a m i n i d a s e - t r e a t e d ROS. I n a l l cases , the ROS plasma membrane was seen ad jacent to the s t acks o f d i s t i n c t d i s c membranes. E l e c t r o n microscopy i n c o n j u n c t i o n w i t h immunocytochemical l a b e l i n g was a l s o used to demonstrate r i c i n l a b e l i n g a long the ROS plasma membrane. I n these s t u d i e s , neuramin ida se - t rea ted ROS were f i x e d w i t h g l u t a r a l d e h y d e and l a b e l e d d i r e c t l y w i t h R i c i n u s communis a g g l u t i n i n conjugated to 12 nm g o l d p a r t i c l e s i n the presence and absence o f the i n h i b i t o r ga lac tose ( F i g . 4 7 ) . Go ld p a r t i c l e s were found evenly d i s t r i b u t e d a long the plasma membrane o f ROS w h i l e few p a r t i c l e s were observed i n the c o n t r o l . P r e v i o u s l y , u n t r e a t e d ROS were shown to l a b e l s p a r s e l y w i t h r i c i n conjugated to g o l d - d e x t r a n p a r t i c l e s (Molday and Molday, 1987a). These r e s u l t s taken together w i t h the r i c i n wes tern b l o t a n a l y s i s i n d i c a t e t h a t e i t h e r the 230kD p r o t e i n or the HOkD p r o t e i n , or b o t h , must be exposed on the plasma membrane f o r r i c i n l a b e l i n g i n neuramindase- treated membranes. INHIBITION OF PHAGOCYTOSIS BY TRYPSIN-TREATED ROS SUPERNATANTS I n the event t h a t a m i l d t r y p s i n treatment o f i n t a c t ROS c leaves the l i g a n d ( s ) r e s p o n s i b l e f o r receptor-media ted b i n d i n g o f ROS, there e x i s t e d the p o s s i b i l i t y t h a t a t r y p s i n i z e d - f r a g m e n t o f the l i g a n d may have been r e l e a s e d i n t o the supernatant . To exp lore t h i s p o s s i b i l i t y , supernatants from u n t r e a t e d and t r y p s i n - t r e a t e d (1 ug/mL) ROS were p r e i n c u b a t e d w i t h bov ine RPE c e l l s i n an attempt to i n h i b i t ROS b i n d i n g and i n g e s t i o n . As shown i n F i g u r e 48, the t r y p s i n - t r e a t e d ROS supernatant from 0.24 mg ROS i n h i b i t e d ROS b i n d i n g by 30% and i n g e s t i o n by 25%. Moreover, a f i v e t imes more concent ra ted t r y p s i n - t r e a t e d ROS supernatant , o b t a i n e d from 1.2 mg ROS, - 1 3 6 -Figure 46. Transmi s s ion e l e c t r o n micrographs o f u n t r e a t e d and enzyme-treated ROS. Unt rea ted ROS ( A ) ; t r y p s i n - t r e a t e d (1 ug/mL) ROS (B) and neuraminidase-t r e a t e d ROS (C) were g l u t a r a l d e h y d e - f i x e d and prepared f o r t r a n s m i s s i o n e l e c t r o n microscopy . Note tha t the enzymatic treatments f a i l e d to a l t e r the genera l m o r p h o l o g i c a l s t r u c t u r e o f the ROS. Bar = 0 .2 um -137-Figure 47. Transmi s s ion e l e c t r o n micrographs o f bov ine ROS t r e a t e d w i t h neuraminidase , f i x e d i n g lu tara ldehyde and d i r e c t l y l a b e l e d w i t h R i c i n u s  communis a g g l u t i n i n - g o l d - d e x t r a n p a r t i c l e s (12 nm d i a m e t e r ) : A) i n the absence o r B) i n the presence o f the i n h i b i t o r g a l a c t o s e . The ROS plasma membrane i s noted by the arrows. Bar = 0.5 urn. -138-F i g u r e 48. I n h i b i t i o n o f ROS b i n d i n g (A) and i n g e s t i o n (B) by t r y p s i n - t r e a t e d ROS supernatant s . Conf luent c u l t u r e s o f bovine RPE c e l l s were p r e i n c u b a t e d w i t h supernatants from u n t r e a t e d ROS (a ) ; t r y p s i n - t r e a t e d (1 ug/mL) ROS (0.24 mg/mL) (b) and t r y p s i n - t r e a t e d (1 ug/mL) ROS (1.2 mg/mL) (c) f o r 3, h a t 3 7 ° C . Subsequently , the c u l t u r e s were t r e a t e d w i t h bov ine ROS (1 x 10 ROS/wel l ) f o r an a d d i t i o n a l 3 h . T h e ^ P E c e l l s were washed, f i x e d and sur face bound ROS were de tec ted by I - l a b e l e d rho 4D2 ant ibody b i n d i n g . ROS i n g e s t i o n was determined by f i r s t permeal j ) j l iz ing the f i x e d RPE c e l l s w i t h T r i t o n X-100 p r i o r to d e t e c t i o n w i t h I - l a b e l e d rho 4D2 ant ibody as p r e v i o u s l y d e s c r i b e d . I n ( A ) , va lues represent mean + S .D. where n = 3 g l a s s c o v e r s l i p s . The mean d i f f e r e n c e between (a) and (b) was not s t a t i s t i c a l l y s i g n i f i c a n t a c c o r d i n g to a s tudent t - t e s t (P> 0 . 0 5 ) . - 1 3 9 -i n h i b i t e d ROS b i n d i n g by 50% and i n g e s t i o n by approx imate ly 65%. These r e s u l t s support the hypothes i s t h a t a p o l y p e p t i d e fragment capable o f p a r t i a l l y i n h i b i t i n g ROS phagocytos i s was r e l e a s e d from the ROS by t r y p s i n . T h i s p o l y p e p t i d e ( s ) may be d e r i v e d from the 230kD or HOkD plasma membrane s p e c i f i c g l y c o p r o t e i n . RCA I AFFINITY CHROMATOGRAPHY The p r e v i o u s o b s e r v a t i o n t h a t the 230kD g l y c o p r o t e i n was a r i c i n b i n d i n g g l y c o p r o t e i n suggested t h a t r i c i n a f f i n i t y chromatography may be capable o f s u c c e s s f u l l y p u r i f y i n g t h i s g l y c o p r o t e i n from n e u r a m i n i d a s e - t r e a t e d ROS. Consequent ly , n e u r a m i n i d a s e - t r e a t e d , T r i t o n X-100 s o l u b i l i z e d ROS were sub jec ted to RCA I a f f i n i t y chromatography. Precolumn ROS p r o t e i n s ( F i g . 49, lane a) and g a l a c t o s e - e l u t e d ROS p r o t e i n s ( F i g . 49, l ane b) were, i n t u r n , ana lyzed by SDS-gel e l e c t r o p h o r e s i s and western b l o t t i n g . Coomassie b lue s t a i n i n g bands o f apparent M r= 230,000; 160,000; 110,000; 68,000; 38,000 and 34,000 were e l u t e d from the RCA I a f f i n i t y column by excess ga lac tose ( lane 125 b ) . Western b l o t a n a l y s i s u s i n g I - l a b e l e d RCA I I i d e n t i f i e d the 230kD band as a s p e c i f i c RCA I I b i n d i n g g l y c o p r o t e i n . Other l i g h t l y RCA I I -l a b e l e d bands were a l s o de tec ted at apparent M = 160,000 and 110,000. L i t t l e rhodops in was de tec ted at apparent M^ = 34,000 or 68,000 by an a n t i r h o d o p s i n rho 1D4 ant ibody i n the RCA I a f f i n i t y i s o l a t e d p r o t e i n p r e p a r a t i o n . PROTEIN RECONSTITUTION STUDIES ROS p r o t e i n p r e p a r a t i o n s were r e c o n s t i t u t e d i n t o p h o s p h o l i p i d (DOPC/DOPE) membrane v e s i c l e s and used i n phagocytos i s s t u d i e s i n an attempt to i d e n t i f y the l i g a n d ( s ) i n v o l v e d i n receptor-media ted phagocytos i s o f ROS by bov ine RPE c e l l s . Conf luent c u l t u r e s o f bovine RPE c e l l s were shown to -140-a b a b a b Figure 49. A n a l y s i s o f RCA I a f f i n i t y chromatography by SDS-gel e l e c t r o p h o r e s i s and western b l o t t i n g . Bovine ROS (a) and RCA I a f f i n i t y p u r i f i e d , ga lac tose e l u t e d p r o t e i n s (b) were ana lyzed by SDS-gel e l e c t r o p h o r e s i s on a 6-15% grad ient po lyac ry l amide (0.4% b i s ) s l a b g e l f o l l o w e d by coomassie b l u e (CB) s t a i n i n g o r ^ ^ a n s f e r to n i t r o c e l l u l o s e papers . T r a n s f e r papers were t r e a t e d w i t h . j ^ I ' l a b e l e d - R C A I I or rho 1D4 a n t i r h o d o p s i n ant ibody i n c o n j u n c t i o n w i t h I - l a b e l e d goat antimouse Ig an t ibody . - I n -e f f e c t i v e l y phagocyt ize I - l a b e l e d ROS ( F i g . 50a) . Both p r o t e i n - f r e e 125 v e s i c l e s and r e c o n s t i t u t e d rhodops in v e s i c l e s f a i l e d to i n h i b i t I - l a b e l e d ROS phagocytos i s ( F i g . 5 0 b , c ) . However, two separate p r e p a r a t i o n s (A and B) o f r i c i n a f f i n i t y p u r i f i e d ROS p r o t e i n s , when r e c o n s t i t u t e d i n t o 125 p h o s p h o l i p i d v e s i c l e s , e f f e c t i v e l y i n h i b i t e d I - l a b e l e d ROS phagocytos i s by 55-60% ( F i g . 5 0 d , e ) . When one o f these same r i c i n a f f i n i t y p u r i f i e d r e c o n s t i t u t e d p r o t e i n v e s i c l e p r e p a r a t i o n s was t r e a t e d w i t h m i l d t r y p s i n (1 125 ug/mL), the e f f e c t i v e n e s s to i n h i b i t I - l a b e l e d ROS phagocytos i s was reduced to 30% ( F i g . 5 0 f ) . I n t h i s t r y p s i n - t r e a t e d , v e s i c l e p r e p a r a t i o n no attempt was made to separate any t r y p s i n i z e d p o l y p e p t i d e fragments from the r e c o n s t i t u t e d v e s i c l e s p r i o r to per forming the i n h i b i t i o n s tudy . ROS FICOLL DISC MEMBRANES a) R i c i n B i n d i n g A n a l y s i s ROS d i s c membranes are r o u t i n e l y prepared by h y p o t o n i c l y s i s and f l o a t a t i o n on F i c o l l (Smith et a l . , 1975) . O r i g i n a l l y these membrane p r e p a r a t i o n s were thought to c o n s i s t p r i m a r i l y o f ROS d i s c s w i t h l i t t l e or no ROS plasma membrane present (Smith et a l . , 1975). However, a t t h i s time there was no s p e c i f i c ROS plasma membrane marker a v a i l a b l e to c o n f i r m t h i s assumption. L a t e r , Molday and Molday (1987a) i d e n t i f i e d the ROS 230kD and HOkD r i c i n b i n d i n g g l y c o p r o t e i n s as b e i n g s p e c i f i c to the ROS plasma membrane. I n an attempt to e l u c i d a t e the nature o f F i c o l l i s o l a t e d ROS membranes, p r e p a r a t i o n s o f dark-adapted , u n t r e a t e d and n e u r a m i n i d a s e - t r e a t e d ROS were compared to u n t r e a t e d ROS F i c o l l d i s c membranes, n e u r a m i n i d a s e - t r e a t e d ROS F i c o l l d i s c membranes and f rozen/ thawed, n e u r a m i n i d a s e - t r e a t e d F i c o l l d i s c -142-a b c d e f F i g u r e 50. I n h i b i t i o n o f ROS phagocytos i s by membrane v e s i c l e s r e c o n s t i t u t e d w i t h rhodops in and RCA I a f f i n i t y column i s o l a t e d p r o t e i n s . Conf luent c u l t u r e s o f bovine RPE c e l l s were p re incuba ted w i t h c u l t u r e f l u i d ( a ) ; p r o t e i n - f r e e v e s i c l e s ( b ) ; p e p t i d e - e l u t e d , r e c o n s t i t u t e d r h o d o p s i n v e s i c l e s ( c ) ; RCA a f f i n i t y column i s o l a t e d , r e c o n s t i t u t e d p r o t e i n v e s i c l e s : P r e p a r a t i o n A (d ) ; RCA a f f i n i t y column i s o l a t e d , r e c o n s t i t u t e d p r o t e i n v e s i c l e s : P r e p a r a t i o n B ( e ) ; or RCA a f f i n i t y column i s o l a t e d , r e c o n s t i t u t e d , t r y p s i n - t r e a t e d p r o t e i n v e s i c l e s obta ined^f jgm: P r e p a r a t i o n B ( f ) . A f t e r 3 h , the c u l t u r e s were incubated w i t h 1 x 10 I - l a b e l e d ROS/wel l f o r an a d d i t i o n a l 3 h . The g la s s c o v e r s l i p s were washed and counted i n a Gamma 8000 counter . Values represent mean + S .D. n = 5-6 g l a s s c o v e r s l i p s . -143-membranes by SDS-polyacr lyamide g e l e l e c t r o p h o r e s i s ( F i g . 