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Molecular cloning, expression and characterization of photoreceptor cell peripherin : the defective protein… Connell, Gregory James 1990

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MOLECULAR CLONING, EXPRESSION AND CHARACTERIZATION OF PHOTORECEPTOR CELL PERIPHERIN - THE DEFECTIVE PROTEIN RESPONSIBLE FOR THE RETINAL DEGENERATION SLOW (rds) DEFECT BY GREGORY JAMES CONNELL B.Sc,  The U n i v e r s i t y o f B r i t i s h Columbia, 1985  A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY in THE FACULTY OF GRADUATE STUDIES (Department o f Biochemistry)  We accept t h i s t h e s i s as conforming t o t h e r e q u i r e d standard  THE UNIVERSITY OF BRITISH COLUMBIA June 1990  Gregory James C o n n e l l , 1990  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  ^L^cfi  c &r&V  The University of British Columbia Vancouver, Canada  Date  DE-6 (2/88)  Y/^/tffi  - i i -  Abstract  P e r i p h e r i n , a membrane p r o t e i n w i t h an apparent m o l e c u l a r weight o f 34 kDa, has been p r e v i o u s l y l o c a l i z e d t o the r i m r e g i o n o f t h e v e r t e b r a t e r o d p h o t o r e c e p t o r d i s k membrane u s i n g monoclonal a n t i b o d i e s and immunocytochemical l a b e l l i n g t e c h n i q u e s . As an i n i t i a l  step i n d e t e r m i n i n g t h e  s t r u c t u r e and f u n c t i o n o f t h i s p r o t e i n , cDNA c o n t a i n i n g i t s c o d i n g sequence has been c l o n e d and sequenced. A bovine retinal / g t l l  e x p r e s s i o n l i b r a r y was screened  with  a n t i p e r i p h e r i n monoclonal a n t i b o d i e s , and a 583 base p a i r c l o n e was i n i t i a l l y  i s o l a t e d . The remaining p a r t o f t h e  c o d i n g sequence was o b t a i n e d from subsequent r e s c r e e n i n g s o f the same l i b r a r y and an independent  XgtlO l i b r a r y . A C-  t e r m i n a l CNBr fragment o f p e r i p h e r i n was p u r i f i e d by immunoaffinity chromatography and r e v e r s e phase h i g h performance l i q u i d chromatography. The amino a c i d sequence of  t h e i s o l a t e d C-terminal p e p t i d e and t h e N-terminal  sequence a n a l y s i s o f immunoaffinity p u r i f i e d p e r i p h e r i n a r e i n agreement w i t h t h e cDNA sequence. At t h e amino a c i d l e v e l , t h e sequence o f p e r i p h e r i n has 92.5%  sequence i d e n t i t y t o t h e gene proposed  t o be  r e s p o n s i b l e f o r t h e r e t i n a l degeneration slow d e f e c t i n mice ( T r a v i s e t al.(1989)  Nature 338, 70-73)  The d i f f e r e n c e s  between t h e two sequences can be a t t r i b u t e d t o s p e c i e s d i f f e r e n c e s . The i d e n t i t y o f t h e r e t i n a l d e g e n e r a t i o n  slow  -iii-  gene product and  i t s i n t r a c e l l u l a r l o c a l i z a t i o n were  p r e v i o u s l y unknown. The  cDNA sequence o f p e r i p h e r i n p r e d i c t s t h a t t h e r e  p o s s i b l y f o u r transmembrane domains. On the b a s i s  of  immunocytochemical s t u d i e s and  the  h y d r o p h i l i c C-terminal  sequence a n a l y s i s ,  segment c o n t a i n i n g the  antigenic  s i t e s f o r the a n t i p e r i p h e r i n monoclonal a n t i b o d i e s has l o c a l i z e d on the c y t o p l a s m i c  are  been  s i d e of the d i s k membrane.  There are t h r e e consensus sequences f o r a s p a r a g i n e l i n k e d glycosylation. Deglycosylation a t l e a s t one  s t u d i e s have i n d i c a t e d t h a t  of these s i t e s i s u t i l i z e d . The  sequence o f p e r i p h e r i n was  expressed i n COS-1  complete  coding  c e l l s . Western  b l o t a n a l y s i s of the expressed p e r i p h e r i n suggest t h a t i t e x i s t s as a homodimer i n the absence o f a r e d u c i n g The  agent.  p o s s i b l e f u n c t i o n of p e r i p h e r i n i n r e l a t i o n t o i t s  primary s t r u c t u r e i s d i s c u s s e d .  -iv-  TABLE OF CONTENTS page I.  ABSTRACT  i i  I I . TABLE OF CONTENTS —  :  III.  LIST OF FIGURES  IV.  LIST OF ABBREVIATIONS  V.  iv •  v i i viii  ACKNOWLEDGEMENT —  ix  VI. INTRODUCTION A. The V e r t e b r a t e  Retina  1  B. The Outer Segment  3  C. V i s u a l E x c i t a t i o n  •  5  D. Rod Outer Segment P r o t e i n s  10  E. Photoreceptor C e l l P e r i p h e r i n  11  F. R e t i n a l Degeneration Slow  14  G. T h e s i s I n v e s t i g a t i o n  17  V I I . MATERIALS  AND METHODS  A. M a t e r i a l s B. Screening  :  18  of the Retinal L i b r a r i e s  19  C. I s o l a t i o n o f Phage DNA and Sequencing  21  D. Northern B l o t A n a l y s i s  22  E. Immunoaffinity Column P r e p a r a t i o n  23  F. Rod Outer Segment P r e p a r a t i o n  23  G. P u r i f i c a t i o n f o r N-terminal Sequence Analysis  24  H. I s o l a t i o n o f a CNBr P e r i p h e r i n f o r Amino A c i d Sequencing  24  I. D e g l y c o s y l a t i o n  of Peripherin  Peptide 25  -v-  VIII.  J . SDS - P o l y a c r y l a m i d e 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  26  K. Subcloning o f t h e Complete Coding Sequence o f P e r i p h e r i n i n t o pAX 111 a COS C e l l E x p r e s s i o n V e c t o r  26  L. COS C e l l Maintenance  27  M. T r a n s f e c t i o n o f COS-1 C e l l s w i t h t h e pAX 111 V e c t o r  29  N. H a r v e s t i n g  30  of the Transfected  COS C e l l s  RESULTS A. P u r i f i c a t i o n o f P e r i p h e r i n f o r N-terminal Sequence A n a l y s i s  31  B. I s o l a t i o n o f P e r i p h e r i n cDNA Clones  33  C. I s o l a t i o n o f a C-terminal CNBr Fragment o f P e r i p h e r i n f o r Amino A c i d Sequence A n a l y s i s -3 5 D. Amino A c i d Sequence o f P e r i p h e r i n  35  E. B l o t H y b r i d i z a t i o n A n a l y s i s  43  F. P r o t e i n Homologies  IX.  •  43  G. S y n t h e s i s o f a C o n s t r u c t C o n t a i n i n g the Complete Open Reading Frame o f Peripherin  43  G. E x p r e s s i o n  49  DISCUSSION  X. CONCLUSION  o f P e r i p h e r i n i n COS C e l l s  52 63  -vi-  XI REFERENCES  65  -vii-  L I S T OF  FIGURES Page  Figure Figure  1. 2.  Neural Cells of the Retina S t r u c t u r e o f t h e Rod C e l l  2 4  Figure  3.  Model  f o r Rod O u t e r Segment F o r m a t i o n  6  Figure  4.  Model  f o r Visual Transduction  9  Figure  5.  Localization  Figure  6.  Identification SDS  of Peripherin  12  o f P e r i p h e r i n on  - polyacrylamide  gel  15  111 V e c t o r  28  Figure  7.  Schematic  Figure Figure  8. 9.  Purification of Peripherin N-terminal Sequence A n a l y s i s o f P e r i p h e r i n and Design o f a Degenerate O l i g o n u c l e o t i d e Probe  Figure Figure  o f t h e pAX  an  1 0 . R e s t r i c t i o n Map a n d S e q u e n c i n g S t r a t e g y o f C l o n e d cDNA E n c o d i n g P e r i p h e r i n 11. P u r i f i c a t i o n of  of a C-terminal  32  34 36  Fragment  Peripherin  37  Figure  12. Amino A c i d Sequence o f P e r i p h e r i n  40  Figure  13. D e g l y c o s y l a t i o n  42  Figure  14. N o r t h e r n B l o t A n a l y s i s o f P e r i p h e r i n Messages 15. An A l i g n m e n t o f t h e Amino A c i d Sequence of P e r i p h e r i n with the Proposed rds Sequence  46  16. A C o n s t r u c t C o n t a i n i n g t h e C o m p l e t e Coding Sequence o f P e r i p h e r i n  47  Figure  Figure  of Peripherin  44  Figure  17. W e s t e r n B l o t A n a l y s i s o f t h e P e r i p h e r i n E x p r e s s e d w i t h i n t h e COS-1 C e l l s  51  Figure  18. A S t r u c t u r a l  57  Model o f P e r i p h e r i n  -viii-  LIST OF ABBREVIATIONS bp  base  BSA  bovine serum albumin  DEAE  diethylaminoethyl  EDTA  ethylenediamine t e t r a a c e t a t e  Endo H  endo-6-N-acetylglucosaminidase H.  HPLC  high-performance l i q u i d  Ig  pair  chromatography  immunoglobin  kb  kilobase  Mr  apparent m o l e c u l a r weight  PTH  penylthiohydantoin  rd  r e t i n a l degeneration  rds  r e t i n a l d e g e n e r a t i o n slow  ROS  r o d o u t e r segment  SDS  sodium dodecyl  TEMED  —  UV  —•  Tris  sulfate  N,N,N',N'-Tetramethylethylenediamine  :  ultraviolet — • —  tris(hydroxymethyl)aminomethane  -ix-  ACKNOWLEDGEMENT I would l i k e t o thank Dr. Robert Molday f o r h i s e x c e l l e n t supervision,  encouragement and support o f t h i s p r o j e c t . I  would l i k e t o extend my g r a t i t u d e and thanks t o Dr. Ross M a c G i l l i v r a y and t h e many members o f h i s l a b who have me a s s i s t a n c e  given  over t h e course o f these s t u d i e s . I would a l s o  l i k e t o thank Dr. Rob Kay f o r t h e use o f h i s pAX 111 expression  v e c t o r and f o r h e l p f u l d i s c u s s i o n s  concerning the  COS c e l l t r a n s f e c t i o n procedure. F i n a l l y , I would l i k e t o thank L a u r i e Molday, Drs. Simon and D e l y t h Wong, Dr. J e f f Leung, Dr. Robert McMaster, Dr. Dale L a i r d , Shu-Chan Hsu and Dr. Rod Mclnnes f o r many h e l p f u l  discussions.  -1-  INTRODUCTION  The V e r t e b r a t e R e t i n a  The  r e t i n a , l o c a t e d a t t h e p o s t e r i o r end o f t h e  e y e b a l l , transduces  l i g h t into neural signals, integrates  these s i g n a l s and then t r a n s m i t s them through t h e o p t i c nerve t o t h e b r a i n . The p h o t o r e c e p t o r  c e l l s , i n which the  transduction of l i g h t into neural s i g n a l s occurs, are the layer of r e t i n a l lens  n e u r a l c e l l s f u r t h e s t removed from t h e eye  ( F i g u r e 1 ) . These c e l l s a r e c l a s s i f i e d as r o d o r cone  c e l l s on t h e b a s i s o f t h e i r p h o t o s e n s i t i v i t y and morphology. The p h o t o s e n s i t i v e pigment o f t h e p h o t o r e c e p t o r  cell  c o n s i s t s o f a p r o t e i n component, o p s i n , t h a t i s c o v a l e n t l y l i n k e d t o 11-cis r e t i n a l .  I t i s the i n t e r a c t i o n o f t h e  d i f f e r e n t o p s i n molecules  w i t h t h e r e t i n a l t h a t determines  the a b s o r p t i o n maxima (Nathans e t a l . , 1986). The human b l u e , green,  and r e d s e n s i t i v e cone pigments have a b s o r p t i o n  maxima a t 420, 530 and 560 nm r e s p e c t i v e l y . Rhodopsin, the rod p h o t o s e n s i t i v e molecule has an a b s o r p t i o n maxima a t 500 nm. In t h e human r e t i n a t h e r e a r e approximately rod c e l l s and 6.5 m i l l i o n cone c e l l s  120 m i l l i o n  (Pirenne, 1967). The  cone c e l l s a r e l o c a t e d p r i m a r i l y i n t h e fovea, t h e c e n t r e o f the v i s u a l f i e l d , whereas t h e r o d c e l l s a r e l o c a t e d i n the  -2-  F i g u r e 1. S c h e m a t i c d i a g r a m o f s o m e o f t h e d i f f e r e n t n e u r a l c e l l t y p e s i n t h e r e t i n a . T h e r o d (R) a n d c o n e (C) p h o t o r e c e p t o r c e l l s a r e i n d i c a t e d . The h o r i z o n t a l (H) a n d b i p o l a r (B) n e u r a l c e l l s s y n a p s e d i r e c t l y w i t h t h e p h o t o r e c e p t o r s . T h e a m a c r i n e (A) a n d g a n g l i o n (G) n e u r o n s a r e a l s o i l l u s t r a t e d . The a r r o w i n d i c a t e s t h e d i r e c t i o n o f l i g h t (modified a f t e r Dowling,1970).  -3-  more p e r i p h e r a l r e g i o n s o f the r e t i n a . Both types o f c e l l s c o n s i s t o f a s y n a p t i c t e r m i n a l , an i n n e r segment and an o u t e r segment chemical  ( F i g u r e 2 ) . The s y n a p t i c t e r m i n a l makes  s y n a p t i c connections  h o r i z o n t a l neural c e l l s c o n t a i n s the nucleus  with the b i p o l a r and  ( F i g u r e 1 ) . The i n n e r segment  and mitochondria  of the c e l l . A l l  p r o t e i n s y n t h e s i s takes p l a c e i n the i n n e r segment. The o u t e r segments o f cone and rod c e l l s a r e s p e c i a l i z e d o r g a n e l l e s which c o n t a i n the o p s i n molecules and t h e other p r o t e i n s r e q u i r e d f o r the t r a n s d u c t i o n o f l i g h t i n t o electrical  s i g n a l s as p a r t o f the v i s u a l e x c i t a t i o n p r o c e s s .  The o u t e r segment i s attached t o the i n n e r segment by a connecting  The Outer  cilium.  Segment  The o u t e r segment o f the r o d c e l l  c o n t a i n hundreds of  c l o s e l y stacked membrane d i s k s t h a t a r e d i s c o n t i n u o u s with a surrounding  plasma membrane throughout most o f t h e r o d outer  segment. These d i s k s a r e organized array  into a highly  ordered  ( F i g u r e 2 ) . In v e r t e b r a t e s the d i s k s a l s o have  i n f o l d i n g s o r i n c i s u r e s . The cone o u t e r segment d i f f e r s the r o d o u t e r segment i n t h a t the d i s k s a r e continuous  from with  the plasma membrane throughout the e n t i r e s t r u c t u r e . It  i s not y e t c l e a r how the r o d outer segment d i s k  membranes a r e formed. S t e i n b e r g e t al.(1980) have proposed a model f o r the formation o f r o d o u t e r segments t h a t i s based  disk  F i g u r e 2. a ) A s c h e m a t i c o f a l o n g i t u d i n a l c r o s s s e c t i o n t h r o u g h a r o d p h o t o r e c e p t o r c e l l ( p r o v i d e d b y R. M o l d a y ) . b) A t h r e e d i m e n s i o n a l r e p r e s e n t a t i o n o f t h e r o d o u t e r segment. The cone p h o t o r e c e p t o r c e l l d i f f e r s from t h e r o d c e l l i n t h a t t h e membrane d i s k s a r e c o n t i n u o u s w i t h t h e p l a s m a membrane t h r o u g h o u t t h e o u t e r s e g m e n t ( F e i n a n d Szuts, 1982).  -5-  on e l e c t r o n m i c r o s c o p i c o b s e r v a t i o n s . In t h e i r model t h e d i s k s r e s u l t from an outgrowth o f t h e c i l i a r y  plasma  membrane. There a r e components i n t h e c i l i a r y  membrane t h a t  are  proposed t o i n t e r a c t w i t h b i n d i n g s i t e s e i t h e r  on t h e c i l i u m o r w i t h a c y t o s k e l e t a l element associated with the c i l i u m  directly  closely  ( F i g u r e 3 ) . A c t i n has  been found t o be a s s o c i a t e d w i t h t h e d i s t a l r e g i o n o f t h e cilium  (Chaitin et a l .  1984 and C h a i t i n and Bok 1986). I t  has been suggested t h a t an a c t i n mediated  contractile  mechanism c o u l d be t h e cause f o r t h e outward membrane f o l d i n g back on i t s e l f .  growing  Treatment o f r a b b i t  retina  i n v i v o w i t h c y t b c h a l a s i n D, an i n h i b i t o r o f a c t i n p o l y m e r i z a t i o n , r e s u l t s i n t h e f o r m a t i o n o f l a r g e membrane outgrowths i n s t e a d o f normal o u t e r segment d i s k s  (Vaughan &  F i s h e r , 1989). Rim f o r m a t i o n , which s e a l s t h e f o l d e d membrane i n t o an i n t a c t d i s k i s proposed t o s t a r t a t t h e attachment s i t e near t h e c i l i u m and move outwards  towards  the  plasma membrane. Upon f o r m a t i o n o f t h e completed r i m ,  the  d i s k becomes detached from t h e plasma membrane.  Visual  Excitation  A model f o r t h e v i s u a l e x c i t a t i o n p r o c e s s i n t h e r o d o u t e r segment i s o u t l i n e d i n f i g u r e 4. In t h e dark N a to  a l e s s e r degree C a  cGMP dependent  + 2  +  and  a r e a b l e t o e n t e r t h e c e l l through a  channel l o c a t e d i n t h e o u t e r segment  (Hodgkin  -6-  rim  mature rl disk oluter -^segment  \  ^formation  oo  O O  membrane vesicle;;  INNER SEGMENT  F i g u r e 3. A h y p o t h e t i c a l model f o r t h e f o r m a t i o n o f t h e r o d o u t e r segment d i s k membranes, a) Membrane v e s i c l e s a r e i n s e r t e d i n t o t h e e v a g i n a t i n g membrane near t h ebase o f t h e c i l i u m . B i n d i n g s i t e s f o r a component i n t h e membrane a r e i n d i c a t e d o n t h e c i l i u m (#.) . T h e o u t g r o w t h o f t h e m e m b r a n e i s hypothesized t o be l i m i t e d by an inward d i r e c t e d a c t i n mediated c o n t r a c t i l e mechanism ^ ^_ ) . b ) R i m f o r m a t i o n t a k e s p l a c e between a d j a c e n t f o l d s o f t h e membrane, c ) T h i s somehow l e a d s to. t h e s e p a r a t i o n o f t h e d i s k membrane f r o m t h e p l a s m a membrane.  -7-  et  a l . , 1985). The Ca  through a N a \ C a +  i s removed from t h e o u t e r segment  exchanger. F o r every  + 2  are taken i n t o t h e o u t e r segment a K extruded  +  four Na  +  ions that  i o n and a C a  (Cervetto e t a l . , 1989). The N a  the c e l l by a N a \ K  +  +  + 2  i o n are  i s removed from  ATPase t h a t i s l o c a t e d i n t h e i n n e r  +  segment ( S t a h l & Baskin,  1984). The r o d i n n e r segment N a \ K +  +  ATPase has n o t been t h a t w e l l c h a r a c t e r i z e d , b u t t h e ATPase of t h e human e r y t h r o c y t e pumps t h r e e N a cell  f o r every two K  +  +  i o n s out o f t h e  i o n s t h a t a r e taken i n (Sen & Post,  1964). T h i s i o n flow i n t o t h e outer segment and out through the i n n e r segment i s known as t h e dark c u r r e n t . Absorption  o f a photon by t h e 1 1 - c i s r e t i n a l group o f  the o p s i n causes a s e r i e s o f c o n f o r m a t i o n a l  changes t h a t  e v e n t u a l l y l e a d s t o i t s c o n v e r s i o n t o t h e a l l t r a n s form and to  i t s r e l e a s e from t h e o p s i n . During t h e p r o c e s s t h e  conformation  o f t h e o p s i n i s a l t e r e d so t h a t i t i s a b l e t o  i n t e r a c t and a c t i v a t e t r a n s d u c i n o r G - p r o t e i n . c o n s i s t s o f an oc subunit(39 subunit(8  kDa),  Transducin  a 6 subunit(36  kDa),  and a 2f  kDa) (Fung, 1983). The a c t i v a t i o n i n v o l v e s t h e  exchange o f GDP on t h e i n a c t i v e form o f t h e oc s u b u n i t f o r GTP.  