51) . As p r e v i o u s l y shown, t r a n s d u c i n subuni t s (apparent M^= 39,000 and 37 ,000) , phosphodies terase (apparent M r= 88,000-90,000) (Baehr e t a l . , 1979) and rhodops in (apparent M^= 34,000) appeared as major coomassie b l u e s t a i n i n g bands i n ROS and neuramin ida se - t rea ted ROS ( lane a , b ) . I n F i c o l l d i s c membrane p r e p a r a t i o n s rhodops in was s t i l l p resent as the major band, but the membrane-associated p r o t e i n s , phosphodies terase and t r a n s d u c i n , were m i s s i n g ( l ane c , d , e ) . T h i s was i n genera l agreement w i t h Smith e t a l . (1975) 125 s t u d i e s . Western b l o t a n a l y s i s u s i n g I - l a b e l e d RCA I I i d e n t i f i e d an i n t e n s e l y l a b e l e d band at 230kD i n n e u r a m i n i d a s e - t r e a t e d ROS ( lane b ) . 125 Moreover , I - l a b e l e d RCA I I more i n t e n s e l y l a b e l e d a band a t 230kD i n f rozen/ thawed , neuramin ida se - t rea ted F i c o l l d i s c membranes ( l ane e) than i n n e u r a m i n i d a s e - t r e a t e d F i c o l l d i s c membranes ( lane d ) . These r e s u l t s i n d i c a t e d t h a t the plasma membrane s p e c i f i c 230kD g l y c o p r o t e i n , i d e n t i f i e d by Molday and Molday (1987a), was a l s o found i n F i c o l l d i s c membranes, and fur thermore , F i c o l l d i s c membranes were a c c e s s i b l e to neuraminidase-125 d i g e s t i o n and I - l a b e l e d RCA I I b i n d i n g . The p o p u l a t i o n o f membrane 125 v e s i c l e s t h a t were a v a i l a b l e f o r neuraminidase d i g e s t i o n and I - l a b e l e d RCA I I b i n d i n g was i n c r e a s e d by f r eeze- thawing the membrane p r e p a r a t i o n . When n i t r o c e l l u l o s e t r a n s f e r papers were t r e a t e d w i t h neuraminidase 125 p r i o r to the i n c u b a t i o n w i t h I - l a b e l e d RCA I I , a d d i t i o n a l bands were l a b e l e d w i t h the r a d i o i o d i n a t e d l e c t i n ( F i g . 51) . I n u n t r e a t e d ROS and u n t r e a t e d F i c o l l d i s c membranes a band o f apparent M^= 250,000 was i n t e n s e l y 125 l a b e l e d w i t h I - l a b e l e d RCA I I ( lane a , c ) , w h i l e a band a t 250kD i n p r e v i o u s l y neuramin ida se - t rea ted ROS was o n l y s l i g h t l y l a b e l e d ( lane b ) . These r e s u l t s i n d i c a t e d t h a t the r i c i n b i n d i n g band a t 250kD migra ted f u r t h e r i n t o the g e l when the s i a l i c a c i d re s idues were c l e a v e d from the g l y c o p r o t e i n . I n essence, the 230kD r i c i n b i n d i n g g l y c o p r o t e i n i s the same -144-Figure 51. A n a l y s i s o f ROS and F i c o l l d i s c membrane p r e p a r a t i o n s by SDS-gel e l e c t r o p h o r e s i s and r i c i n l a b e l i n g . Equal amounts o f u n t r e a t e d ROS membranes ( A ) ; n e u r a m i n i d a s e - t r e a t e d ROS memb ranes (B) j u n t r e a t e d F i c o l l d i s c membranes (C) ; neuramin ida se - t rea ted F i c o l l d i s c membranes (D) and f rozen/ thawed, neuramin ida se - t rea ted F i c o l l d i s c membranes (E) were sub jec ted to SDS-gel e l e c t r o p h o r e s i s on a 5-15% g r a d i e n t p o l y a c r y l a m i d e (0.2% b i s ) g e l . P o l y p e p t i d e s were ana lyzed by coomassie b l u e (CB) s t a i n i n g or by t r a n s f e r a l : 0 n i t r o c e l l u l o s e paper. T r a n s f e r papers were e i t h e r d i r e c t l y l a b e l e d w i t h I - l a b e l e d RCA I I ^ R i c i n ) or t r e a t e d w i t h neuraminidase (NMA) f o r 1 h p r i o r to l a b e l i n g w i t h I - l a b e l e d RCA I I (NMA R i c i n ) . - 1 4 5 -g l y c o p r o t e i n as the 250kD p r o t e i n minus the s i a l i c a c i d r e s i d u e s . A d d i t i o n a l r i c i n l a b e l i n g a t 250kD i n F i c o l l d i s c and f rozen/ thawed , F i c o l l d i s c membrane p r e p a r a t i o n s i n d i c a t e d t h a t a l a r g e f r a c t i o n o f F i c o l l d i s c s were i n s i d e out or were o r g i n a l l y i n a c c e s s i b l e to neuraminidase d i g e s t i o n ( lane d , e ) . Based on g e l scan i n t e n s i t i e s , approx imate ly t w o - t h i r d s o f the plasma membrane component i n F i c o l l d i s c membrane p r e p a r a t i o n s were i n s i d e out , however, i n f rozen/ thawed , F i c o l l d i s c membrane p r e p a r a t i o n s approx imate ly o n e - t h i r d o f the ROS plasma membrane d e r i v e d membrane v e s i c l e s were i n s i d e o u t . b) I n h i b i t i o n o f ROS phagocytos i s by ROS F i c o l l D i s c Membranes The presence o f ROS plasma membranes i n F i c o l l d i s c membrane p r e p a r a t i o n s suggested t h a t these p r e p a r a t i o n s may e f f e c t i v e l y i n h i b i t the 125 phagocytos i s o f I - l a b e l e d ROS by RPE c e l l s . When RPE c e l l s were p r e i n c u b a t e d w i t h 2 . 5 - f o l d excess F i c o l l d i s c ( F i g . 52b) or f rozen/ thawed , 125 F i c o l l d i s c ( F i g . 52c) membranes, the phagocytos i s o f I - l a b e l e d ROS was i n h i b i t e d by approx imate ly 40%. Moreover, when F i c o l l d i s c membranes were m i l d l y d i g e s t e d w i t h t r y p s i n (1 ug/mL), washed by c e n t r i f u g a t i o n to remove 125 s o l u b l e components, and used to i n h i b i t the phagocytos i s o f I - l a b e l e d ROS, no i n h i b i t i o n o f phagocytos i s was observed ( F i g . 52d) . 125 I n h i b i t i o n o f I - l a b e l e d ROS phagocytos i s by F i c o l l d i s c s was a l s o 125 shown to be c o n c e n t r a t i o n dependent ( F i g . 53) . The i n h i b i t i o n o f I -l a b e l e d ROS phagocytos i s by ROS F i c o l l d i s c membranes reached s a t u r a t i o n at an approximate p r o t e i n c o n c e n t r a t i o n o f 0.4 mg/mL. When these F i c o l l d i s c s were t r e a t e d w i t h m i l d t r y p s i n (1 ug/mL) and washed by c e n t r i f u g a t i o n , a dramat ic l o s s i n the a b i l i t y to i n h i b i t ROS phagocytos i s was observed. -146-F i g u r e 52. 125 The e f f e c t o f F i c o l l d i s c membranes on the phagocytos i s o f I -l a b e l e d ROS by RPE c e l l s . C u l t u r e d bovine RPE c e l l s were p r e i n c u b a t e d f o r 2.5 h a t 37°C w i t h RPE c e l l c u l t u r e f l u i d ( a ) ; F i c o l l d i s c membranes (400 ug) ( b ) ; f rozen/ thawed , F i c o l l d i s c membranes (400 ug) ( c ) ; or t r y p s i n -t r e a t e d (1 ug/mL), washed, F i c o l l dis<j^membranes (400 ug) ( d ) . ^The RPE c e l l s were washed and incubated w i t h I - l a b e l e d ROS (1.5 x 10 ROS/mL) f o r 3 h a t 3 7 ° C . The c o v e r s l i p s were washed, d r i e d and counted i n a Gamma 8000 c o u n t e r . Values represent mean + S.D. n = 3 g l a s s c o v e r s l i p s . The mean d i f f e r e n c e between (a) and (b) was not s t a t i s t i c a l l y s i g n i f i c a n t a c c o r d i n g to a s tudent t - t e s t (P> 0 . 0 5 ) . -147-F i g u r e 53. 125 I n h i b i t i o n o f I - l a b e l e d ROS phagocytos i s by F i c o l l d i s c and t r y p s i n - t r e a t e d , F i c o l l d i s c membranes. I n c r e a s i n g c o n c e n t r a t i o n s o f F i c o l l d i s c membranes ( £ ) and t r y p s i n - t r e a t e d (1 ug/mL^, washed, F i c o l l d i s c membranes ( | ) were p re incuba ted f o r 2.5 h a t 37 C w i t h c o n f l u e n t c u l t u r e s o J 5 R P E c e l l s . The RPE c e l j s were washed i n dPBS and t h e i r a b i l i t y to b i n d I - l a b e l e d ROS (1 x 10 ROS/mL) was measured. Each p o i n t i s the average o f two g l a s s c o v e r s l i p s . -148-IODINATION AND TRYPSINIZATION OF INTACT ROS I n an e a r l y s tudy , C l a r k and H a l l (1982) c h a r a c t e r i z e d the p r o t e i n s on the sur face o f o s m o t i c a l l y i n t a c t ROS which were a c c e s s i b l e to l a c t o p e r o x i d a s e - c a t a l y z e d r a d i o i o d i n a t i o n . They c o n s i s t a n t l y observed rhodops in to be r a d i o l a b e l e d as w e l l as p r o t e i n s o f apparent M^ = 226,000; 110,000 and 66,000. I n these s t u d i e s , C l a r k and H a l l (1982) suggested t h a t the 226kD and the HOkD p r o t e i n s were components o f the ROS plasma membrane. L a t e r , the r a d i o i o d i n a t e d ROS 226kD plasma membrane g l y c o p r o t e i n was shown 125 by D r . F iona M i l l a r to be the same p r o t e i n t h a t was de tec ted w i t h I -l a b e l e d RCA I I (unpubl i shed r e s u l t s ) . The p o s s i b i l i t y e x i s t e d t h a t the p r o t e o l y t i c fragment(s) r e l e a s e d from t r y p s i n - t r e a t e d ROS i s d e r i v e d from the 230kD g l y c o p r o t e i n and encompasses a r i c i n b i n d i n g s i t e and a t l e a s t one r a d i o i o d i n a t i o n s i t e . I n t a c t , dark-adapted , neuramin ida se - t rea ted ROS, r a d i o i o d i n a t e d by a l a c t o p e r o x i d a s e - c a t a l y z e d r e a c t i o n and d i g e s t e d w i t h t r y p s i n (1 ug/mL) were ana lyzed by SDS-gel e l e c t r o p h o r e s i s and autoradiography i n an attempt to i d e n t i f y the s o l u b l e r a d i o i o d i n a t e d p o l y p e p t i d e fragments r e l e a s e d i n t o the supernatant ( F i g . 54) . The coomassie b lue s t a i n i n g p a t t e r n s f o r u n l a b e l e d ( lane a ) , r a d i o i o d i n a t e d ( lane b) and r a d i o i o d i n a t e d , t r y p s i n - t r e a t e d ( lane c) ROS were i d e n t i c a l . The supernatant from the t r y p s i n d i g e s t i o n conta ined l i g h t s t a i n i n g bands between apparent M = 25,000-95,000 ( lane d ) . The i n t e n s e l y s t a i n e d band at apparent M^ = 21,500 was soybean t r y p s i n i n h i b i t o r . Autoradiography i d e n t i f i e d major r a d i o i o d i n a t e d ROS bands a t apparent M r= 230,000; 110,000; 90,000; 66,000 and 34,000 ( F i g . 54, lane b ) . I n agreement w i t h C l a r k and H a l l ' s (1982) s t u d i e s , the major change observed by autoradiography a f t e r t r y p s i n treatment o f the i n t a c t r a d i o i o d i n a t e d ROS was a s u b s t a n t i a l decrease i n the i n t e n s i t y o f the 230kD band ( lane c ) . The t r y p s i n - t r e a t e d ROS supernatant had s e v e r a l s m a l l r a d i o i o d i n a t e d p o l y p e p t i d e -149-F i g u r e 54. 125 A n a l y s i s o f neuraminidase and t r y p s i n t r e a t e d I - l a b e l e d ROS by SDS-gel e l e c t r o p h o r e s i s and autoradiography. Bovine ROS ( A ) ; neuraminidase -^jjgated, I - l a b e l e d ROS (B ) ; neuraminidase and t r y p s i n - t r e a t e d (1 ug/mL), I - l a b e l e ^ ROS (C) and the supernatant from neuraminidase and t r y p s i n -t r e a t e d , I - l a b e l e d ROS (D) were sub jec ted to e l e c t r o p h o r e s i s on a 7-17% g r a d i e n t p o l y a c r y l a m i d e (0.4% b i s ) g e l and e i t h e r s t a i n e d w i t h coomassie b l u e (CB) ( l e f t - s i d e ) or t r a n s f e r r e d to n i t r o c e l l u l o s e paper . The t r a n s f e r paper was sub jec ted to autoradiography ( r i g h t - s i d e ) . -150-bands a t apparent M r= 34,000; 31,000; 24,000; 16,000; and 7,000 ( lane d ) . I t would appear t h a t one or more o f these p o l y p e p t i d e fragments was d e r i v e d from t r y p s i n cleavage o f the 230kD g l y c o p r o t e i n . I n an attempt to i s o l a t e the ga lac tose c o n t a i n i n g 230kD p o l y p e p t i d e fragment, the r a d i o i o d i n a t e d , t r y p s i n - t r e a t e d ROS supernatant was sub jec ted to RCA I a f f i n i t y chromatography. As shown i n F i g u r e 55, the m a j o r i t y o f the 125 I - l a b e l was de tec ted i n f r a c t i o n s 1-4, however, when the RCA I column was 125 s p e c i f i c a l l y e l u t e d w i t h g a l a c t o s e , a peak o f I r a d i o a c t i v i t y was observed a t f r a c t i o n 10. F r a c t i o n s from t h i s RCA I column were sub jec ted to SDS-gel e l e c t r o p h o r e s i s and autoradiography ( F i g . 56) . The unbound p r o t e i n fragments washed through the RCA I a f f i n i t y column were i d e n t i f i e d by coomassie b l u e s t a i n i n g and autoradiography at apparent = 31,000; 16,000 and 7,000 ( lane d ) . However, when the ga lac tose e l u t e d f r a c t i o n was s u b j e c t e d to e l e c t r o p h o r e s i s , no bands were v i s i b l e by coomassie b l u e s t a i n i n g , but two f a i n t , d i f f u s e d bands a t apparent M^= 34,000 and 24,000 were d e t e c t e d by autoradiography ( lane e ) . I n a p a r a l l e l experiment , neuramin ida se - t rea ted ROS were t r e a t e d w i t h t r y p s i n (1 ug/mL) and the supernatant was sub jec ted to RCA I a f f i n i t y chromatography. Both the unbound p r o t e i n fragment f r a c t i o n and the s p e c i f i c ga l ac to se e l u t e d f r a c t i o n were used i n an attempt to i n h i b i t the 125 phagocytos i s o f I - l a b e l e d ROS by RPE c e l l s ( F i g . 57) . The s p e c i f i c ga l ac to se e l u t e d p r o t e i n f r a c t i o n i n h i b i t e d phagocytos i s by approx imate ly 50% ( F i g . 57c) w h i l e a 3 times h i g h e r c o n c e n t r a t i o n o f unbound p r o t e i n from the RCA I a f f i n i t y column i n h i b i t e d phagocytos i s by o n l y 35% ( F i g . 57b) . These r e s u l t s suggest t h a t a p r o t e i n fragment s p e c i f i c a l l y e l u t e d from the r i c i n a f f i n i t y column p a r t i a l l y i n h i b i t s ROS p h a g o c y t o s i s . However, o ther t r y p s i n - c l e a v e d fragments appear to a l s o have some a b i l i t y to i n h i b i t ROS -151-F i g u r e 55. S e p a r a t i o n o f g a l a c t o s e - c o n t a i n i n g , r a d i o i o d i n a t e d p o l y p e p t i d e s RCA I a f f i n i t y chromatography. The supernatant from neuraminidase and t r y p s i n - t r e a t e d (1 ug/mL), r a d i o i o d i n a t e d ROS was passed through a RCA I a f f i n i t y column and bound p o l y p e p t i d e s were e l u t e d w i t h 0.2 M g a l a c t o s e . Column f r a c t i o n s (3 mL) were counted i n a Beckman Gamma 8000 c o u n t e r . - 1 5 2 -220K 34K 230 K -34 K -24K -16 K 7 K A B C D E A B C D E Figure 56, S^Jjj -ge l e l e c t r o p h o r e s i s and autoradiography o f neuraminidase -t r e a t e d , I - l a b e l e d ROS and RCA I a f f i n i t y column f r a c t i o n s . Neuramin ida se - t rea ted , r a d i o i o d i n a t e d ROS ( A ) ; neuraminidase and t r y p s i n t r e a t e d , r a d i o i o d i n a t e d ROS (B) ; supernatant from neuraminidase and t r y p s i n t r e a t e d , r a d i o i o d i n a t e d ROS (C) ; unbound RCA I a f f i n i t y column f r a c t i o n from the neuraminidase and t r y p s i n t r e a t e d , r a d i o i o d i n a t e d ROS supernatant (D) ; and the 0.2 M ga l ac to se e l u t e d f r a c t i o n from the RCA I a f f i n i t y column ( E ) ; were sub jec ted to e l e c t r o p h o r e s i s on a 7-17% g r a d i e n t SDS-polyacry lamide (0.4% b i s ) g e l . G e l s l i c e s were e i t h e r s t a i n e d w i t h coomassie b l u e (CB) ( l e f t - s i d e ) or t r a n s f e r r e d to n i t r o c e l l u l o s e paper f o r autorad iography ( r i g h t - s i d e ) . - 1 5 3 -a b F i g u r e 57. I n h i b i t i o n o f ROS phagocytos i s by unbound and g a l a c t o s e - e l u t e d p o l y p e p t i d e s o b t a i n e d from a RCA I a f f i n i t y column. The supernatant from neuraminidase and t r y p s i n t r e a t e d ROS was sub jec ted to RCA I a f f i n i t y omatography. The a b i l i t y o f c u l t u r e d bovine RPE c e l l s to phagocyt i ze I - l a b e l e d ROS (1 x 10 ROS/mL) a f t e r a 2 h p r e i n c u b a t i o n w i t h u n t r e a t e d ROS supernatant (150 u l ) ( a ) ; 27 ug o f unbound p o l y p e p t i d e s from the RCA I a f f i n i t y column ( b ) ; or 9 ug o f 0.2 M g a l a c t o s e - e l u t e d , p o l y p e p t i d e s from the RCA I a f f i n i t y column ( c ) ; was measured. A f t e r 3 h the g l a s s c o v e r s l i p s were washed, d r i e d and counted i n a Beckman Gamma 8000 c o u n t e r . Values r epre sent mean + S.D. n = 4 g la s s c o v e r s l i p s . -154-p h a g o c y t o s i s . ROS 1B3 MONOCLONAL ANTIBODY A monoclonal an t ibody , des ignated ROS 1B3, r e c e n t l y generated by D r . D e l y t h R e i d (unpubl i shed r e s u l t s ) was found to s p e c i f i c a l l y b i n d to the ROS 230kD g l y c o p r o t e i n by SDS-gel e l e c t r o p h o r e s i s and immunoblott ing techniques ( F i g . 58 ) . The ROS 1B3 ant ibody l a b e l e d a band a t an apparent M r= 250,000 i n u n t r e a t e d ROS ( lane a ) . I n neuramin ida se - t rea ted ROS ( lane c ) , the ROS 1B3 a n t i b o d y i n t e n s e l y l a b e l e d a band a t apparent M r = 230,000 and l i g h t l y l a b e l e d bands a t apparent M r = 135,000; 110,000 and a doub le t a t 80,000-82,000, as w e l l as o ther membrane-bound p r o t e o l y t i c fragments . T h i s suggested t h a t the neuraminidase p r e p a r a t i o n may have been contaminated w i t h a p r o t e a s e , or i n t r i n s i c ROS proteases may have been r e l e a s e d d u r i n g the neuraminidase t rea tment . When ROS were m i l d l y d i g e s t e d w i t h t r y p s i n (1 ug/mL) , ROS 1B3 an t ibody b i n d i n g to the 230kD band was comple te ly e l i m i n a t e d , but major membrane-bound p o l y p e p t i d e fragments a t apparent M^= 135,000; 110,000 and a double t a t 80,000-82,000 were i n t e n s e l y l a b e l e d ( lane b ) . T h i s suggested t h a t the ROS 1B3 ant ibody was b i n d i n g to the same plasma membrane s p e c i f i c 230kD g l y c o p r o t e i n which was p r e v i o u s l y shown to b i n d r i c i n and to undergo l a c t o p e r o x i d a s e - c a t a l y z e d r a d i o i o d i n a t i o n on the sur face o f ROS. The ROS 1B3 ant ibody was f u r t h e r c h a r a c t e r i z e d by c o m p e t i t i v e i n h i b i t i o n assays ( F i g . 59 ) . The ROS 1B3 ant ibody bound 5 t imes more e f f e c t i v e l y to u n f i x e d ROS than g l u t a r a l d e h y d e - f i x e d ROS. Moreover, s o l u b i l i z e d ROS were approx imate ly 2 t imes more e f f e c t i v e i n i n h i b i t i n g ROS 1B3 b i n d i n g to i m m o b i l i z e d ROS than i n t a c t ROS. A ROS plasma membrane p r e p a r a t i o n competed 200 times more e f f e c t i v e l y f o r the ROS 1B3 ant ibody than ROS d i s c s membranes, sugges t ing t h a t the 230kD p r o t e i n i s p r i n c i p a l l y i n the ROS -155-Figure 58. SDS-gel e l e c t r o p h o r e s i s and immunoblot a n a l y s i s o f ROS membranes t r e a t e d w i t h t r y p s i n and neuraminidase . Unt rea ted bov ine ROS ( a ) , neuraminidase f o l l o w e d by t r y p s i n - t r e a t e d ROS (b) and n e u r a m i n i d a s e - t r e a t e d ROS (c) were sub jec ted to SDS-gel e l e c t r o p h o r e s i s on a 7-15% g r a d i e n t p o l y a c r y l a m i d e (0.4% b i s ) g e l . Ge l s l i c e s were e i t h e r s t a i n e d w i t h coomassie b lue (CB) or t r a n s f e r r e d to n i t r o c e l l u l o s e paper . The t r a n s f e r paper was l a b e l e d w i t h ROS 1B3 monoclonal a n t i b o ^ v ^ c u l t u r e f l u i d f o r 30 min . A f t e r washing, an t ibody b i n d i n g was detec ted by I - l a b e l e d goat antimouse Ig a n t i b o d y . -156-Competing Antigen (mg/mL) F i g u r e 59. Compet i t ive i n h i b i t i o n o f ROS 1B3 monoclonal ant ibody b i n d i n g to the ROS 230kD g l y c o p r o t e i n by ROS membrane p r e p a r a t i o n s . The supernatant from ROS 1B3 hybridoma c e l l c u l t u r e s was p r e i n c u b a t e d w i t h s e r i a l d i l u t i o n s o f g l u t a r a l d e h y d e - f i x e d ROS membranes ( ^ ) ; ROS d i s c membranes ( A ) ; ROS membranes (O) I T r i t o n X-100 s o l u b i l i z e d ROS (f~J) and ROS plasma membrane ( 0 ) and subsequent ly t e s t e d f o r the c a p a c i t y to b i n d to T r i t o n X-100 l ^ u b i l i z e d , i m m o b i l i z e d bovine ROS by the i n d i r e c t radioimmune assay u s i n g I - l a b e l e d goat antimouse I g . -157-plasma membrane. 