The o n l y known f u n c t i o n o f t h e R and .jr'subunits i s t o  p o s i t i o n the oc subunit so t h a t i t can i n t e r a c t  with  rhodopsin. The  a c t i v a t e d =X subunit  phosphodiesterase. subunit(88 subunit  kDa),  i n t u r n can a c t i v a t e  The phospodiesterase a 6 subunit(84  consists of a n ° ^  kDa) and an i n h i b i t o r y  &  (11 kDa) (Baehr e t a l . , 1979). A c t i v a t i o n i s thought  -8-  t o i n v o l v e the displacement of the i n h i b i t o r y s u b u n i t (Hurley & S t r y e r , 1982). Upon a c t i v a t i o n ,  phosphodiesterase  i s a b l e t o h y d r o l y z e cGMP. The decreased l e v e l s o f cGMP i n the o u t e r segment r e s u l t s i n the c l o s i n g of some of the sodium channels the N a \ K +  +  ATPase i s s t i l l  (Fesenko e t a l . ,  1985). S i n c e  o p e r a t i n g i n the i n n e r segment,  the c l o s i n g of the channels r e s u l t i n the h y p e r p o l a r i z a t i o n of the p h o t o r e c e p t o r membrane. T h i s h y p e r p o l a r i z a t i o n m o d i f i e s the s y n a p t i c output t o the c o n n e c t i n g neurons.  The  e n t i r e e x c i t a t i o n process occurs w i t h i n m i l l i s e c o n d s of the i l l u m i n a t i o n o f the p h o t o r e c e p t o r  cell.  There are s e v e r a l mechanisms used t o d e a c t i v a t e the v i s u a l cascade. S h o r t l y a f t e r a c t i v a t i o n , kinase(68 kDa)  rhodopsin  b i n d s t o the c y t o p l a s m i c f a c e of rhodopsin  and p h o s p h o r y l a t e s up t o n i n e s e r i n e and t h r e o n i n e r e s i d u e s (Wilden & Kuhn, 1982). T h i s p h o s p h o r y l a t i o n decreases the a f f i n i t y o f rhodopsin f o r t r a n s d u c i n and i n c r e a s e s the a f f i n i t y f o r a r r e s t i n . A r r e s t i n ( 4 8 kDa)  competes f o r the  same b i n d i n g s i t e on rhodopsin as t r a n s d u c i n , and by b i n d i n g t o r h o d o p s i n i t prevents  a c t i v a t i o n of further transducin  molecules. In a d d i t i o n , the  s u b u n i t of t r a n s d u c i n has a  GTPase a c t i v i t y t h a t h y d r o l y z e s i t s bound GTP  t o GDP  r e t u r n s any a c t i v a t e d p r o t e i n t o i t s i n a c t i v e  state.  which  A r r e s t i n i s a l s o thought t o be a b l e t o d i r e c t l y i n h i b i t a c t i v a t e d phosphodiesterase  (Zuckerman e t a l . 1985).  the  -9-  Plastna  P0£  Membrane  Disc  Memtxane  F i g u r e 4: A m o d e l f o r v i s u a l t r a n s d u c t i o n . T h e c G M P g a t e d c a t i o n channel i s open i n t h e dark. Upon i l l u m i n a t i o n , rhodopsin undergoes a conformational change t h a t p e r m i t s i t to a c t i v a t e t r a n s d u c i n o r G-protein. The a c t i v a t e d transducin i nturn activates a phosphodiesterase that h y d r o l y z e s cGMP. T h e d e c r e a s e d l e v e l s o f cGMP r e s u l t i n t h e c l o s i n g o f some o f t h e c h a n n e l s a n d t h e h y p e r p o l a r i z a t i o n o f t h e c e l l ( p r o v i d e d b y R. M o l d a y ) .  -10-  Rod  Outer Segment P r o t e i n s  The p h o t o a c t i v e  p r o t e i n , rhodopsin,  i s t h e major  c o n s t i t u e n t o f both the plasma membrane and t h e d i s k membrane o f t h e r o d outer segment. The C-terminus o f rhodopsin  and t h e F ^ I ^ loop which j o i n s t h e f i f t h and s i x t h  membrane spanning h e l i c e s a r e o r i e n t e d on t h e c y t o p l a s m i c f a c e o f t h e membranes where they a r e a b l e t o i n t e r a c t w i t h o t h e r components o f t h e v i s u a l cascade such as k i n a s e and t r a n s d u c i n  rhodopsin  (Kuhn, 1981; Hargrave, 1982). The N-  t e r m i n a l segment which c o n t a i n s two asparagine l i n k e d carbohydrate c h a i n s  i s o r i e n t a t e d on t h e e x t r a c e l l u l a r  s u r f a c e o f t h e plasma membrane and i n t h e lumen o f t h e d i s k ( R o h l i c h , 1976; Hargrave, 1977; C l a r k & Molday, 1979). In a d d i t i o n t o rhodopsin,  t h e plasma membrane c o n t a i n s a  unique s e t o f p r o t e i n s t h a t a r e not p r e s e n t  i n the disk  membrane. S e v e r a l 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 were i d e n t i f i e d t h a t a r e s p e c i f i c f o r t h e plasma membrane  (Molday  & Molday, 1987a). These p r o t e i n s were used as markers i n the p u r i f i c a t i o n o f t h e plasma membrane from t h e d i s k membrane u s i n g a d e n s i t y p e r t u r b a t i o n method (Molday & Molday 1987b). The  63 kDa cGMP - gated c a t i o n channel and t h e 220 kDa  sodium - c a l c i u m exchanger were subsequently shown t o be r e s t r i c t e d t o t h e plasma membrane (Bauer, 1988; Cook e t a l . , 1989,  Reid e t a l . , 1990).  The  d i s k membrane, i t s e l f ,  can be d i v i d e d i n t o two  domains w i t h unique p r o t e i n compositions.  F a l k and F a t t  -11-  (1969) have shown t h a t an osmium t e t r o x i d e s o l u t i o n d i s s o l v e d the l a m e l l a e of the d i s k s but not the  rims.  Papermaster e t al.(1978) used a p o l y c l o n a l antibody t o l o c a l i z e a 290 f r o g ROS.  kDa  p r o t e i n t o the d i s k i n c i s u r e s and  T h i s p o l y c l o n a l serum does not, however,  r e a c t w i t h any  component p r e s e n t  rims of  cross  i n the mammalian  p h o t o r e c e p t o r . Molday e t a l . (1987) l o c a l i z e d a 33  kDa  p r o t e i n , p e r i p h e r i n , s p e c i f i c a l l y t o the rim r e g i o n o f bovine d i s k membrane u s i n g monoclonal a n t i b o d i e s  the  and  immunocytochemical l a b e l i n g techniques ( F i g u r e 5 ) . A 22 0 concanavalin  A - binding  p r o t e i n s are a l s o present  g l y c o p r o t e i n and  kDa  s e v e r a l minor  i n the mammalian d i s k membrane,  but they have not y e t been l o c a l i z e d t o e i t h e r domain (Molday & Molday 1987b).  Photoreceptor C e l l  Peripherin  P e r i p h e r i n cannot be e x t r a c t e d w i t h c h e l a t i n g and  chaotropic  detergent f o r s o l u b i l i z a t i o n  from the d i s k membrane  agents, but  i t requires a  (Molday e t a l . , 1987).  p r o t e i n migrates as a dimer on an SDS  - polyacrylamide  i n the absence of 2 - mercaptoethanol s u g g e s t i n g e x i s t as two  subunits  held together  The  by one  gel  t h a t i t may  o r more d i s u l f i d e  bonds (Molday e t a l . , 1987). I t i s not known whether the subunits  are i d e n t i c a l . In the presence o f 2 -  mercaptoethanol, p e r i p h e r i n migrates as a r e l a t i v e l y  sharp  band d i r e c t l y beneath rhodopsin a t a m o l e c u l a r weight of 3 3  -12-  F i g u r e 5. The l o c a l i z a t i o n o f p e r i p h e r i n w i t h i n t h e r o d o u t e r segment, a) A l o n g i t u d i n a l s e c t i o n through a r o d outer segment t h a t had been l a b e l l e d w i t h 3B6, an a n t i p e r i p h e r i n monoclonal antibody, f o l l o w e d by goat antimouse I g - g o l d dextran p a r t i c l e s . L a b e l l i n g i s shown t o be c o n c e n t r a t e d along t h e p e r i p h e r a l r e g i o n o f t h e outer segment where t h e d i s k membranes come i n t o c l o s e c o n t a c t w i t h t h e plasma membrane. Bar=0.25um. b) An i s o l a t e d d i s k showing t h e b i n d i n g o f 3B6 p r e f e r e n t i a l l y along t h e r i m r e g i o n . Bar=.05um (Molday e t a l . , 1987).  -13-  -14-  kDa  ( F i g u r e 6). The d e t e r m i n a t i o n o f the number of  p e r i p h e r i n molecules p r e s e n t i n the rod o u t e r segment has been complicated by the d i f f i c u l t y i n p u r i f y i n g i t . P e r i p h e r i n appears,  though, t o c o n s t i t u t e l e s s than 5% of  the t o t a l d i s k p r o t e i n . T h i s c o u l d s t i l l make i t , a f t e r rhodopsin, one of the more abundant membrane p r o t e i n s of the photoreceptor  cell.  R e t i n a l Degeneration  Slow  R e t i n a l degeneration slow (rds) i s a c o n g e n i t a l r e t i n a l d i s o r d e r t h a t has been i d e n t i f i e d i n a s t r a i n of mice Nie e t a l . ,  (van  1978). Those mice t h a t are homozygous f o r the  d e f e c t are i n i t i a l l y c h a r a c t e r i z e d by an absence of photoreceptor c e l l The  o u t e r segments (Sanyal & Jansen,  i n n e r segments of the p h o t o r e c e p t o r c e l l s and  1981).  the  neurons t h a t synapse w i t h them i n i t i a l l y appear t o be normal (Jansen & Sanyal, 1984). Membrane v e s i c l e s have been shown to  accumulate c l o s e t o the r e g i o n where o u t e r segment  formation would normally occur. At 2 t o 3 weeks a f t e r b i r t h , t h e r e i s a gradual l o s s of the remaining p o r t i o n of the p h o t o r e c e p t o r c e l l s , and a f t e r one year, the p h o t o r e c e p t o r s are completely degenerated.  The o t h e r r e t i n a l c e l l  types  remain u n a f f e c t e d . The d e f e c t i v e gene i s c l o s e l y l i n k e d t o the H2 gene complex, and i t maps t o chromosome 17 al.,  1979).  (Demant e t  -15-  F i g u r e 6. R o d o u t e r s e g m e n t p r o t e i n s w e r e s e p a r a t e d b y SDS p o l y a c r y l a m i d e g e l e l e c t r o p h o r e s i s i n t h e p r e s e n c e (+ME) or i n t h e a b s e n c e (-ME) o f 2 - m e r c a p t o e t h a n o l . The l a n e s o f t h e g e l w e r e e i t h e r s t a i n e d d i r e c t l y w i t h C o o m a s s i e B l u e (CB) o r u s e d i n i m m u n o b l o t s . The a n t i p e r i p h e r i n m o n o c l o n a l a n t i b o d i e s 2B6 a n d 3B6 l a b e l a r e l a t i v e l y s h a r p b a n d t h a t i n the presence of 2-mercaptoethanol runs underneath the broad r h o d o p s i n b a n d a t 34 k D a . I n t h e a b s e n c e o f 2 mercaptoethanol the a n t i p e r i p h e r i n monoclonal antibodies l a b e l a b a n d c l o s e r t o 68 k D a . T h e a n t i r h o d o p s i n m o n o c l o n a l a n t i b o d y 1D4 l a b e l s t h e b r o a d 34 k D a b a n d b o t h i n t h e p r e s e n c e a n d a b s e n c e o f t h e r e d u c i n g a g e n t . A 68 k D a b a n d , w h i c h i s p r o b a b l y a g g r e g a t e d r h o d o p s i n , i s a l s o l a b e l l e d by t h e 1D4 a n t i b o d y . T h e i n t e n s i t y o f t h i s b a n d i s i n d e p e n d e n t of t h e presence o f 2-mercaptoethanol (Molday e t a l . , 1987).  -16-  T r a v i s e t a l . (1989) have r e c e n t l y i d e n t i f i e d a c a n d i d a t e gene f o r the rds d e f e c t . By c a r r y i n g out s u b t r a c t i v e and d i f f e r e n t i a l colony s c r e e n i n g of w i l d - t y p e mice minus a s t r a i n homozygous f o r r e t i n a l (rd), rd  degeneration  p h o t o r e c e p t o r s p e c i f i c cDNA c l o n e s were o b t a i n e d .  The  d e f e c t , which i s u n r e l a t e d t o rds and maps t o chromosome  5, i s c h a r a c t e r i z e d by a r a p i d d e g e n e r a t i o n o f the p h o t o r e c e p t o r c e l l s t h a t i s complete w i t h i n f o u r weeks a f t e r birth  (Carter-Dawson e t a l . ,  1978). A l l o t h e r r e t i n a l  cell  types remain u n a f f e c t e d . A panel o f mouse x hamster h y b r i d c e l l - l i n e DNAs was  used t o determine  the chromosome  assignments o f the d i f f e r e n t p h o t o r e c e p t o r s p e c i f i c cDNA c l o n e s . One  c l o n e t h a t mapped t o chromosome 17, the same  l o c a t i o n as the rds d e f e c t , was  i d e n t i f i e d . Genomic c l o n e s  were i s o l a t e d from l i b r a r i e s made from both w i l d type rds  and  mice u s i n g t h i s cDNA c l o n e as a probe. The genomic c l o n e  from the rds s t r a i n was  found t o c o n t a i n 10 kb of  DNA  i n s e r t e d i n t o i t t h a t are not p r e s e n t i n the w i l d - t y p e gene. Northern b l o t a n a l y s i s r e v e a l e d t h a t normal s i z e d t r a n s c r i p t s were not produced  from t h i s gene. On t h i s b a s i s ,  the p h o t o r e c e p t o r s p e c i f i c cDNA c l o n e mapping t o chromosome 17 was  i d e n t i f i e d as b e i n g the gene r e s p o n s i b l e f o r the rds  d e f e c t . N e i t h e r the gene product nor i t s l o c a l i z a t i o n w i t h i n the p h o t o r e c e p t o r c e l l had been  determined.  -17-  Thesis  Investigation  The l o c a l i z a t i o n o f p h o t o r e c e p t o r c e l l  peripherin  s p e c i f i c a l l y t o t h e r i m r e g i o n o f t h e d i s k membrane introduced  several questions.  How does t h i s l o c a t i o n i n t h e  d i s k membrane r e l a t e t o i t s f u n c t i o n ?  Is i t a s t r u c t u r a l  p r o t e i n t h a t i s somehow i n v o l v e d i n t h e formation  of the  ordered a r r a y o f d i s k membranes w i t h i n t h e o u t e r  segment?  Does i t have anything  t o do w i t h r i m formation  on t h e d i s k  membranes? The d i f f u s i o n o f rhodopsin throughout t h e d i s k membrane i s necessary f o r i t s f u n c t i o n  (Leibman & Pugh,  1981). I s p e r i p h e r i n sequestered i n t o t h e r i m r e g i o n o f the d i s k membrane so t h a t i t w i l l not i n t e r f e r e w i t h rhodopsin? Does p e r i p h e r i n have a r o l e i n the v i s u a l e x c i t a t i o n p r o c e s s ? How does p r o t e i n s o r t i n g take p l a c e among t h e r i m and  lamellar regions  o f t h e d i s k membrane and t h e plasma .  membrane o f t h e outer segment?  T h i s study i s an i n i t i a l  attempt t o p a r t i a l l y answer some o f these q u e s t i o n s . The work i n v o l v e s t h e m o l e c u l a r c l o n i n g , e x p r e s s i o n and characterization of peripherin.  -18-  MATERIALS and METHODS  Materials General l a b o r a t o r y reagents were o b t a i n e d from Chemicals (Toronto, O n t a r i o ) , F i s h e r S c i e n t i f i c  BDH  (Ottawa,  Ontario) o r Sigma Chemical Company (St. L o u i s , MO). r e s t r i c t i o n enzymes and the o t h e r DNA  The  m o d i f y i n g enzymes were  purchased from Bethesda Research L a b o r a t o r i e s  (Burlington,  O n t a r i o ) , Boehringer Mannheim (Dorval, Quebec),  Pharmacia  (Baie d'Urfe, Quebec) o r The U n i t e d S t a t e s B i o c h e m i c a l Corporation  ( C l e v e l a n d , OH). The .XgtlO and the / g t l l  l i b r a r i e s were the generous g i f t s o f Dr. Jeremy Nathans (John Hopkins) and Dr. Dan Oprian (Brandeis) r e s p e c t i v e l y . The BA85 S c h l e i c h e r & S c h u e l l n i t r o c e l l u l o s e f i l t e r s  that  were used f o r the plaque l i f t s were o b t a i n e d from Chemonics Scientific  L t d . (Richmond,  B.C.), and the n y l o n  (Hybond) were o b t a i n e d from Amersham ( O a k v i l l e ,  filters Ontario).  The Gene Clean k i t was purchased from B i o 101 Inc. (La J o l l a , CA). O l i g o ( d T ) - c e l l u l o s e from C o l l a b o r a t i v e Research  (type 3) was  purchased  (Bedford, MA). A l l  o l i g o n u c l e o t i d e s were s y n t h e s i z e d by Dr. Tom A t k i n s o n ( U . B . C ) . The r a d i o a c t i v e i s o t o p e s and the Quanta I I I i n t e n s i f y i n g screens were o b t a i n e d from Dupont (Markham, Ontario). Bovine r e t i n a were e i t h e r purchased f r o z e n from Hormel ( A u s t i n , MN)  o r o b t a i n e d f r e s h from J & L Meats (Surrey,  B.C.). Cyanogen bromide and  HPLC grade a c e t o n i t r i l e were  -19-  purchased from F i s h e r S c i e n t i f i c . The HPLC grade trifluoroacetic  a c i d was o b t a i n e d from P i e r c e (Rockford,  I L ) . Acrylamide, ammonium p e r s u l f a t e , N-N'  methylene  b i s a c r y l a m i d e and TEMED were o b t a i n e d from B i o - Rad (Richmond,  CA). The Immobilon  paper and the r e v e r s e phase  C18 HPLC column were o b t a i n e d from M i l l i p o r e  (Mississauga,  O n t a r i o ) . N - Glycanase F was o b t a i n e d from Genzyme (Boston, MA)  and Endo H from Seikagaku Kogyo (Tokyo, J a p a n ) . C e l l  c u l t u r e media,  f e t a l c a l f s e r a , t r y p s i n , p e n i c i l l i n and  s t r e p t o m y c i n were o b t a i n e d from Gibco ( B u r l i n g t o n , DEAE - d e x t r a n (MW  Ontario).  500,000) was o b t a i n e d from Pharmacia and  c h l o r o q u i n e diphosphate from Sigma.  Screening of the retinal libraries A bovine r e t i n a l cDNA l i b r a r y t h a t was p r e p a r e d i n the phage e x p r e s s i o n v e c t o r jLgt 11 ( B a r r e t t e t a l . , 1985) screened u s i n g monoclonal a n t i b o d i e s T h i s l i b r a r y was  was  (Young & Davis, 1983).  a m p l i f i e d from 3.5 x 1 0  5  independent  c l o n e s . The average s i z e o f the o l i g o dT primed cDNA i n s e r t s t h a t were used i n the c o n s t r u c t i o n o f t h e l i b r a r y was l k b . One complete l i b r a r y was p l a t e d on E s c h e r i c h i a c o l i Y1090 [ lacU169  Ion p r o A  180 phage/cm  2  +  araD139 s t r A supF  (pMC9)] a t a d e n s i t y o f  and i n c u b a t e d a t 42°C f o r 3 hours.  N i t r o c e l l u l o s e f i l t e r s t h a t had been s a t u r a t e d w i t h i s o p r o p y l - l - t h i o - B - D - g a l a c t o p y r a n o s i d e were o v e r l a i d on the plaques, and the p l a t e s were i n c u b a t e d a t 37°C f o r 5 t o 8 hours. The f i l t e r s were quenched  i n 3% BSA and then  -20-  i n c u b a t e d w i t h a mixture o f t h e a n t i p e r i p h e r i n  monoclonal  a n t i b o d i e s 2B6 and 3B6 (Molday e t a l . , 1987). The  filters  were r e a c t e d w i t h goat antimouse Ig t h a t had been  labelled  with  1  2  5  i  to a s p e c i f i c a c t i v i t y of 4xl0  dpm/mg u s i n g t h e  9  chloramine T method (Hunter & Greenwood, 1962). A f t e r both the  primary and secondary a n t i b o d y i n c u b a t i o n s , t h e f i l t e r s  were washed 5 times w i t h a s o l u t i o n c o n t a i n i n g 150 mM NaCl, 10 mM Na HP0 2  4  and 0.05% Tween 20. Autoradiography was  c a r r i e d out f o r approximately 18 hours. P o s i t i v e  signals  were plaque p u r i f i e d by f o u r a d d i t i o n a l rounds o f s c r e e n i n g s a t s u c c e s s i v e l y lower phage  dilutions.  