125 I n an attempt to use t h i s ant ibody to b l o c k ROS p h a g o c y t o s i s , I -l a b e l e d ROS were coated w i t h p u r i f i e d ROS 1B3 ant ibody p r i o r to i n c u b a t i n g w i t h bov ine RPE c e l l s . As shown i n F i g u r e 60, o n l y a 20% i n h i b i t i o n i n phagocytos i s was observed when ROS were coated w i t h ROS 1B3 a n t i b o d y , w h i l e 125 no i n h i b i t i o n i n phagocytos i s was observed f o r I - l a b e l e d ROS incubated w i t h rho 4D2 a n t i b o d y . The ex tent o f phagocytos i s i n h i b i t i o n observed f o r ROS l a b e l e d w i t h ROS 1B3 ant ibody was not s t a t i s t i c a l l y s i g n i f i c a n t . ROS 1B3 IMMUNOAFFINITY CHROMATOGRAPHY I n an attempt to i s o l a t e the ROS 230kD g l y c o p r o t e i n (which was e a r l i e r shown to be the same as the 250kD g l y c o p r o t e i n minus the s i a l i c a c i d r e s i d u e s ) , ROS were s o l u b i l i z e d i n CHAPS and sub jec ted to chromatography on a ROS 1B3 ant ibody immunoaf f in i ty column. As shown i n F i g u r e 61, when samples ob ta ined from immunoaf f in i ty chromatography were probed f o r ROS 1B3 b i n d i n g u s i n g a s o l i d - p h a s e radioimmune assay, no b i n d i n g was de tec ted i n the f i r s t 6 f r a c t i o n s where the m a j o r i t y o f the p r o t e i n was found. However, e l u t i o n o f the column a t low pH y i e l d e d c o n s i d e r a b l e ROS 1B3 b i n d i n g a c t i v i t y i n f r a c t i o n s 24-32. SDS-gel e l e c t r o p h o r e s i s and immunoblott ing techniques were used to ana lyze the ROS 1B3 immunoaf f in i ty column p u r i f i e d p r o t e i n s ( F i g . 62) . Coomassie b l u e s t a i n i n g o f the unbound ROS p r o t e i n s ( lane b) i n comparison w i t h the precolumn ROS ( lane a ) , i n d i c a t e d t h a t the m a j o r i t y o f the ROS p r o t e i n s loaded on the column f a i l e d to b i n d to the immunoaf f in i ty column. When the column was e l u t e d w i t h low pH, a p r o t e i n which s t a i n e d l i g h t l y w i t h coomassie b l u e was observed at an apparent M^ = 34,000 ( lane c ) . By immunoblot t ing , ROS 1B3 ant ibody l a b e l e d a g l y c o p r o t e i n a t an apparent M = -158-F i g u r e 60. 125 I n h j b ^ t i o n o f I - l a b e l e d ROS, phagocytos i s by monoclonal a n t i b o d i e s . I - l a b e l e d ROS (1.5 x 10 ROS/mL) were p r e i n c u b a t e d w i t h b u f f e r a ) , 4 - f o l d molar excess a n t i r h o d o p s i n (rho 4D2) monoclonal ant ibody (b) or g r e a t e r than 1 5 - f o l d molar excess ant i -230kD g l y c o p r o t e j n c ( R O S 1B3) monoclonal ant ibody (c) f o r 1.5 h . Subsequently , the t r e a t e d , I - l a b e l e d ROS were incuba ted w i t h c u l t u r e d bovine RPE c e l l s f o r 3 h . Va lues represent mean + S .D. n = 3 g l a s s c o v e r s l i p s . The mean d i f f e r e n c e between (a) and (c) was not s t a t i s t i c a l l y s i g n i f i c a n t based on a s tudent t - t e s t (P> 0 . 0 5 ) . -159-E c o CO CM <D O C co -O k. O co n < O C B Q O O C X D O O O O Q - 6 - 4 - 3 - 2 r 1 CO I X T3 C o m E a TJ 10 15 20 25 30 35 Fraction Number Figure 61. I s o l a t i o n o f the 230kD g l y c o p r o t e i n by immunoaf f in i ty chromatography as determined by s o l i d - p h a s e radioimmune assay. CHAPS -s o l u b i l i z e d ROS were sub jec ted to immunoaf f in i ty chromatography on an a n t i -230kD antibody-Sepharose column. The column was washed i n PBS c o n t a i n i n g 0.5% CHAPS f o l l o w e d by e l u t i o n ( s t a r t i n g a t f r a c t i o n 23) w i t h 50 mM g l y c i n e , pH 2 . 8 , c o n t a i n i n g 300 mM NaCl and 0.5% CHAPS. The p r o t e i n c o n c e n t r a t i o n was measured i n each f r a c t i o n by absorbance a t 280 nm (O) • A l s o , 25 uL from each 1 mL f r a c t i o n was d r i e d on a U bottom m i c r o t i t e r p l a t e and probed w i t h ROS 1B3 ant ibody by s o l i d - p h a s e radioimmune assay ( 0 ) . -160-Figure 62. SDS-gel e l e c t r o p h o r e s i s and immunoblot a n a l y s i s o f ROS sub jec ted to ant i -230kD g l y c o p r o t e i n immunoaf f in i ty chromatography. S o l u b i l i z e d ROS (a ) ; unbound p r o t e i n s from an ant i-230kD g l y c o p r o t e i n an t ibody (ROS 1B3) Sepharose immunoaf f in i ty column (b ) ; and low pH e l u t e d immunoa f f in i ty i s o l a t e d p r o t e i n s ( c ) ; were sub jec ted to e l e c t r o p h o r e s i s on a 5-15% g r a d i e n t SDS-polyacrylamide (0.4% b i s ) g e l . Ge l s l i c e s were e i t h e r s t a i n e d w i t h coomassie b lue (CB) or t r a n s f e r r e d to n i t r o c e l l u l o s e papers . T r a n s f e r papers were^J-abeled w i t h ROS 1B3, ROS 4B2 or rho 4D2 monoclonal a n t i b o d i e s f o l l o w e d by I - l a b e l e d goat antimouse I g . - 1 6 1 -250,000 i n the precolumn ROS, as w e l l as i n the low pH e l u t e d p r o t e i n f r a c t i o n , w h i l e o n l y a t r a c e amount o f a degraded 250kD product was observed i n the unbound p r o t e i n f r a c t i o n . The rho 4D2 a n t i r h o d o p s i n monoclonal ant ibody i n t e n s e l y l a b e l e d rhodops in and i t s aggregate forms (M^ = 34,000; 68,000; 102,000 and 136,000) i n the precolumn and unbound p r o t e i n f r a c t i o n s . However, o n l y low l e v e l s o f rhodops in was de tec ted i n the low pH e l u t e d p r o t e i n f r a c t i o n . F i n a l l y , a monoclonal a n t i b o d y , de s igna ted ROS 4B2, s p e c i f i c f o r a s p e c t r i n - l i k e p r o t e i n (Wong and Molday, 1986), l a b e l e d a band a t apparent M^= 240,000 and a degraded band i n the precolumn and unbound p r o t e i n f r a c t i o n s o n l y . ROS p r o t e i n p r e p a r a t i o n s from the ROS 1B3 immunoaf f in i ty column were r e c o n s t i t u t e d i n t o p h o s p h o l i p i d v e s i c l e s and used i n an attempt to i n h i b i t 125 the b i n d i n g o f I - l a b e l e d ROS to RPE c e l l s . As shown i n F i g u r e 63, when 10 ug/mL o f the immunoaf f in i ty i s o l a t e d p r o t e i n f r a c t i o n was r e c o n s t i t u t e d i n t o p h o s p h o l i p i d v e s i c l e s , the r e s u l t i n g v e s i c l e p r e p a r a t i o n was capable o f i n h i b i t i n g phagocytos i s by n e a r l y 40%. I n c o n t r a s t , when 10 ug/mL o f the unbound p r o t e i n f r a c t i o n , which was devo id o f the 230kD g l y c o p r o t e i n , was r e c o n s t i t u t e d i n t o l i p i d v e s i c l e s , no i n h i b i t i o n o f ROS phagocytos i s was observed. However, h i g h e r c o n c e n t r a t i o n s (150 ug/mL) o f the unbound 125 r e c o n s t i t u t e d p r o t e i n f r a c t i o n i n h i b i t e d the phagocytos i s o f I - l a b e l e d ROS by approx imate ly 40%. -162-50 Reconstituted Protein (ug/weR) F i g u r e 63. I n h i b i t i o n o f ROS phagocytos i s by membrane v e s i c l e s r e c o n s t i t u t e d w i t h immunoaf f in i ty i s o l a t e d ROS p r o t e i n s . Immunoaf f in i ty i s o l a t e d 230kD g l y c o p r o t e i n s ( 0 ) and unbound p r o t e i n s from the ROS 1B3 immunoaf f in i ty column (H) were r e c o n s t i t u t e d i n t o p h o s p h o l i p i d v e s i c l e s and p re incuba ted with. , c u l t u r e d bov ine RPE c e l l s f o r 3 h a t 37 C p r i o r to the a d d i t i o n o f 1 x 10 I - l a b e l e d ROS/mL and a f u r t h e r i n c u b a t i o n f o r 3 h a t 3 7 ° C . The c u l t u r e d RPE c e l l s were washed, d r i e d and counted i n a Gamma 8000 c o u n t e r . Each p o i n t was the average o f 2-3 g l a s s c o v e r s l i p s . -163-DISCUSSION The b i n d i n g and i n g e s t i o n o f ROS i s g e n e r a l l y b e l i e v e d to i n v o l v e s p e c i f i c ROS l i g a n d ( s ) and RPE r e c e p t o r ( s ) s i n c e c u l t u r e d RPE c e l l s show a s e l e c t i v e pre ference f o r ROS over RBC, b a c t e r i a , yea s t and o ther p a r t i c l e s ( H a l l and Mayerson, 1986). The RPE sur face r e c e p t o r ( s ) and the ROS l i g a n d ( s ) i n v o l v e d i n t h i s process remain unknown. I n Chapter 3 we have p r o v i d e d evidence t h a t rhodops in l o c a l i z e d i n the ROS plasma membrane does not act as the l i g a n d i n s p e c i f i c ROS b i n d i n g by RPE c e l l s . I t i s more l i k e l y t h a t a ROS plasma membrane s p e c i f i c p r o t e i n ( s ) would ac t i n such a c a p a c i t y . R e c e n t l y , Molday and Molday (1987a) have demonstrated by g e l e l e c t r o p h o r e s i s and western b l o t t i n g t h a t the ROS plasma membrane c o n t a i n s two g l y c o p r o t e i n s (apparent M r = 230,000 and 110,000) which are not p re sent i n the ROS d i s c membrane. Both o f these ROS plasma membrane s p e c i f i c g l y c o p r o t e i n s bound the l e c t i n r i c i n (which i s s p e c i f i c f o r ga lac tose r e s i d u e s ) i n neuraminidase-t r e a t e d ROS. Neuraminidase treatment o f ROS was r e q u i r e d f o r the h y d r o l y s i s of a c c e s s i b l e s i a l i c a c i d re s idues from complex g l y c o p r o t e i n s , thus produc ing t e r m i n a l ga lac tose re s idues f o r r i c i n b i n d i n g . I n the m a j o r i t y o f complex g l y c o p r o t e i n s the nature o f the o l i g o s a c c h a r i d e i s as shown i n F igure 64. -164-Fucose * 1 , 6 MAN i i 9 QLcNAc GAL Sialic acid M " y (31,4 <*2,3 I 0 * 1 , 3 ^ MAN « l \ 0 1 - 2 / MAN /31 .4\ ASN GLcNAc GLcNAc MAN „ , „ . .. . . (31,4 01 ,4 v GLcNAc — GAL — g Sialic acid GLcNAc GAL Sialic acid (31,4 a 2,3 F i g u r e 64. S t r u c t u r e o f a r e p r e s e n t a t i v e a s p a r a g i n e - l i n k e d o l i g o s a c c h a r i d e from a complex g l y c o p r o t e i n . The i d e n t i f i c a t i o n o f these two r i c i n b i n d i n g g l y c o p r o t e i n s (230kD and HOkD) as ROS plasma membrane s p e c i f i c markers was l a t e r u t i l i z e d i n a ROS plasma membrane p u r i f i c a t i o n scheme. I n these s t u d i e s , Molday and Molday (1987b) t r e a t e d i n t a c t ROS w i t h neuraminidase and l a b e l e d the ROS plasma membrane w i t h r i c i n - g o l d - d e x t r a n p a r t i c l e s . A f t e r a m i l d t r y p s i n treatment o f the r i c i n - g o l d - d e x t r a n l a b e l e d ROS membranes, they were ab le to separate the ROS plasma membrane from the ROS d i s c membrane on a sucrose g r a d i e n t . One d i s t i n g u i s h i n g f ea ture o f ROS d i s c membranes was t h a t they l a c k e d the s i a l o g l y c o p r o t e i n s (230kD and HOkD) which b i n d r i c i n a f t e r neuraminidase t rea tment . Molday and Molday (1987b) r e s u l t s , i n summary, showed t h a t not o n l y were there p r o t e i n s s p e c i f i c f o r the ROS plasma membrane, but a l s o the ROS plasma membrane has a much more complex molecu la r compos i t ion than the d i s c membrane. I t was p o s t u l a t e d t h a t the l i g a n d ( s ) r e s p o n s i b l e f o r -165-i n i t i a t i n g ROS receptor-media