cDNA t h a t was used as probes i n l a t e r s c r e e n i n g s o f t h e same  Agtll  l i b r a r y and a ^ g t l O l i b r a r y  1983) was l a b e l l e d  (Nathans & Hogness,  u s i n g c < - p - A T P and random hexamer 32  primers a c c o r d i n g t o t h e method o f F e i n b e r g and V o g e l s t e i n (1983). The s p e c i f i c a c t i v i t y o f t h e s e probes was approximately  5 x 10  7  cpm/pmol. The ^ g t l O  a m p l i f i e d from 2.5 x 1 0  5  l i b r a r y had been  independent c l o n e s . O l i g o dT primed  cDNAs g r e a t e r than 500 bp were used i n i t s c o n s t r u c t i o n . The degenerate o l i g o n u c l e o t i d e mixture t h a t was used t o s c r e e n theyCgtlO  l i b r a r y was 5' end l a b e l l e d  u s i n g 2 f - P - A T P and T4 32  p o l y n u c l e o t i d e k i n a s e ( M a n i a t i s e t a l . , 1982). On average, the  e n d - l a b e l l e d probes had a s p e c i f i c a c t i v i t y o f 1-2 x 1 0  cpm/pmol. Hybond n y l o n membrane f l t e r s make t h e plaque l i f t s  (Amersham) were used t o  f o r t h e DNA h y b r i d i z a t i o n s .  After  d e n a t u r a t i o n , t h e DNA was UV c r o s s - l i n k e d t o t h e f i l t e r s .  6  -21-  The f i l t e r s were p r e h y b r i d i z e d f o r 2 hours a t t h e h y b r i d i z a t i o n temperature i n a s o l u t i o n c o n t a i n i n g 6 x SSC (1 x SSC = 0.15 M NaCl, 0.015 M sodium c i t r a t e pH 7.0), 2 x Denhardts  (1 x Denhardts = 0.02% F i c o l l  400, 0.02%  p o l v i n y l p y r r o l i d o n e 360, 0.02% BSA), 0.5% SDS and 1 mM EDTA. The p r e h y b r i d i z a t i o n s o l u t i o n was removed and r e p l a c e d w i t h f r e s h s o l u t i o n c o n t a i n i n g 0.5 - 1 x 1 0  6  cpm p e r ml o f  l a b e l l e d probe. The h y b r i d i z a t i o n s w i t h t h e l a b e l l e d cDNA probes were c a r r i e d out a t 68° C f o r 12 hours, and t h e h y b r i d i z a t i o n s w i t h t h e l a b e l l e d degenerate o l i g o n u c l e o t i d e mixture were c a r r i e d out a t 40° C f o r 18 hours. The f i l t e r s were washed a t t h e h y b r i d i z a t i o n temperature f o r s e v e r a l hours w i t h s e v e r a l changes o f a s o l u t i o n c o n t a i n i n g 6 x SSC, .5% SDS and 1 mM EDTA. Autoradiography was done a t -60° C using the i n t e n s i f y i n g screens.  Isolation of phage DNA and sequencing Recombinant phage DNA was i s o l a t e d by t h e procedure o f M a n i a t i s e t a l (1982). EcoRl d i g e s t e d  DNA was f r a c t i o n a t e d  on an agarose g e l , and t h e i n s e r t DNA was p u r i f i e d w i t h t h e Gene C l e a n k i t which uses t h e g l a s s powder e l u t i o n method of V o g e l s t e i n and G i l l e s p i e  (1979). P u r i f i e d i n s e r t was  l i g a t e d i n t o M13mpl8 o r M13mpl9 f o r sequencing by t h e d i d e o x y n u c l e o t i d e c h a i n t e r m i n a t i o n method (Sanger e t a l . , 1977) u s i n g e i t h e r t h e Klenow fragment o f DNA polymerase I or m o d i f i e d T7 DNA polymerase. Both s t r a n d s o f t h e r e g i o n  -22-  coding  f o r t h e open r e a d i n g f r a m e were s e q u e n c e d  to  completion.  Northern blot analysis T o t a l RNA  was  isolated  from b o v i n e r e t i n a s  t h e method o f C h i r g w i n e t a l . ( 1 9 7 9 ) . The dissected  according to  retinas  from t h e eyes a t t h e s l a u g h t e r house  immediately  frozen  in liquid  n i t r o g e n . The  (10 g) were p l a c e d  i n 100 ml  of homogenizing  g u a n i d i n e - HC1, dithiothreitol) a Polytron  0.025M s o d i u m  citrate  and  and  frozen  10  and d i s r u p t e d w i t h t h r e e 15 s e c o n d b u r s t s  f o r RNA  w i t h one  t h i r d v o l u m e o f 95% e t h a n o l f o l l o w e d by  successively  through several  retinal  RNA  & Leder,  was  RNA  was  was  transferred  t r a n s f e r s were h y b r i d i z e d f o r m a m i d e , 0.1%  SDS,  ( l x SSPE = 180 mM 42°C w i t h 1 0  6  of  was  extraction solution.  from  the column  ug o f p o l y a d e n y l a t e d  Bovine l i v e r  RNA  was  o f W a l t e r Funk.  s e p a r a t e d on a 1.2% and  M  precipitation  t h r o u g h an o l i g o ( d T ) - c e l l u l o s e  1 9 7 2 ) . A p p r o x i m a t e l y 700  gift  formaldehyde  of  then p u r i f i e d  o b t a i n e d f r o m 10 g o f t i s s u e .  the generous RNA  rounds  sample  s m a l l e r volumes o f homogenization  m i x t u r e by p a s s a g e (Aviv  (7.5  mM  ( K i n e m a t i c a ) s e t a t medium s p e e d . The  Polyadenylated  retinas  solution  enriched  in  were  100  NaCl,  dpm/ml o f  agarose g e l c o n t a i n i n g  t o Hybond n y l o n membrane.  i n 5x D e n h a r d t s , ug/ml s a l m o n  10 mM 3 2  P  2.2M  NaH P0 , 2  labelled  membranes were t h e n washed a t 70°C  4  9% d e x t r a n ,  sperm DNA, ImM  The  and  EDTA, pH  cDNA p r o b e s . i n . l x S S P E and  43%  5x  SSPE  7.4)  at  The .1%  SDS.  -23-  Immunoaffinity  Column Preparation  The antiperipherin monoclonal antibody 2B6 was  purified  from ascites f l u i d by p r e c i p i t a t i o n with 50% ammonium s u l f a t e followed by DEAE-Sepharose chromatography. SDS polyacrylamide gel electrophoresis indicated that the antibody was e s s e n t i a l l y pure. The 2B6 monoclonal antibody was covalently coupled to Sepharose 2BC1 using a CNBr a c t i v a t i o n method (Cuatrecasas, 1970). Approximately  2 mg of  antibody was coupled to each ml of packed beads.  Rod Outer Segment Preparation Rod outer segments were prepared under dim red l i g h t from 80 frozen retinas as previously described (Molday & Molday, 1987b). B r i e f l y , 80 frozen retinas were placed i n 30 ml of a homogenization 2 mM MgCl  2  solution (2 0% sucrose, 10 mM glucose,  and 2 0 mM T r i s acetate pH 7.2)  and gently shaken  for 1 minute. This treatment breaks o f f the outer segments from the connecting ciliums. The solution was passed  through  a Teflon screen and then layered onto s i x 24 ml 25 - 60 % (wt/vol) sucrose gradients which also contained 10  mM  glucose, 2 mM MgCl , and 20 mM T r i s - acetate pH 7.2. 2  The  gradients were spun at 25,000 rpm i n a SW - 27 rotor (Beckman Instruments)  f o r one hour. The pink rod outer  segment band was collected and washed several times with homogenizing solution. Approximately obtained from the 80 retinas.  60 mg of protein were  -24-  Purification of peripherin for N-terminal sequence analysis The r o d o u t e r segments were resuspended 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 2mg/ml i n a b u f f e r c o n t a i n i n g 20 mM T r i s a c e t a t e pH 7.4, lOOmM NaCl and 0.4mM 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 . Approximately 8 mg o f t h e resuspended ROS were s o l u b i l i z e d w i t h an equal volume o f 50 mM o c t y l g l u c o s i d e o r 18 mM CHAPS and passed through 1ml o f the  2B6 antibody - Sepharose  2BC1 column. The column was  washed w i t h 20 volumes o f t h e r e s u s p e n s i o n b u f f e r and then e l u t e d w i t h e i t h e r 0.1 M a c e t i c a c i d o r 0.05 M f o r m i c a c i d . During both t h e washing  and e l u t i o n o f t h e column, t h e  d e t e r g e n t was kept a t one h a l f t h e c o n c e n t r a t i o n t h a t was used d u r i n g t h e i n i t i a l s o l u b i l i z a t i o n . The e l u t e d p r o t e i n was  then d i a l y z e d a g a i n s t 0.1% SDS and sequenced  by t h e  P r o t e i n M i c r o c h e m i s t r y Centre a t t h e U n i v e r s i t y o f V i c t o r i a . P r o t e i n q u a n t i t a t i o n was done w i t h t h e BCA p r o t e i n assay r e a g e n t ( P i e r c e ) u s i n g bovine serum albumin as t h e s t a n d a r d .  Isolation of a CNBr peripherin peptide for amino acid sequencing A f t e r washing  i n d i s t i l l e d H 0, lOmg o f t h e ROS 2  p r e p a r a t i o n were resuspended i n 0.7ml o f 98% f o r m i c a c i d , and t h e volume was a d j u s t e d t o 1ml w i t h d i s t i l l e d H 0. CNBr 2  was  added t o a f i n a l c o n c e n t r a t i o n o f 0.05g/ml, and t h e  d i g e s t was l e f t f o r 14 hours a t room temperature  i n the  dark. The mixture was d r i e d under vacuum, and t h e p e l l e t was e x t r a c t e d s e v e r a l times w i t h 0.05M NH HC0 4  3  pH 8.0.  The e x t r a c t e d CNBr c l e a v e d p e p t i d e s were passed through 2ml o f t h e 2B6 antibody - Sepharose  2BC1 column, and t h e  -25-  column was washed w i t h 15 volumes o f 0.05M NH HC0 4  3  pH 7.0.  The column was then e l u t e d w i t h 5 volumes o f 0.05M f o r m i c a c i d , and t h e e l u a t e was d r i e d under vacuum. The sample was resuspended  i n lOOul o f d i s t i l l e d H 0 c o n t a i n i n g 0.05% 2  t r i f l u o r o a c e t i c a c i d and loaded onto a r e v e r s e phase C18 HPLC column. The HPLC column was e l u t e d w i t h a d i s t i l l e d water - a c e t o n i t r i l e g r a d i e n t t h a t c o n t a i n e d 0.05% t r i f l u o r o a c e t i c a c i d . The g r a d i e n t r a n from 10% t o 80% a c e t o n i t r i l e over a p e r i o d o f 100 minutes.  Peptide e l u t i o n  was d e t e c t e d by m o n i t o r i n g t h e column e f f l u e n t a t 215 nm. Peak f r a c t i o n s were assayed f o r t h e presence o f t h e p e r i p h e r i n p e p t i d e by dot b l o t t i n g onto Immobilon paper and s c r e e n i n g w i t h t h e 2B6 monoclonal described  antibody as p r e v i o u s l y  (Molday e t a l . , 1987). The peak f r a c t i o n a s s a y i n g  p o s i t i v e f o r 2B6 antibody b i n d i n g was sequenced by Dr. Ruedi Aebersold  (Biomedical Research Center, U . B . C ) .  Deglycosylation of peripherin ROS were s o l u b i l i z e d 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 3mg/ml i n 2% SDS i n t h e presence and absence o f 0.1M d i t h i o t h r e i t o l . The s o l u b i l i z e d ROS were d i l u t e d 1:9 w i t h e i t h e r N-Glycanase F b u f f e r 1.5%  (0.2M sodium phosphate pH 8.6,  Nonidet P-40, 0.4mM 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  lOmM p h e n a n t h r o l i n e ) o r Endo H b u f f e r  (0.05M sodium  phosphate pH 5.5, 1.5% Nonidet P-40 and 0.4mM 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 ) . The d i l u t e d ROS were i n c u b a t e d a t 37° f o r 24 hours w i t h e i t h e r 9 u n i t s / m l NGlycanase F o r 0.06 u n i t s / m l Endo H. The r e a c t i o n p r o d u c t s  -26-  were separated was  detected  on an 8% p o l y a c r y l a m i d e  g e l , and p e r i p h e r i n  by western b l o t t i n g as p r e v i o u s l y  described  (Molday e t a l . , 1987).  SDS  - P o l y a c r y l a m i d e 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 The  p r o t e i n samples were combined w i t h an equal volume  of g e l l o a d i n g b u f f e r and  (5% SDS, 40% sucrose,  lOmM T r i s pH 6.8  10% 2-mercaptoethanol), and upto a maximum o f 20 ug o f  p r o t e i n was a p p l i e d t o t h e g e l w e l l s i n a maximum volume o f 2 0 u l . The r a t i o o f acrylamide t o N-N' methylene bisacrylamide was  i n the gels  (0.75mm t h i c k n e s s X 3.0 cm length)  30 t o 0.8. E l e c t r o p h o r e s i s was c a r r i e d out by u s i n g t h e  b u f f e r system o f Laemmli (1970). The p r o t e i n s were 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 from t h e p o l y a c r y l a m i d e t o Immobilon paper u s i n g a Hoefer t r a n s b l o t apparatus  gels (model  TE22). The t r a n s f e r s were c a r r i e d out f o r 30 minutes a t 0.30 amps i n a b u f f e r c o n t a i n i n g 20 mM T r i s pH 7.4, 2 mM EDTA, 0.01%  SDS and 10% methanol.  Subcloninq  o f t h e Complete Coding Sequence o f P e r i p h e r i n  i n t o pAX 111 - a COS C e l l E x p r e s s i o n  Vector  A schematic diagram showing t h e s i g n i f i c a n t f e a t u r e s of the pAX 111 v e c t o r  (R. Kay & R.K. Humphries - p e r s o n a l  communication) i s shown on f i g u r e 7. The v e c t o r  contains  both t h e C o l E l and SV40 o r i g i n s o f r e p l i c a t i o n . The DNA coding  f o r t h e complete open r e a d i n g  frame o f p e r i p h e r i n was  l i g a t e d i n t o t h e BamHI c l o n i n g s i t e s which p u t t h e  -27-  e x p r e s s i o n of p e r i p h e r i n under the c o n t r o l of the cytomegalo v i r u s promoter. The v e c t o r c o n t a i n i n g the p e r i p h e r i n i n s e r t was  i n t o E . c o l i DH5»<p3,  transformed  a d e r i v a t i v e o f E.  DH5«*. c o n t a i n i n g the p 3 plasmid. The p 3 plasmid c o n s t i t u t i v e kanamycin r e s i s t a n c e , and  coli  contains  i t a l s o c a r r i e s amber  mutants of a m p i c i l l i n and t e t r a c y c l i n e r e s i s t a n c e . These amber mutants become a c t i v e f o l l o w i n g t r a n s f o r m a t i o n pAX  1 1 1 which c o n t a i n s the Sup Approximately  with  F gene.  2 0 0 c o l o n i e s were o b t a i n e d  from the  t r a n s f o r m a t i o n of competent DH5o<p3 c e l l s w i t h 4 . 5 ng o f the l i g a t e d v e c t o r . The t r a n s f o r m a t i o n mixture presence of 4 0 ug/ml a m p i c i l l i n ,  was  p l a t e d i n the  2 5 ug/ml kanamycin and 8  ug/ml t e t r a c y c l i n e . The o r i e n t a t i o n of the i n s e r t e d p e r i p h e r i n sequence r e l a t i v e t o the cytomegalo promoter  was  determined by r e s t r i c t i o n mapping u s i n g a B g l l l d i g e s t and an EcoRI - Kpnl double d i g e s t . Transformants c o n t a i n i n g both o r i e n t a t i o n s of the i n s e r t were i s o l a t e d , and the DNA was  C s C l p u r i f i e d a c c o r d i n g t o the method d e s c r i b e d by  Maniatis et a l . ( 1 9 8 2 ) . plasmid  COS  plasmid  DNA was  Approximately  1 0 0 ug o f p u r i f i e d  obtained from a 2 0 0 ml c u l t u r e .  C e l l Maintenance COS-1  cells  (Gluzman, 1 9 8 1 ) were grown on 1 0 cm  p e t r i p l a t e s i n medium A t h a t was  (Dulbecco's  supplemented with 10 % heat  minutes) f e t a l c a l f serum, 2 mM pH7.2  , streptomycin  diameter  m o d i f i e d E a g l e s medium i n a c t i v a t e d ( 5 6 ° f o r 30  glutamine,  10 mM  Hepes  ( 1 0 0 u n i t s / m l ) , and p e n i c i l l i n  (100  -28-  /3-GLOBIN POLY A SITE  F i g u r e 7. A s c h e m a t i c d i a g r a m s h o w i n g t h e s i g n i f i c a n t f e a t u r e s o f t h e pAX 1 1 1 v e c t o r . T h e BamHI a n d S m a l c l o n i n g s i t e s are adjacent t o the c y t o m e g a l o v i r u s p r o m o t e r (CMV). The v e c t o r c o n t a i n s b o t h t h e C o l E l and SV40 o r i g i n s o f replication (ORI).  -29-  u n i t s / m l ) ) . Upon r e a c h i n g 60 t o 80% c o n f l u e n c y , the c e l l s were r i n s e d once w i t h PBS KH P0 , 8 mM 2  of  Na HP0  4  0.05%  2  (137 mM  pH 7.3)  4  NaCl, 2.7 mM  K C l , 1.5  mM  and then i n c u b a t e d w i t h 2 ml  t r y p s i n f o r 3 minutes. The t r y p s i n was  quenched  w i t h 8 ml of medium A, and the c e l l s t h a t were loosened from the p e t r i p l a t e were then spun a t approximately 400g i n a IEC t a b l e - t o p c e n t r i f u g e . The c e l l p e l l e t was resuspended of  cell  either  i n f r e s h medium A f o r r e p l a t i n g or i n 2 t o 3 ml  f r e e z i n g media (medium A c o n t a i n i n g 8.3%  dimethyl  s u l f o x i d e and 10% heat i n a c t i v a t e d f e t a l c a l f serum).  The  c e l l s t h a t were t o be s t o r e d f r o z e n were a l i q u o t e d i n t o 1 ml c r y o g e n i c tubes, p l a c e d i n an i n s u l a t e d box a t -60° o v e r n i g h t and then t r a n s f e r r e d t o l i q u i d  T r a n s f e c t i o n of COS-1  N. 2  C e l l s w i t h the pAX  The t r a n s f e c t i o n procedure t h a t was Hammarskjold e t a l . (1986). The COS  111 V e c t o r used was  taken from  c e l l s were d i v i d e d  into  10 cm diameter p e t r i p l a t e s one or two days p r i o r t o the t r a n s f e c t i o n so t h a t they would be about 60% c o n f l u e n t when t r a n s f e c t e d . About 7.5 added t o 2 ml TS Na HP0 , 1 mM 2  of  4  (140 mM  MgCl  2  NaCl, 25 mM  pH 7.5)  111 p l a s m i d DNA T r i s , 5 mM  and t h i s was  + 2  or C a  + 2  was  K C l , 0.5  and then once w i t h TD  c e l l s were  (TS without  any  ) . The c e l l s were then i n c u b a t e d w i t h the DNA  DEAE-dextran s o l u t i o n a t room temperature then a t 37°C f o r 40 minutes. The DNA  mM  then added t o 2ml  TS c o n t a i n i n g 1.0 mg/ml DEAE-dextran. The COS  washed once w i t h TS Mg  ug o f the pAX  f o r 10 minutes  and  - DEAE-dextran s o l u t i o n  -30-  was removed and 4 ml o f 20% g l y c e r o l i n TS was added. The s o l u t i o n was g e n t l y s w i r l e d o f f and on t h e c e l l s minutes.  for 2  The g l y c e r o l s o l u t i o n was a s p i r a t e d o f f , and t h e  c e l l s were washed s u c c e s s i v e l y w i t h TS f o l l o w e d by medium A. The p l a t e s were then incubated f o r 5 hours a t 37°C w i t h 100 uM c h l o r o q u i n e diphosphate  i n medium A. The s o l u t i o n was  a s p i r a t e d o f f and r e p l a c e d w i t h medium A. The c e l l s were l e f t t o grow f o r 60 t o 70 hours w i t h f r e s h medium A b e i n g added t o t h e c e l l s  daily.  H a r v e s t i n g o f t h e T r a n s f e c t e d COS  Cells  The p l a t e s were washed once w i t h PBS, and then 1 ml o f l y s i s buffer  (1% CHAPS and 0.2 mg/ml  p h e n y l m e t h a n e s u l f o n y l f l u o r i d e i n PBS) was added. The p l a t e s were scraped w i t h a rubber policeman and t h e l y s i s  solution  was spun i n a microfuge f o r 15 minutes a t 4°C. The p o s t n u c l e a r supernatant  (about 1.2 ml) was t r a n s f e r r e d t o a  f r e s h tube and f r o z e n a t -60°C. A n a l y s i s o f t h e expressed p e r i p h e r i n product was done by western  blotting.  -31-  RESULTS  P u r i f i c a t i o n of P e r i p h e r i n f o r N - t e r m i n a l Sequence A n a l y s i s P e r i p h e r i n was  p u r i f i e d from rod o u t e r segments by  immunoaffinity chromatography. Western b l o t t i n g  and  radioimmune assays both i n d i c a t e d t h a t t h e r e was r h o d o p s i n contamination w i t h i n the p u r i f i e d  still  some  sample.  Depending on the p u r i f i c a t i o n , the degree of t h i s contamination was  estimated t o make up between 10% t o 50% of  the t o t a l p r o t e i n i n the p r e p a r a t i o n . The sample shown i n f i g u r e 8 i s the b e s t p r e p a r a t i o n t h a t was  o b t a i n e d , and the  r h o d o p s i n content i s approximately 10%. S i n c e r h o d o p s i n has an a c e t y l a t e d N - terminus  (Hargrave, 1977),  i t was  reasoned  t h a t t h i s i m p u r i t y would have no e f f e c t on the sequencing. The r e s u l t from the N-terminal sequence a n a l y s i s i s shown i n f i g u r e 9. The y i e l d of PTH-amino a c i d s from each c y c l e of sequencing was  approximately 10 t o 20 pmol.  I n i t i a l l y , t h e r e was  not a h i g h degree o f c o n f i d e n c e i n  the sequence a n a l y s i s . The y i e l d o f amino a c i d s c a l c u l a t e d from each c y c l e of sequencing was 100 l e s s than what was  approximately a f a c t o r of  expected. P e r i p h e r i n forms v e r y l a r g e  aggregates under non r e d u c i n g c o n d i t i o n s , and i t i s p o s s i b l e t h a t a l a r g e aggregate o f p e r i p h e r i n may  not have been  a c c e s s i b l e t o the a n a l y s i s . There i n i t i a l l y was though,  t h a t p e r i p h e r i n may  the sequence t h a t was  concern,  have a b l o c k e d N-terminus,  and  o b t a i n e d c o u l d have been from a minor  -32-  F i g u r e 8. P u r i f i c a t i o n o f p e r i p h e r i n by i m m u n o a f f i n i t y c h r o m a t o g r a p h y . A p p r o x i m a t e l y 10 mg o f r o d o u t e r s e g m e n t p r o t e i n s w e r e r e s u s p e n d e d i n 0.5 m l o f b u f f e r A (lOmM H e p e s p H 7 . 2 , 100 mM N a C l ) a n d a d d e d d r o p w i s e u n d e r d i m r e d l i g h t t o 10 m l o f b u f f e r A c o n t a i n i n g 18 mM C H A P S . T h e solubilized m a t e r i a l was d i l u t e d w i t h 8 m l b u f f e r A a n d i n c u b a t e d w i t h 2 m l o f t h e p a c k e d 2B6 a n t i b o d y - S e p h a r o s e 2 B C 1 b e a d s f o r one h o u r on a r o t a t i n g p l a t f o r m . The b e a d s w e r e t h e n p o u r e d i n t o a c o l u m n a n d w a s h e d w i t h 15 c o l u m n v o l u m e s o f b u f f e r A c o n t a i n i n g 9 mM C H A P S . T h e c o l u m n w a s e l u t e d w i t h 0.05 M f o r m i c a c i d w h i c h a l s o c o n t a i n e d 9 mM C H A P S . T o t a l r o d o u t e r s e g m e n t p r o t e i n s ( l a n e a) a n d t h e i m m u n o a f f i n i t y p u r i f i e d p e r i p h e r i n f r a c t i o n s ( l a n e s b & c ) w e r e s e p a r a t e d b y SDS p o l y a c r y l a m i d e g e l e l e c t r o p h o r e s i s i n t h e p r e s e n c e o f 2mercaptoethanol. A p p r o x i m a t e l y 10 u g o f t h e r o d o u t e r segment p r o t e i n s w e r e l o a d e d o n t o l a n e a, a p p r o x i m a t e l y 0.2 u g o f t h e f i r s t c o l u m n e l u t i o n f r a c t i o n was l o a d e d o n t o l a n e b , a n d a p p r o x i m a t e l y 0.8 u g o f t h e p e a k p e r i p h e r i n e l u t i o n f r a c t i o n was l o a d e d o n t o l a n e c. The t o t a l y i e l d o f t h e p u r i f i e d p e r i p h e r i n was a p p r o x i m a t e l y 220 u g . The l a n e s o f the g e l were e i t h e r s t a i n e d d i r e c t l y w i t h Coomassie Blue (CB) o r u s e d i n i m m u n o b l o t s . T h e i m m u n o b l o t s w e r e l a b e l l e d w i t h e i t h e r 2B6, an a n t i p e r i p h e r i n m o n o c l o n a l a n t i b o d y , o r w i t h 1C5, a n a n t i r h o d o p s i n m o n o c l o n a l a n t i b o d y .  -33-  contaminant i n the sample. The background n o i s e of t h e a n a l y s i s was  also high.  I s o l a t i o n of p e r i p h e r i n cDNA c l o n e s A mixture 2B6  and  of two  3B6 was  a n t i p e r i p h e r i n monoclonal a n t i b o d i e s  used t o screen the >*gtll bovine  l i b r a r y . Plaque s c r e e n i n g of 350  000  r e t i n a l cDNA  recombinants l e d t o the  i s o l a t i o n o f 3 c l o n e s which r e a c t e d t o both a n t i b o d i e s . Clone JL.5,  w i t h the l a r g e s t i n s e r t , has a 105 bp open  r e a d i n g frame ( F i g u r e 10) t h a t i s i n frame w i t h the £ g a l a c t o s i d a s e gene i n the / g t l l The  l i b r a r y was  from JK.5. primary  Over 300  vector.  rescreened with the complete  insert  p o s i t i v e s i g n a l s were o b t a i n e d from the  s c r e e n . Twenty o f these p o s i t i v e s were chosen a t  random and plaque p u r i f i e d . Clones A.4  and J..8,  f i g u r e 10, c o n t a i n the l a r g e s t i n s e r t s . The complete sequence o v e r l a p with X.5 o t h e r a t the 3' u n t r a n s l a t e d end.  l i b r a r y . Two  two  c l o n e s have  but d i v e r g e from each The  c o n t i n u e s t o the 5' end o f c l o n e J-.8. fragment a t the 5' end ofX.8 was  shown i n  open r e a d i n g frame The  249  bp  EcoRI-Hpall  used t o r e s c r e e n the same  a d d i t i o n a l c l o n e s X.11  and / .17 were o b t a i n e d .  These c l o n e s have complete sequence o v e r l a p w i t h the H p a l l probe but d i v e r g e from each other a t the 5' ( F i g u r e 10). An i n d e p e n d e n t / g t 1 0 bovine screened  with the 499  EcoRl-  ends  retinal library  was  bp EcoRI - P s t I fragment f r o m / . 17  and  w i t h a degenerate o l i g o n u c l e o t i d e d e r i v e d from the p u t a t i v e N-terminal  sequence of p e r i p h e r i n ( F i g u r e 9 ) . From a screen  -34-  a)  1 5 10 15 A l a Leu Leu Lys V a l Lys Phe Asp G i n Lys Lys A r g V a l Lys Leu 20 A l a G i n G l y Leu ### He  25 30 Leu Met Asn Trp Phe Ser V a l Leu A l a G l y  He  b) 5' CCA ATT CAT CAG CCA CAG G T T A A G G  CCC CTG 3' T A G  F i g u r e 9. a)The N-terminal amino a c i d sequence was determined from immunoaffinity p u r i f i e d p e r i p h e r i n . One o f t h e tryptophan r e s i d u e s , as determined from the cDNA sequence, was d e s t r o y e d d u r i n g the p r o t e i n sequencing (###) and was not d e t e c t e d , b) The degenerate o l i g o n u c l e o t i d e mixture t h a t was used t o s c r e e n a X g t l O l i b r a r y was d e r i v e d from amino a c i d s 17 t o 24.  -35-  of  500  X-18  000 recombinants,  ( F i g u r e 10), was  Clone  o n l y one p o s i t i v e s i g n a l ,  clone  o b t a i n e d which r e a c t e d t o both  J^. 18 has complete sequence o v e r l a p w i t h X. 17  bp b e f o r e d i v e r g i n g from i t a t i t s 5'  probes. f o r 234  end.  I s o l a t i o n of a C-terminal CNBr fragment o f p e r i p h e r i n f o r amino a c i d sequencing T o t a l rod o u t e r segment p r o t e i n s were d i g e s t e d w i t h CNBr, and the d i g e s t e d products were s e p a r a t e d on a r e v e r s e phase HPLC column (11a). I n d i v i d u a l column f r a c t i o n s were assayed  f o r the presence  of the C - t e r m i n a l CNBr fragment of  p e r i p h e r i n by dot b l o t t i n g onto Immobilon paper and s c r e e n i n g w i t h the 2B6 monoclonal antibody. A f t e r the d i g e s t was  passed through a 2B6  immunoaffinity column, o n l y 3 major  peaks were v i s i b l e on the chromatogram ( F i g u r e l i b ) . these chromatogram peaks was  One  of  i d e n t i f i e d as the C - t e r m i n a l  CNBr fragment of p e r i p h e r i n , one o f the peaks was  identified  as the N-terminal CNBr fragment of rhodopsin, and the o t h e r remaining peak was  left  unidentified.  P r e d i c t e d Amino a c i d sequence o f p e r i p h e r i n The sequencing  s t r a t e g y used f o r the d i f f e r e n t cDNA  c l o n e s i s o u t l i n e d i n f i g u r e 10. The complete amino a c i d sequence of p e r i p h e r i n i s shown i n f i g u r e 12A. of  the f i r s t  32 amino a c i d s of p e r i p h e r i n was  the N - t e r m i n a l sequence a n a l y s i s  The  sequence  obtained  ( F i g u r e 12A). The  from  initial  amino a c i d i n the mature p r o t e i n i s a l a n i n e i n d i c a t i n g t h a t e i t h e r a l e a d e r sequence or the i n i t i a t o r methionine  was  -36Clones J  E  I  X.S  J  E  _J  E  L.  I  J  X.4  1.8  TQA  P _i  K i  H  J..17  X.18 100 bp  F i g u r e 10. R e s t r i c t i o n map and sequencing s t r a t e g y of c l o n e d cDNA encoding p e r i p h e r i n . The r e s t r i c t i o n map shows o n l y the r e l e v a n t s i t e s f o r EcoRI (E) , H p a l l (H) , Kpnl (K) , and P s t I (P) . The i n s e r t from c l o n e X.5, o b t a i n e d from s c r e e n i n g the cDNA r e t i n a l l i b r a r y w i t h the a n t i p e r i p h e r i n monoclonal a n t i b o d i e s , was used t o r e s c r e e n the l i b r a r y . Clones J. . 