ted phagocytos i s by RPE c e l l s may be plasma membrane s p e c i f i c . I n t h i s chapter , s t u d i e s were performed w i t h the a i d o f i n v i t r o phagocytos i s assays i n an attempt to de f ine the ROS plasma membrane l i g a n d ( s ) i n v o l v e d i n ROS p h a g o c y t o s i s . An immunochemical l a b e l i n g assay was developed f o r s t u d y i n g the k i n e t i c s o f ROS phagocytos i s by bovine RPE c e l l s . The b i n d i n g o f ROS by bovine RPE c e l l s appeared to be a s a t u r a b l e process w i t h r e spec t to c o n c e n t r a t i o n . Moreover, ROS i n g e s t i o n by bovine RPE c e l l s was r a p i d and reached a maximum a f t e r approx imate ly 2 h . These k i n e t i c s t u d i e s were i n g e n e r a l agreement w i t h p r e v i o u s a n a l y s i s on the b i n d i n g and i n g e s t i o n o f r a t ROS by r a t RPE c e l l s ( C h a i t i n and H a l l , 1983; H a l l and Abrams, 1987). H a l l and Abrams (1987), u s i n g a double l a b e l i n g f l u o r e s c e n t a s say ing procedure , showed tha t ROS b i n d i n g and i n g e s t i o n were s a t u r a b l e processes w i t h s a t u r a t i o n b e i n g reached a f t e r approx imate ly 4 h . I n agreement w i t h our o b s e r v a t i o n s , H a l l and Abrams (1987) a l s o showed t h a t s a t u r a t i o n was not due to a complete c o v e r i n g o f the RPE c e l l s w i t h ROS as l i g h t microscopy r e v e a l e d ample sur face area f o r f u r t h e r ROS b i n d i n g . These r e s u l t s support the v iew t h a t the process o f ROS b i n d i n g and i n g e s t i o n i s mediated by s p e c i f i c - r e c e p t o r s on the RPE c e l l s u r f a c e . S a t u r a t i o n o f ROS phagocytos i s may be r e g u l a t e d by a feedback mechanism as the RPE c e l l s become e s s e n t i a l l y f u l l o f i n g e s t e d ROS fragments , or a l t e r n a t i v e l y , RPE c e l l sur face r ecep tor s may c l u s t e r and i n t e r n a l i z e w i t h the bound ROS r e s u l t i n g i n the eventua l d e p l e t i o n o f s p e c i f i c r e c e p t o r s from the c e l l s u r f a c e . I n summary, the k i n e t i c s o f ROS phagocytos i s by bov ine RPE c e l l s was found to be s i m i l a r to t h a t observed f o r r a t RPE c e l l s ( C h a i t i n and H a l l , 1983; H a l l and Abrams, 1987). Thus, i t appears t h a t c u l t u r e d bovine RPE c e l l s have r e t a i n e d the p r o p e r t i e s o f t y p i c a l RPE c e l l s -166-and may e f f e c t i v e l y be used to s tudy the components i n v o l v e d i n ROS p h a g o c y t o s i s . Moreover, the radioimmunochemical l a b e l i n g method f o r a n a l y z i n g ROS phagocytos i s was shown to s u c c e s s f u l l y d i s t i n g u i s h between surface-bound ROS and inge s ted ROS. I n i t i a l l y , H a l l (1978) r e p o r t e d t h a t r a t RPE c e l l s i n g e s t e d 2-10 times more l i g h t - a d a p t e d ROS than dark-adapted ROS. However, i n a reassessment of these s t u d i e s , C o l l e y and H a l l (1986) r e p o r t e d t h a t there was no d i f f e r e n c e between the b i n d i n g and i n g e s t i o n o f dark-adapted and l i g h t - a d a p t e d ROS by r a t RPE c e l l s . The d i sc repancy i n t h e i r r e s u l t s was a t t r i b u t e d to crude p r e p a r a t i o n s o f ROS b e i n g used i n the i n i t i a l s tudy . I n our s t u d i e s , l i g h t -adapted and dark-adapted ROS were e f f e c t i v e l y and e q u a l l y bound by bovine RPE c e l l s . These r e s u l t s i n d i c a t e t h a t the r e c e p t o r - l i g a n d i n t e r a c t i o n s between RPE c e l l s and ROS are s t a b l e to b l e a c h i n g . ROS b i n d i n g to the RPE c e l l sur face was shown to be temperature dependent as no ROS b i n d i n g was observed at 4 ° C . H a l l and Abrams (1987) d i d an e l a b o r a t e s tudy on the e f f e c t o f temperature on ROS b i n d i n g and i n g e s t i o n by r a t RPE c e l l s . I n agreement w i t h the r e s u l t s shown h e r e , H a l l and Abrams (1987) demonstrated t h a t no ROS b i n d i n g or i n g e s t i o n was de tec ted a t temperatures below 10°C which i s i n c o n t r a s t to macrophages which are capable o f b i n d i n g p a r t i c l e s a t 4 °C ( S i l v e r s t e i n e t a l . , 1977). H a l l and Abrams (1987) a l s o i d e n t i f i e d a second t r a n s i t i o n a l temperature a t 17°C where the r a t RPE c e l l s were capable o f b i n d i n g ROS, but o n l y h i g h e r temperatures a l l o w e d f o r ROS i n g e s t i o n . The e f f e c t o f low temperatures on ROS b i n d i n g and i n g e s t i o n by RPE c e l l s c o u l d be s t be e x p l a i n e d by a l o s s i n membrane f l u i d i t y . I t i s p o s s i b l e t h a t RPE c e l l r ecep tor s are unable to adequate ly c l u s t e r on the c e l l sur face f o r h i g h a f f i n i t y ROS b i n d i n g . At h i g h e r temperatures (17 °C) p a r t i a l membrane f l u i d i t y i s p re served to a l l o w f o r adequate ROS attachment, but proper transmembrane s i g n a l i n g to the -167-c y t o s k e l e t a l system f o r ROS i n g e s t i o n may be l a c k i n g . A l t e r n a t i v e l y , the RPE c e l l c y t o s k e l e t a l system may not be p r o p e r l y a c t i v a t e d at 1 7 ° C , r e s u l t i n g i n a l o s s o f the c a p a c i t y to i n g e s t ROS. I n an attempt to de f ine the molecu la r p r o p e r t i e s o f the l i g a n d on the sur face o f the ROS which i s i n v o l v e d i n r e c o g n i t i o n by bov ine RPE c e l l s , ROS were t r e a t e d w i t h m i l d c o n c e n t r a t i o n s o f t r y p s i n and neuraminidase . Neuramin ida se - t rea ted ROS were found to b i n d more e f f e c t i v e l y than unt rea ted ROS to c u l t u r e d bovine RPE c e l l s . T h i s may be due to an a r t i f i c i a l l y induced change i n sur face charge. A l t e r n a t i v e l y , changes i n sur face s i a l i c r e s idues o f ROS may s p e c i f i c a l l y f a c i l i t a t e ROS b i n d i n g to RPE c e l l s . Cohen and N i r (1984) observed a l o s s o f c o l l o d i a l i r o n b i n d i n g ( s p e c i f i c f o r s i a l i c r e s i d u e s ) on shed ROS fragments i n d y s t r o p h i c r a t s , sugges t ing a s p e c i f i c r o l e f o r s i a l i c r e s i d u e s . However, Tarnowski and McLaugh l in (1987) demonstrated t h a t s i a l a t e d g l y c o p r o t e i n coated beads were not p r e f e r e n t i a l l y p h a g o c y t i z e d by RPE c e l l s . Thus, the involvement o f s i a l i c r e s idues on ROS sur face g l y c o p r o t e i n s remains u n c l e a r . The more i n t e r e s t i n g o b s e r v a t i o n from our s t u d i e s was the decrease i n b i n d i n g and i n g e s t i o n o f t r y p s i n -t r e a t e d ROS by bovine RPE c e l l s . The observed decrease i n ROS phagocytos i s 125 was not a r t i f i c i a l l y due to a l o s s i n I - l a b e l e d rho 4D2 b i n d i n g to t r y p s i n - t r e a t e d ROS. Western b l o t a n a l y s i s o f neuraminidase and t r y p s i n t r e a t e d ROS r e v e a l e d t h a t the major r i c i n b i n d i n g p r o t e i n a t 230kD, and to a l e s s e r e x t e n t , the HOkD g l y c o p r o t e i n were c l eaved by low c o n c e n t r a t i o n s o f t r y p s i n . Under these c o n d i t i o n s , t r y p s i n treatment o f ROS was observed by coomassie b lue s t a i n i n g o f SDS-polyacrylamide ge l s to have no other e f f e c t . Moreover , Con A l a b e l i n g o f t r y p s i n and neuraminidase t r e a t e d ROS conf i rmed t h a t the 230kD g l y c o p r o t e i n was d i s t i n c t from the prominent 220kD (ROS 1.2) g l y c o p r o t e i n . -168-Immunocytochemical l a b e l i n g s t u d i e s l o c a l i z e d the r i c i n - g o l d - d e x t r a n b i n d i n g to the ROS plasma membrane o f neuramin ida se - t rea ted ROS (Molday and Molday, 1987a). F u r t h e r s t u d i e s r e v e a l e d t h a t the po lypept ide- f ragments c l e a v e d from the ROS sur face by t r y p s i n were r e l e a s e d i n t o the supernatant . Moreover, these fragments were capable o f i n h i b i t i n g ROS phagocytos i s by 50-65%. The s o l u b l e p o l y p e p t i d e fragment(s) r e s p o n s i b l e f o r the i n h i b i t i o n o f r e c e p t o r -mediated ROS phagocytos i s were p o s t u l a t e d to have been d e r i v e d from the plasma membrane s p e c i f i c 230kD g l y c o p r o t e i n . N e v e r t h e l e s s , the p o s s i b i l i t y can not be exc luded t h a t these i n h i b i t o r y g l y c o p o l y p e p t i d e ( s ) may have been d e r i v e d from the somewhat l e s s s e n s i t i v e HOkD r i c i n b i n d i n g g l y c o p r o t e i n . I n t h i s c o n t e x t , the p a r t i a l r e t e n t i o n o f ROS phagocytos i s seen w i t h t r y p s i n - t r e a t e d ROS may be due to an incomplete d i g e s t i o n o f the HOkD g l y c o p r o t e i n . Thus, the l e s s t r y p s i n s e n s i t i v e HOkD g l y c o p r o t e i n must be c o n s i d e r e d as a candidate f o r the ROS l i g a n d i n v o l v e d i n ROS/RPE i n t e r a c t i o n s . I n order to exp lore t h i s h y p o t h e s i s , an attempt was made to i s o l a t e the 230kD g l y c o p r o t e i n from s o l u b i l i z e d , neuramin ida se - t rea ted ROS on a r i c i n a f f i n i t y column. R i c i n a f f i n i t y chromatography y i e l d e d an e n r i c h e d p r e p a r a t i o n o f 230kD g l y c o p r o t e i n which o n l y c o n t a i n e d a t r a c e amount o f r h o d o p s i n . However, the r i c i n a f f i n i t y column p r e p a r a t i o n c o n t a i n e d p r o t e i n s a t apparent M r = 160,000; 110,000; 68,000 and 38,000. T h i s p r e p a r a t i o n was r e c o n s t i t u t e d i n t o p h o s p h o l i p i d v e s i c l e s and s u c c e s s f u l l y used as an i n h i b i t o r o f ROS phagocy to s i s . I n c o n t r a s t , a f f i n i t y p u r i f i e d rhodops in r e c o n s t i t u t e d i n t o p h o s p h o l i p i d v e s i c l e s f a i l e d to i n h i b i t ROS p h a g o c y t o s i s . These r e s u l t s suggest t h a t a component o ther than r h o d o p s i n , i s o l a t e d from the r i c i n a f f i n i t y column, i s i n v o l v e d i n l i g a n d - r e c e p t o r i n t e r a c t i o n s between ROS and RPE c e l l s . Moreover, t h i s component was t r y p s i n s e n s i t i v e as the a b i l i t y o f the r i c i n a f f i n i t y i s o l a t e d p r o t e i n s to i n h i b i t ROS -169-phagocytos i s was p a r t i a l l y removed upon t r y p s i n - t r e a t m e n t . The s o l u b l e t r y p s i n - c l e a v e d p o l y p e p t i d e fragment(s) were not separated from the v e s i c l e s , consequent ly , some i n h i b i t i o n o f ROS phagocytos i s was r e t a i n e d . Prev ious experiments have shown t h a t the 230kD g l y c o p r o t e i n i s h i g h l y s e n s i t i v e to t r y p s i n , thus , i t i s the most l i k e l y candidate i n the r i c i n a f f i n i t y column p r e p a r a t i o n f o r a d i r e c t involvement w i t h the RPE c e l l s . I n order to f u r t h e r study the e f f e c t o f t r y p s i n on ROS phagocytos i s a p r e p a r a t i o n o f h y p o t o n i c a l l y s w e l l e d ROS membranes was i s o l a t e d by f l o a t a t i o n on F i c o l l (Smith et a l . , 1975). These membranes termed " F i c o l l d i s c membranes" were o r i g i n a l l y thought to be d e r i v e d from ROS d i s c membranes (Smith et a l . , 1975). However, the i d e n t i f i c a t i o n o f a r i c i n l a b e l e d , plasma membrane s p e c i f i c 230kD g l y c o p r o t e i n conf i rmed t h a t the ROS membranes i s o l a t e d on a F i c o l l g r a d i e n t were composed o f ROS d i s c membranes and ROS plasma membranes. Approx imate ly a t h i r d o f these ROS plasma membrane v e s i c l e s were r i g h t - s i d e out based on t h e i r a c c e s s i b i l i t y to neuraminidase . When these F i c o l l d i s c membranes were f r o z e n and thawed s e v e r a l t imes an enrichment o f neuraminidase a c c e s s i b l e plasma membrane fragments was o b t a i n e d . S ince the 230kD plasma membrane s p e c i f i c g l y c o p r o t e i n was present i n F i c o l l d i s c membranes, i t was p r e d i c t e d t h a t t h i s membrane p r e p a r a t i o n would e f f e c t i v e l y i n h i b i t ROS p h a g o c y t o s i s , and t h i s i n h i b i t i o n would be t r y p s i n s e n s i t i v e . F i c o l l d i s c membranes and f rozen/ thawed , F i c o l l d i s c membranes d i d indeed i n h i b i t ROS p h a g o c y t o s i s , an i n h i b i t i o n which was e l i m i n a t e d upon t r y p s i n treatment o f the F i c o l l d i s c membranes and removal o f the s o l u b l e p o l y p e p t i d e ( s ) . The i n h i b i t i o n o f ROS phagocytos i s by F i c o l l d i s c membranes, as w i t h i n t a c t ROS, was not g r e a t e r than 70%. The i n a b i l i t y to approach a 100% i n h i b i t i o n o f ROS phagocytos i s may be due to RPE recep tor i n t e r n a l i z a t i o n and r e c y c l i n g to the c e l l -170-sur face d u r i n g the r a d i o i o d i n a t e d ROS chase p e r i o d . H a l l and Abrams (1987) demonstrated t h a t a f t e r a s a t u r a t i n g i n t a k e o f ROS, i n h i b i t o r s o f p r o t e i n s y n t h e s i s f a i l e d to prevent a recovery o f ROS phagocyt i c a b i l i t y . Thus, they suggest t h a t i t i s u n l i k e l y t h a t new recep tor s are s y n t h e s i z e d d u r i n g the ROS chase p e r i o d . I n an attempt to de f ine the p o l y p e p t i d e ( s ) b e i n g r e l e a s e d i n t o the supernatant o f t r y p s i n - t r e a t e d ROS, i n t a c t , n e u r a m i n i d a s e - t r e a t e d , r a d i o i o d i n a t e d ROS were t r y p s i n - t r e a t e d and the supernatant was sub jec ted to r i c i n a f f i n i t y chromatography. I f the ROS p o l y p e p t i d e fragment r e l e a s e d from the 230kD g l y c o p r o t e i n by t r y p s i n c o n t a i n e d at l e a s t one a c c e s s i b l e r a d i o i o d i n a t i o n s i t e and a r i c i n b i n d i n g s i t e , i t shou ld be s u c c e s s f u l l y i s o l a t e d by t h i s approach. I n genera l agreement w i t h C l a r k and H a l l (1982), r a d i o i o d i n a t i o n o f i n t a c t ROS r e s u l t e d i n the in tense l a b e l i n g o f p r o t e i n bands a t apparent M r = 230,000; 110,000; 66,000 and 34,000 w i t h an a d d i t i o n a l band a t apparent M r = 90,000. The 230kD r a d i o i o d i n a t e d p r o t e i n i d e n t i f i e d i n our g e l system appeared to be the same as the 226kD r a d i o i o d i n a t e d p r o t e i n i d e n t i f i e d by C l a r k and H a l l (1982). The r a d i o i o d i n a t e d plasma membrane s p e c i f i c 230kD g l y c o p r o t e i n was a l s o shown p r e v i o u s l y to be n e u r a m i n i d a s e - s e n s i t i v e and capable o f b i n d i n g r i c i n (F iona M i l l a r , unpub l i shed r e s u l t s ) . When the r a d i o i o d i n a t e d ROS were t r e a t e d w i t h t r y p s i n , the m a j o r i t y o f the r a d i o l a b e l was c l e a v e d from the 230kD g l y c o p r o t e i n . The t r y p s i n - t r e a t e d ROS supernatant c o n t a i n e d s e v e r a l s m a l l r a d i o a c t i v e p o l y p e p t i d e fragments, but o n l y two ga l ac to se c o n t a i n i n g p o l y p e p t i d e fragments o f apparent M r = 34,000 and 24,000 were s p e c i f i c a l l y e l u t e d from a r i c i n a f f i n i t y column. Hence, the exper imenta l evidence suggests t h a t these two g l y c o p r o t e i n s are d e r i v e d from the 230kD g l y c o p r o t e i n . I n a p a r a l l e l experiment , n o n - r a d i o l a b e l e d , neuramin ida se - t rea ted ROS -171 -were t r y p s i n i z e d and the supernatant was sub jec ted to r i c i n a f f i n i t y chromatography. P o l y p e p t i d e s which f a i l e d to b i n d to the r i c i n a f f i n i t y column, as w e l l as ga lac tose e l u t e d p o l y p e p t i d e s , were e f f e c t i v e i n i n h i b i t i n g ROS phagocytos i s by RPE c e l l s . I t i s p o s s i b l e t h a t some of the p o l y p e p t i d e fragments which f a i l e d to b i n d to the r i c i n column may have been d e r i v e d from the ga lac tose e l u t e d 34kD or 24kD p o l y p e p t i d e fragments . These s m a l l e r p o l y p e p t i d e fragments were g e n e r a l l y l e s s e f f e c t i v e than the s p e c i f i c a l l y e l u t e d p o l y p e p t i d e fragments i n i n h i b i t i n g ROS phagocy to s i s . An attempt was made to separate the s o l u b l e p o l y p e p t i d e fragments r e l e a s e d from ROS by t r y p s i n on a HPLC C^g column. However, the p o l y p e p t i d e s appeared to b i n d i r r e v e r s i b i l y to the column m a t r i x . I n an attempt to study the plasma membrane o f ROS more f u l l y , p u r i f i e d p r e p a r a t i o n s o f ROS plasma membranes were i n j e c t e d i n t o mice f o r the p r o d u c t i o n o f monoclonal a n t i b o d i e s . One monoclonal ant ibody (ROS 1B3) was shown by immunoblott ing techniques to be s p e c i f i c f o r the neuraminidase-s e n s i t i v e ROS 230kD g l y c o p r o t e i n . Th i s ant ibody competed e f f e c t i v e l y w i t h ROS, s o l u b i l i z e d ROS and p u r i f i e d ROS plasma membrane as measured by radioimmune assay. The f a c t t h a t ROS d i s c membranes competed w i t h the a n t i -230kD g l y c o p r o t e i n ant ibody o n l y a t h i g h c o n c e n t r a t i o n s aga in demonstrates t h a t the 230kD g l y c o p r o t e i n i s a plasma membrane s p e c i f i c p r o t e i n . The s l i g h t c o m p e t i t i o n w i t h ROS d i s c s membranes may be due to a contamina t ion o f the d i s c membrane p r e p a r a t i o n w i t h ROS plasma membrane, or a l t e r n a t i v e l y , a low l e v e l o f the 230kD g l y c o p r o t e i n may be present i n d i s c membranes. The i n h i b i t i o n o f ROS 1B3 ant ibody b i n d i n g by i n t a c t ROS suggests t h a t the ant ibody b inds to the e x t r a c e l l u l a r sur face o f bovine ROS. Moreover, t r y p s i n treatment o f i n t a c t ROS r e s u l t s i n ROS 1B3 ant ibody b i n d i n g to s m a l l e r membrane-bound p r o t e o l y t i c fragments. Consequently , the a n t i g e n i c -172-determinant f o r the ROS 1B3 ant ibody was not the s o l u b l e fragment r e l e a s e d from the 230kD g l y c o p r o t e i n by t r y p s i n , and as p r e d i c t e d , ROS 1B3 ant ibody coated ROS were as e f f e c t i v e l y phagocyt i zed as u n t r e a t e d ROS. An immunoaf f in i ty column u s i n g the ant i -230kD g l y c o p r o t e i n ant ibody (ROS 1B3) was prepared and s u c c e s s f u l l y used to i s o l a t e the 230kD g l y c o p r o t e i n . SDS-gel e l e c t r o p h o r e s i s and immunoblott ing demonstrated t h a t the 230kD g l y c o p r o t e i n migra ted a t a s l i g h t l y h i g h e r molecu la r weight (250kD) i n ROS p r o t e i n p r e p a r a t i o n s which were not t r e a t e d w i t h neuraminidase . The p r o t e i n f r a c t i o n which f a i l e d to b i n d to the immunoaf f in i ty column was devo id o f any d e t e c t a b l e 230kD g l y c o p r o t e i n , w h i l e the low pH e l u t e d f r a c t i o n was e n r i c h e d i n 230kD g l y c o p r o t e i n w i t h some contamina t ing rhodops in and l i t t l e or no contaminat ing s p e c t r i n - l i k e p r o t e i n . The i s o l a t e d 230kD ROS g l y c o p r o t e i n was much more e f f e c t i v e i n i n h i b i t i n g ROS phagocytos i s than the unbound f r a c t i o n from the ROS 1B3 immunoa f f in i ty column. However, the maximum i n h i b i t i o n o f ROS phagocytos i s o b t a i n e d was o n l y 40%. I t i s p o s s i b l e t h a t e i t h e r the low pH e l u t i o n o f the immunoa f f in i ty column may have i r r e v e r s i b l y denatured the 230kD g l y c o p r o t e i n , or a l t e r n a t i v e l y , the 230kD g l y c o p r o t e i n may b i n d to RPE r e c e p t o r s i n concer t w i t h another ROS l i g a n d ( s ) f o r maximum i n h i b i t i o n o f ROS p h a g o c y t o s i s . I t was a l s o apparent t h a t the 230kD g l y c o p r o t e i n was v e r y s e n s i t i v e to protease degradat ion i n the membrane, and p a r t i c u l a r l y , when i s o l a t e d . I t i s p o s s i b l e t h a t i n h i b i t i o n o f ROS phagocytos i s was not g rea ter than 40% due to l i g a n d degrada t ion d u r i n g the p r e i n c u b a t i o n p e r i o d w i t h the RPE c e l l s . Another e x p l a n a t i o n f o r the moderate l e v e l o f i n h i b i t i o n may be due to the b i n d i n g and i n t e r n a l i z a t i o n o f the p u t a t i v e l i g a n d by s p e c i f i c RPE c e l l sur face recep tor s d u r i n g the p r e i n c u b a t i o n p e r i o d . Consequent ly , when ROS were added to the RPE c e l l s f o r a f u r t h e r 3 h , the e f f e c t i v e c o n c e n t r a t i o n o f the 230kD p r o t e i n may have been s i g n i f i c a n t l y decreased -173-w h i l e r e c y c l e d RPE recep tor s became a v a i l a b l e f o r a d d i t i o n a l b i n d i n g . The i n h i b i t i o n o f ROS phagocytos i s by a ROS f r a c t i o n d e v o i d o f d e t e c t a b l e 230kD g l y c o p r o t e i n may be a n o n s p e c i f i c e f f e c t induced by the h i g h c o n c e n t r a t i o n o f ROS p r o t e i n s incubated w i t h the RPE c e l l s . A l t e r n a t i v e l y , another ROS component necessary f o r s p e c i f i c ROS b i n d i n g to RPE c e l l s may be found i n t h i s f r a c t i o n . I n summary, the phagocytos i s o f ROS by c u l t u r e d bov ine RPE c e l l s has been s u c c e s s f u l l y ana lyzed by a d i r e c t ROS r a d i o i o d i n a t i o n method and by a radioimmunochemical l a b e l i n g method. U t i l i z i n g these assay procedures , the ROS c e l l sur face components r e s p o n s i b l e f o r s p e c i f i c receptor -media ted phagocytos i s were i n v e s t i g a t e d . When i n t a c t ROS were m i l d l y t r y p s i n -t r e a t e d , two s o l u b l e g lycopept ide s (34kD and 24kD), r e l e a s e d i n t o the supernatant , were shown to be capable o f i n h i b i t i n g ROS p h a g o c y t o s i s . One or more o f these g l y c o p e p t i d e s was b e l i e v e d to have been d e r i v e d from the 230kD plasma membrane s p e c i f i c g l y c o p r o t e i n . R i c i n a f f i n i t y chromatography and immunoaf f in i ty chromatography were used to i s o l a t e the 230kD g l y c o p r o t e i n from ROS. These a f f i n i t y i s o l a t e d p r o t e i n p r e p a r a t i o n s , when r e c o n s t i t u t e d i n t o p h o s p h o l i p i d v e s i c l e s , were e f f e c t i v e l y used to i n h i b i t the phagocytos i s o f ROS. I n essence, we have i d e n t i f i e d and i s o l a t e d a ROS 230kD plasma membrane s p e c i f i c g l y c o p r o t e i n which may ac t as a l i g a n d i n s p e c i f i c l i g a n d - r e c e p t o r i n t e r a c t i o n s between ROS and RPE c e l l s . THESIS SUMMARY Rod photoreceptor c e l l s undergo a c o n t i n u a l renewal process o f rod outer segment d i s c membrane (Young, 1967). ROS d i s c s formed a t the base o f the ROS p r o g r e s s i v e l y and s e q u e n t i a l l y move toward the a p i c a l t i p o f the ROS where they are shed from the outer segment as s tacks o f d i s c s encapsula ted i n a ROS plasma membrane (Young, 1976). The u n d e r l i n i n g r e t i n a l pigment e p i t h e l i u m which i n t e r d i g i t a t e s w i t h the outer segments, s p e c i f i c a l l y b inds and i n g e s t s these shed ROS packets (Young and Bok, 1969). I n v e s t i g a t o r s to date have not been able to i d e n t i f y the components i n v o l v e d i n the s p e c i f i c i n t e r a c t i o n s between ROS and RPE c e l l s . I n p a r t i c u l a r , c o n s i d e r a b l e e f f o r t and time has been d e d i c a t e d to d e f i n i n g and i s o l a t i n g the s p e c i f i c RPE c e l l sur face r e c e p t o r r e s p o n s i b l e f o r b i n d i n g and a t t a c h i n g ROS. I n recent y e a r s , many ROS phagocytos i s s t u d i e s have been done w i t h normal RPE c e l l s , as w e l l as RPE c e l l s from a d y s t r o p h i c s t r a i n o f r a t s known as the R o y a l C o l l e g e o f Surgeons (RCS) r a t s (Bourne e t a l . , 1938). These r a t s c a r r y an autosomal r e c e s s i v e muta t ion (Dowling and Sidman, 1962) which r e s u l t s i n t h e i r i n a b i l i t y to phagocyt ize ROS. M u l l e n and L a V a i l (1976) i n a s tudy u s i n g c h i m e r i c r a t s , i d e n t i f i e d the r e t i n a l pigment e p i t h e l i u m as the pr imary muta t ion s i t e i n RCS r a t s . L a t e r , C h a i t i n and H a l l (1983) showed t h a t RPE c e l l s from RCS r a t s were capable o f b i n d i n g ROS but the i n g e s t i o n phase o f phagocytos i s was d e f e c t i v e . I t i s p o s s i b l e t h a t i n d i sea sed RPE c e l l s , s p e c i f i c ROS-binding recep tor s are pre sent but are unable to p r o v i d e adequate transmembrane s i g n a l s to the c y t o s k e l e t a l system f o r proper e x c i t a t i o n and ROS i n g e s t i o n . However, these d y s t r o p h i c RPE c e l l s are capable o f i n g e s t i n g a s m a l l number o f ROS ( C h a i t i n and H a l l , 1983). These r e s u l t s suggest t h a t RPE c e l l s may have two se t s o f r e c e p t o r s ; one set f o r n o n s p e c i f i c phagocytos i s and a second set f o r s p e c i f i c ROS p h a g o c y t o s i s . -175 -T h i s was l a t e r supported by r e s u l t s from Mayerson and H a l l (1986), where normal RPE c e l l s were shown to be able to phagocyt ize v a r i o u s p a r t i c l e s but a t a g r e a t l y reduced r a t e compared to ROS. T h i s s e l e c t i v i t y f o r p a r t i c l e types i s a l s o e v i d e n t i n macrophages where p a r t i c l e s coated w i t h IgG and complement are s p e c i f i c a l l y phagocy t i zed , w h i l e o ther non s p e c i f i c a l l y bound p a r t i c l e s are a l s o phagocy t i zed , but o n l y a t a reduced r a t e ( G r i f f i n e t a l . , 1975; S i l v e r s t e i n e t a l . , 1978). A l though IgG and complement are requirements f o r s p e c i f i c phagocytos i s i n macrophages ( S i l v e r s t e i n e t a l . 1978), these components were shown not to be necessary f o r s p e c i f i c ROS phagocytos i s by RPE c e l l s (Mayerson and H a l l , 1986). The q u e s t i o n e x p l o r e d i n t h i s s tudy was what ROS l i g a n d ( s ) are i n v o l v e d i n s p e c i f i c ROS b i n d i n g to RPE c e l l sur face recep tor s ? An i n i t i a l p roposa l by O ' B r i e n (1976) was t h a t rhodops in l o c a l i z e d i n the ROS plasma membrane may ac t as such a l i g a n d . I n an attempt to answer these q u e s t i o n s , monoclonal a n t i b o d i e s s p e c i f i c f o r rhodops in were r a i s e d and used as b i o c h e m i c a l probes t o , not o n l y e l u c i d a t e the s t r u c t u r e and topography o f r h o d o p s i n , bu t a l s o to exp lore the i n t e r a c t i o n s between ROS and RPE c e l l s . Over the pas t few years s e v e r a l monoclonal a n t i b o d i e s have been r a i s e d which are s p e c i f i c f o r v a r i o u s segments o f bovine rhodops in (MacKenzie and Molday, 1982; Molday and MacKenzie , 1983; MacKenzie e t a l . , 1984; L a i r d , 1984). Monoclonal a n t i b o d i e s s p e c i f i c f o r the C - t e r m i n a l o f rhodops in have been p r e c i s e l y l o c a l i z e d as to t h e i r a n t i g e n i c determinants by c o m p e t i t i v e i n h i b i t i o n a n a l y s i s u s i n g s y n t h e t i c p e p t i d e analogues o f r h o d o p s i n (MacKenzie e t a l . , 1984). Other monoclonal a n t i b o d i e s s p e c i f i c f o r c y t o p l a s m i c loop reg ions and the N-terminus o f rhodops in have been c h a r a c t e r i z e d and l o c a l i z e d by v a r i o u s immunocytochemical and b i o c h e m i c a l techniques as d e s c r i b e d i n t h i s r e p o r t . -17 6-The a c c e s s i b i l i t y o f segmental domains on rhodops in f o r ant ibody b i n d i n g , not o n l y p rov ide s evidence f o r the s t r u c t u r a l arrangement o f rhodops in i n the membrane, but a l s o de f ines the rhodops in segments a v a i l a b l e f o r i n t e r a c t i o n s w i t h o ther ROS p r o t e i n s . A l l a n t i b o d i e s s p e c i f i c f o r the C-t e r m i n a l and c y t o p l a s m i c loop reg ions o f rhodops in were capable o f b i n d i n g to rhodops in i n the membrane. Th i s was not unexpected as the c y t o p l a s m i c sur face o f r h o d o p s i n i n d i s c membranes must be a c c e s s i b l e f o r t r a n s d u c i n and r h o d o p s i n k i n a s e b i n d i n g . On the o ther hand, monoclonal a n t i b o d i e s s p e c i f i c f o r the N - t e r m i n a l o f rhodops in , not o n l y f a i l e d to b i n d rhodops in i n the n a t i v e memthey a l s o competed more e f f e c t i v e l y f o r b leached-s o l u b i l i z e d rhodops in than u n b l e a c h e d - s o l u b i l i z e d rhodops in (Molday and MacKenzie , 1983). A l though no f u n c t i o n has been d i r e c t l y a t t r i b u t e d to the N-terminus o f r h o d o p s i n , i t i s c l e a r t h a t a c o n f o r m a t i o n a l change does occur a t the N-terminus o f s o l u b i l i z e d rhodops in upon b l e a c h i n g . Immunoaf f in i ty p u r i f i e d rhodops in g l y c o p e p t i d e s and a s y n t h e t i c r h o d o p s i n p e p t i d e have been used i n c o m p e t i t i v e i n h i b i t i o n assays to p r e c i s e l y l o c a l i z e f i v e monoclonal a n t i b o d i e s to the N - t e r m i n a l o f r h o d o p s i n . One o f these monoclonal a n t i b o d i e s (rho 4D2) bound to rhodops in l o c a l i z e d i n g l u t a r a l d e h y d e - f i x e d ROS plasma membranes as demonstrated by immunocytochemical l a b e l i n g . Th i s monoclonal ant ibody was s u c c e s s f u l l y used as a b i o c h e m i c a l probe i n s t u d y i n g the i n t e r a c t i o n between ROS and bovine RPE c e l l s . An i n v i t r o assay was e s t a b l i s h e d f o r s t u d y i n g the b i n d i n g and i n g e s t i o n o f i s o l a t e d bov ine ROS by c u l t u r e d bovine RPE c e l l s . The ex tent o f phagocytos i s was measured by e i t h e r p r o b i n g ROS-treated RPE c e l l s w i t h a r a d i o l a b e l e d monoclonal ant ibody (rho 4D2) or by d i r e c t l y t r e a t i n g RPE c e l l s w i t h e n z y m a t i c a l l y r a d i o i o d i n a t e d , i n t a c t ROS. I n a d d i t i o n to q u a n t i t a t i v e assays , e l e c t r o n microscopy was used to c o n f i r m t h a t ROS were e f f e c t i v e l y -177-bound and i n g e s t e d by bovine RPE c e l l s . Rhodopsin was c o n c l u s i v e l y shown not to be the l i g a n d i n v o l v e d i n s p e c i f i c l i g a n d - r e c e p t o r i n t e r a c t i o n between ROS and RPE c e l l s by a v a r i e t y o f s t u d i e s . F i r s t o f a l l , immunoaf f in i ty p u r i f i e d 2-39 rhodops in g l y c o p e p t i d e and a 1-16 s y n t h e t i c rhodops in pept ide analogue f a i l e d to i n h i b i t the phagocytos i s o f ROS. Moreover, ROS plasma membrane f r ee d i s c membranes which were f r o z e n and thawed to expose the i n t r a d i s c a l sur face ( C l a r k and Molday, 1979) were o n l y s l i g h t l y e f f e c t i v e i n i n h i b i t i n g ROS p h a g o c y t o s i s , w h i l e i n t a c t ROS i n h i b i t e d ROS phagocytos i s by as much as 70%. Hence, the increa se i n a c c e s s i b l e N - t e r m i n a l segments o f rhodops in i n f rozen/ thawed , ROS d i s c membranes f a i l e d to e f f e c t i v e l y i n h i b i t the phagocytos i s o f ROS. F u r t h e r s t u d i e s u s i n g immunoaf f in i ty p u r i f i e d r h o d o p s i n , r e c o n s t i t u t e d i n t o p h o s p h o l i p i d v e s i c l e s , a l s o f a i l e d to i n h i b i t ROS p h a g o c y t o s i s . I n another s e r i e s o f exper iments , the p o s s i b i l i t y was e x p l o r e d t h a t plasma membrane l o c a l i z e d rhodops in may have undergone a d d i t i o n a l pos t t r a n s l a t i o n a l p r o c e s s i n g r e s u l t i n g i n i t s a b i l i t y to ac t as a ROS l i g a n d . G l u t a r a l d e h y d e - f i x e d ROS dense ly l a b e l e d w i t h rho 4D2 a n t i r h o d o p s i n ant ibody or rho 4D2 a n t i b o d y - g o l d - d e x t r a n p a r t i c l e s were e f f e c t i v e l y bound and i n t e r n a l i z e d by bovine RPE c e l l s . Thus, i t appears t h a t plasma membrane l o c a l i z e d rhodops in does not a c t as a l i g a n d f o r ROS b i n d i n g to RPE c e l l s . I t i s u n l i k e l y t h a t o ther reg ions o f the rhodops in molecule not d i r e c t l y bound by the ant ibody would ac t as the l i g a n d i n these experiments due to the l a r g e s i z e o f the ant ibody and the a n t i b o d y - g o l d - d e x t r a n p a r t i c l e s . I t would appear t h a t the ROS l i g a n d r e s p o n s i b l e f o r s p e c i f i c l i g a n d - r e c e p t o r i n t e r a c t i o n s between ROS and RPE c e l l s must extend beyond the g o l d - d e x t r a n p a r t i c l e s f o r d i r e c t , h i g h a f f i n i t y b i n d i n g . Other i n v e s t i g a t o r s have shown -178-t h a t carbohydrates found on rhodops in f a i l to i n h i b i t the b i n d i n g o f ROS to RPE c e l l s ( L e n t r i c h i a e t a l . , 1987). I n summary, i t i s c l e a r t h a t rhodops in i s not the l i g a n d i n v o l v e d i n s p e c i f i c ROS-RPE b i n d i n g , moreover, i t i s most l i k e l y t h a t a ROS plasma membrane s p e c i f i c p r o t e i n serves as such a l i g a n d . Recent advancements have been made i n t o unders tanding the molecu la r c o m p o s i t i o n o f ROS by the i d e n t i f i c a t i o n o f two plasma membrane s p e c i f i c r i c i n b i n d i n g g l y c o p r o t e i n s (Molday and Molday, 1987a). Furthermore , the e s t ab l i shment o f a p e r t u r b a t i o n procedure des igned to separate the ROS plasma membrane from the ROS d i s c membrane has been s u c c e s s f u l l y used to c o n f i r m the hypothes i s t h a t the p r o t e i n compos i t ion o f the ROS plasma membrane i s much more complex than t h a t o f the ROS d i s c membrane (Molday and Molday, 1987b). Consequently , there e x i s t many p o t e n t i a l ROS plasma membrane s p e c i f i c p r o t e i n s , one or more o f which may be the l i g a n d ( s ) d i r e c t l y i n v o l v e d i n ROS/RPE i n t e r a c t i o n s . S tud ie s here have shown t h a t , not o n l y were t r y p s i n - t r e a t e d ROS p h a g o c y t i z e d a t a reduced r a t e compared to u n t r e a t e d ROS, but a l s o s o l u b l e components r e l e a s e d i n t o the supernatant were capable o f i n h i b i t i n g ROS p h a g o c y t o s i s . The major change i n t r y p s i n - t r e a t e d ROS as i d e n t i f i e d by SDS-g e l e l e c t r o p h o r e s i s and western b l o t a n a l y s i s , was the l o s s o f r i c i n b i n d i n g to the n e u r a m i n i d a s e - s e n s i t i v e , plasma membrane s p e c i f i c 230kD g l y c o p r o t e i n . I n o ther s t u d i e s , F i c o l l d i s c membranes were e f f e c t i v e l y used to i n h i b i t ROS b i n d i n g to RPE c e l l s . O r i g i n a l l y , Smith e t a l . (1975) had i n d i c a t e d t h a t F i c o l l d i s c membranes were devo id o f ROS plasma membrane, however, r i c i n l a b e l i n g o f the 230kD g l y c o p r o t e i n i n F i c o l l d i s c membrane p r e p a r a t i o n s demonstrated t h a t these p r e p a r a t i o n s c o n t a i n e d membrane fragments from the ROS plasma membrane. I t i s p o s s i b l e t h a t plasma membrane fragments are able to f l o a t on F i c o l l by r e s e a l i n g a f t e r osmotic d i s r u p t i o n , or a l t e r n a t i v e l y , the ROS c y t o s k e l e t a l system may r e t a i n the a s s o c i a t i o n between the plasma -179-membrane fragments and the o s m o t i c a l l y i n t a c t d i s c s . These F i c o l l d i s c membranes were s u c c e s s f u l l y used as a model system f o r s t u d y i n g the i n h i b i t i o n o f ROS phagocytos i s and the e f f e c t o f t r y p s i n on p h a g o c y t o s i s . I t was p o s t u l a t e d t h a t the 230kD g l y c o p r o t e i n s i t e o f g l y c o s y l a t i o n (where a t l e a s t one o l i g o s a c c h a r i d e c h a i n c o n t a i n e d a t e r m i n a l s i a l i c a c i d r e s i d u e ) and the s i t e o f r a d i o i o d i n a t i o n may be l o c a t e d on the same t r y p t i c g l y c o p e p t i d e . Such an assumption a l l o w e d f o r the i s o l a t i o n o f the 125 g a l a c t o s e - c o n t a i n i n g , I - l a b e l e d , p o l y p e p t i d e fragments r e l e a s e d by t r y p s i n on a r i c i n a f f i n i t y column. The 34kD and 24kD g l y c o p o l y p e p t i d e fragments were i s o l a t e d on t h i s premise and shown to e f f e c t i v e l y i n h i b i t ROS 125 b i n d i n g . The major change noted from the t r y p s i n treatment o f I - l a b e l e d ROS was a l o s s o f l a b e l from the 230kD g l y c o p r o t e i n . Consequent ly , i t was concluded t h a t the s o l u b l e r i c i n b i n d i n g 34kD or 24kD g l y c o p e p t i d e , or b o t h , was d e r i v e d from the i n t r i n s i c membrane bound 230kD g l y c o p r o t e i n . I n an attempt to p u r i f y the 230kD g l y c o p e p t i d e from the ROS membranes, s o l u b i l i z e d ROS were sub jec ted to r i c i n a f f i n i t y chromatography. When these a f f i n i t y i s o l a t e d p r e p a r a t i o n s , which were e n r i c h e d w i t h the 230kD g l y c o p r o t e i n , were r e c o n s t i t u t e d i n t o p h o s p h o l i p i d v e s i c l e s and p r e i n c u b a t e d w i t h RPE c e l l s , a 50-60% i n h i b i t i o n o f phagocytos i s was o b t a i n e d . These r e s u l t s suggested t h a t the 230kD g l y c o p r o t e i n was the ROS l i g a n d d i r e c t l y i n v o l v e d i n s p e c i f i c ROS b i n d i n g to RPE c e l l s . However, i t i s p o s s i b l e t h a t o ther ROS p r o t e i n s i s o l a t e d w i t h the 230kD g l y c o p r o t e i n by t h i s procedure may have some d i r e c t involvement i n the b i n d i n g o f ROS to RPE c e l l s . I n order to f u r t h e r study the r o l e o f the 230kD g l y c o p r o t e i n i n ROS/RPE i n t e r a c t i o n s , a monoclonal ant ibody was r a i s e d s p e c i f i c f o r the e x t r a c e l l u l a r sur face o f the i n t r i n s i c 230kD g l y c o p r o t e i n . A l though t h i s -180 -ant ibody f a i l e d to b i n d to the s o l u b l e component o f t r y p s i n - t r e a t e d ROS and i n h i b i t phagocytos i s d i r e c t l y , i t was s u c c e s s f u l l y used to i s o l a t e the 230kD g l y c o p r o t e i n . T h i s immunoaf f in i ty p r e p a r a t i o n of low pH e l u t e d 230kD g l y c o p r o t e i n appeared o n l y to be contaminated w i t h r h o d o p s i n and was much more e f f e c t i v e i n i n h i b i t i n g ROS phagocytos i s than a f r a c t i o n d e v o i d o f any 230kD g l y c o p r o t e i n . However a c a u t i o n a r y note must be taken i n t h a t the ex tent o f i n h i b i t i o n o f ROS phagocytos i s d i d not exceed 40%. I t i s p o s s i b l e t h a t the low pH e l u t i o n o f the immunoaf f in i ty column may have p a r t i a l l y and i r r e v e r s i b l y denatured the 230kD g l y c o p r o t e i n . A l t e r n a t i v e l y , the 230kD g l y c o p r o t e i n may o n l y be one ROS component necessary f o r proper and complete i n h i b i t i o n o f ROS b i n d i n g to RPE c e l l s . I t i s p o s s i b l e t h a t a s tep-wise i n t e r a c t i o n o f l i g a n d s on the ROS sur face w i t h r e c e p t o r s on the RPE c e l l may be r e q u i r e d as d e s c r i b e d i n macrophages ( G r i f f i n e t a l . , 1975; S i l v e r s t e i n e t a l . , 1978). T h i s s tep-wise i n t e r a c t i o n may i n v o l v e one type o f l i g a n d or l i g a n d s o f d i f f e r e n t types . I n c o n c l u s i o n , a d d i t i o n a l s t u d i e s c o u l d be performed u s i n g c o n v e n t i o n a l i o n exchange and g e l e x c l u s i o n chromatographic techniques i n an attempt to i s o l a t e a g r e a t e r q u a n t i t y o f the 230kD g l y c o p r o t e i n f o r f u r t h e r i n h i b i t i o n o f phagocytos i s s t u d i e s . A l though the evidence f o r the involvement o f the 230kD g l y c o p r o t e i n i n ROS/RPE c e l l i n t e r a c t i o n s i s q u i t e s t r o n g , there e x i s t s the p o s s i b i l i t y t h a t a minor p r o t e i n not de tec ted by SDS-gel e l e c t r o p h o r e s i s and immunoblott ing techniques may be contamina t ing the p r e p a r a t i o n s used i n the r e c o n s t i t u t i o n s t u d i e s , and t h u s , be d i r e c t l y r e s p o n s i b l e f o r the i n h i b i t i o n o f ROS phagocytos i s by RPE c e l l s . More c o n v i n c i n g evidence f o r the involvement o f the ROS 230kD g l y c o p r o t e i n i n ROS/RPE i n t e r a c t i o n s c o u l d be ob ta ined by r a i s i n g a monoclonal ant ibody s p e c i f i c f o r the s o l u b l e 230kD g l y c o p r o t e i n - d e r i v e d , g l y c o p e p t i d e r e l e a s e d i n t o the supernatant o f t r y p s i n - t r e a t e d , i n t a c t ROS. Such an a n t i b o d y , when - 1 8 1 -bound to i n t a c t ROS, may d i r e c t l y i n h i b i t ROS p h a g o c y t o s i s . Moreover, an a d d i t i o n a l ant i -230kD g l y c o p r o t e i n ant ibody may be more s u c c e s s f u l l y used i n immunoa f f in i ty columns f o r the p u r i f i c a t i o n o f the 230kD g l y c o p r o t e i n . 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