4 and X.8 were i s o l a t e d . The 5' EcoRI - H p a l l fragment from X.8 was used t o r e s c r e e n the same l i b r a r y . Two a d d i t i o n a l c l o n e s jL.11 and X.17 were o b t a i n e d . The 499 bp EcoRI - P s t I fragment from X.17 and a degenerate o l i g o n u c l e o t i d e probe d e r i v e d from the N-terminal amino a c i d sequence o f p e r i p h e r i n were used t o s c r e e n an independent A g t l O l i b r a r y . Clone X-18 h y b r i d i z e d t o both probes. I d e n t i c a l sequence o v e r l a p among the c l o n e s i s r e p r e s e n t e d by the bold lines and sequence divergence by the narrow lines. The d o t t e d segment above c l o n e X.1% r e p r e s e n t s t h e sequence t h a t i s i n agreement with t h e N-terminal amino a c i d sequence. The sequence coding f o r t h e f i r s t 5 N-terminal amino a c i d s of the mature p r o t e i n i s not p r e s e n t on c l o n e X-18. The arrows r e p r e s e n t the d i r e c t i o n and extent of sequence d e t e r m i n a t i o n . The sequence was determined by the d i d e o x y n u c l e o t i d e method u s i n g the u n i v e r s a l M13 sequencing primer (solid arrow) or s y n t h e s i z e d o l i g o n u c l e o t i d e s (dashed arrow) . The p o s i t i o n o f the t e r m i n a t i o n codon (TGA) on c l o n e A. 11 and the p o s i t i o n o f the s t a r t of the open r e a d i n g frame (^r) on c l o n e X'18 a r e i n d i c a t e d .  -37F i g u r e 11. P u r i f i c a t i o n of a C - t e r m i n a l CNBr fragment of p e r i p h e r i n . a) T o t a l rod o u t e r segment p r o t e i n s were d i g e s t e d w i t h CNBr, and 10 mg of the d i g e s t e d p r o d u c t s were passed through a r e v e r s e phase C18 HPLC column. The HPLC column was e l u t e d w i t h a d i s t i l l e d water - a c e t o n i t r i l e g r a d i e n t t h a t c o n t a i n e d 0.05% t r i f l u o r o a c e t i c a c i d . The g r a d i e n t ran from 10% t o 80% a c e t o n i t r i l e over a p e r i o d of 100 minutes. Peptide e l u t i o n was d e t e c t e d by m o n i t e r i n g the column e f f l u e n t a t 215 nm. b) A f t e r d i g e s t i o n w i t h CNBr, 10 mg of the rod o u t e r segment p r e p a r a t i o n were passed through a 2B6 antibody - Sepharose 2BC1 column. The e l u t e d m a t e r i a l was then run through the HPLC column under the same c o n d i t i o n s d e s c r i b e d i n a. Peak f r a c t i o n s were assayed f o r the presence of the p e r i p h e r i n p e p t i d e by dot b l o t t i n g onto Immobilon paper and s c r e e n i n g w i t h the 2B6 monoclonal antibody as p r e v i o u s l y d e s c r i b e d (Molday e t a l . , 1987). The f r a c t i o n s were a l s o screened w i t h 4D2, a monoclonal antibody d i r e c t e d t o the N-terminus of rhodopsin. The peak f r a c t i o n a s s a y i n g p o s i t i v e f o r 2B6 b i n d i n g was sequenced.  -38-  b  2 B 6  4 D 2 £ r-cj> CM*'.  c— ^ -  o  r- t/-. \ o «  c- c<-j «-  -o a? V <^  CJ <D CD <X-  "O °~  r  1  -39-  removed. Except for the f i r s t f i v e N-terminal amino acids, the coding region of clone ,£.18 i s i n complete agreement with the amino acid sequence data. This indicated that the N-terminal amino acid sequence was that of peripherin and not a contaminant. The remaining amino acid sequence was deduced from the overlapping cDNA c l o n e s ^ . 1 1 and /.17 . As shown i n figure 12A, the amino acid sequence that was obtained d i r e c t l y from the C-terminal  CNBr fragment of  peripherin i s also i n complete agreement with the cDNA sequence. The predicted s i z e of the mature protein i s 345 amino acids (Figure 12A). There are three p o t e n t i a l asparagine linked glycosylation s i t e s (Neuberger & Marshall, 1968) within the sequence (Figure 12A). Treatment of peripherin with Endo H or N-Glycanase F causes a small decrease i n i t s mobility (Figure 13) i n d i c a t i n g the presence of N-linked carbohydrate on at least one of these s i t e s . The hydrophobicity  of the predicted peripherin sequence  was examined. The Kyte D o o l i t t l e p l o t predicts that there are four hydrophobic regions that are long enough to be potential membrane spanning domains (Figure 12B). This i s i n agreement with the s o l u b i l i t y properties of the protein which suggest that peripherin i s an i n t e g r a l membrane protein (Molday et a l . , 1987). The same hydrophobic regions were also predicted to be membrane spanning by the method of Rao and Argos using the parameters proposed i n the o r i g i n a l paper (Rao & Argos, 1986).  F i g u r e 12. Amino a c i d sequence o f p e r i p h e r i n . a) The amino a c i d sequence o f an i s o l a t e d C-terminal p e p t i d e o f p e r i p h e r i n and the N-terminal amino a c i d sequence o f immunoaffinity p u r i f i e d p e r i p h e r i n (parentheses) i s i n agreement with a conceptual t r a n s l a t i o n o f t h e cDNA sequence. Some o f t h e c y s t e i n e and tryptophan r e s i d u e s were d e s t r o y e d d u r i n g t h e p r o t e i n sequencing and were n o t d e t e c t e d . The segments t h a t a r e p r e d i c t e d by t h e h y d r o p h o b i c i t y p l o t (b) t o be p o s s i b l e membrane spanning domains a r e u n d e r l i n e d . There a r e t h r e e p o t e n t i a l a s p a r a g i n e l i n k e d g l y c o s y l a t i o n s i t e s w i t h i n t h e sequence(•) . The p a r t o f t h e sequence c o n t a i n i n g t h e a n t i g e n i c s i t e s f o r t h e a n t i p e r i p h e r i n monoclonal a n t i b o d i e s i s bracketed, b) A K y t e - D o o l i t t l e h y d r o p h o b i c i t y p l o t was done on t h e t r a n s l a t e d sequence. Hydrophobic amino a c i d s a r e p l o t t e d above t h e dashed l i n e and h y d r o p h i l i c r e s i d u e s below t h e line.  -4130 20 10 (Ala Leu Leu Lys Val Lys Phe Asp Gin Lys Lys Arg Val Lys Leu A l a Gln,Gly Leu TrpLeu Met Asn Trp Phe Ser Val Leu A l a Gly V AAA TTT GAC CAG AAG AAG CGG GTC AAG TTG GCC CAA GGG CTC TGG CTC ATG AAC TGG TTC TCC GTG TTG GCT GGT  T  60 50 40 l i e l i e ) H e Phe Gly Leu Gly Leu Phe Leu. Lys H e Glu Leu Arg Lys Arg Ser Asp Val Met Asn Asn Ser Glu Ser His Phe Val Pro TTT GTG CCC ATC ATC ATC TTC GGC TTA GGG CTG TTC CTG AAG ATT GAA CTC CGG AAG AGA AGC GAT GTG ATG AAC AAT TCT GAG AGC CAT 90 80 70 Asn Ser Leu I l e Gly Val Gly Val Leu Ser Cys Val Phe Asn Ser Leu A l a Gly,Lys H e Cys Tyr Asp A l a Leu Asp Pro Ala Lys Tyr AAT TCC TTG ATC GGG GTG GGG GTG CTG TCC TGT GTC TTC AAT TCT CTG GCT GGC AAG ATC TGT TAC GAC GCC CTG GAC CCT GCC AAG TAC 120 110 100 A l a Lys Trp Lys Pro Trp Leu Lys Pro Tyr,Leu A l a Val Cys Val Leu Phe Asn Val Vat Leu Phe Leu Val A l a Leu Cys Cys Fhe Leu GCC AAG TGG AAG CCC TGG CTG AAG CCG TAC CTG GCC GTG TGT GTC CTC TTC AAC GTG GTC CTC TTC CTG GTG GCC CTC TGC TGC TTC CTC 150 140 130 Leu.Arg Gly Ser Leu Glu Ser Thr Leu A l a His Gly Leu Lys Asn Gly Met Lys Phe Tyr Arg Asp Thr Asp Thr Pro Gly -rg Cys Phe CTG CGG GGC TCG CTG GAG AGT ACG CTG GCC CAC GGA CTC AAG AAC GGC ATG AAA TTC TAT CGG GAC ACG GAC ACC CCA GGC CGG TGT TTC 180 170 160 Met Lys Lys Thr H e Asp Met Leu Gin H e Gtu Phe Lys Cys Cys Gly Asn Asn Gly Phe Arg Asp Trp Phe Glu H e Glr. Trp • l eSer ATG AAG AAG ACC ATC GAC ATG CTG CAG ATC GAG TTC AAG TGC TGC GGC AAC AAC GGC TTT CGG GAC TGG TTT GAG ATT CAG TGG ATC AGC 210 190 200 Asn Arg Tyr Leu Asp Phe Ser Ser Lys Glu Val Lys Asp Arg H e Lys Ser Asn Val Asp Gly Arg Tyr Leu Val Asp Gly Val Fro Phe AAC CGC TAT CTG GAT TTT TCC TCC AAA GAA GTC AAA GAT CGC ATC AAG AGC AAT GTG GAC GGG CGG TAC CTG GTG GAC GGT GTC CCC TTC 240 220 230 Ser Cys Cys Asn Pro Asn Ser Pro Arg Pro Cys H e Gin Tyr Gin Leu Thr ATT> Asn Ser A l a His Tyr Ser Tyr Asp His Gin Thr Glu AGC TGC TGC AAC CCC AAC TCA CCG CGG CCC TGC ATC CAG TAC CAG CTC ACC AAC AAC TCT GCG CAC TAC AGC TAC GAT CAC CAG ACG GAG 270 250 260 Glu Leu Asn Leu Trp Leu Arg Gly Cys Arg Ala A l a Leu Leu Ser Tyr Tyr Ser Asn Leu Met ksn Thr Thr Gly A l a Val Thr Leu Leu GAG CTC AAC CTG TGG CTG CGT GGC TGC AGG GCC GCC CTG CTG AGC TAT TAC AGC AAC CTC ATG AAT ACT ACA GGC GCT GTG ACG CTC CTC (  300 280 290 Val Trp Leu Phe Glu Val Thr H e Thr Val Gly leu,Arg Tyr Leu His Thr A l a Leu Glu G l y Met [Ala Asn Pro Glu Asp Pro Glu Cys GTT TGG CTC TTT GAG GTG ACC ATC ACT GTT GGG CTA CGC TAC CTG CAC ACG GCG CTG GAA GGC ATG GCC AAC CCC GAA GAC CCT GAG TGC 330 320 310rGlu Ser Glu G l y Trp Leu)Leu Glu Lys Ser[Val Pro Glu Thr Trp Lys A l a Phe Leu Glu Ser Val Lys Lys Leu G l y Lys G l y Asn Gin GAG AGT GAG GGC TGG CTT CTG GAG AAG AGC GTG CCG GAG ACC TGG AAG GCC TTT CTG GAG AGT GTG AAG AAG CTG GGC AAG GGC AAC CAG Val G l u A l a G l u G l y G l u Asp A l a Gly G i n A l a Pro A l a A l a GlyJEND GTG GAA GCC GAG GGC GAG GAC GCA GGC CAG GCC CCG GCG GCA GGC TGA CGGCCCTGCGGCCCCCTCCCCTCTGCACACTGAAAAGTAGTGGACTCCAGG- -3'  B se -  1  60  128  188  A m i n o acid number  Z48  388  -42-  DTT  DTT  +  33 kDa -  -  +  - +  Endo H  Endo H  F i g u r e 1 3 . D e g l y c o s y l a t i o n o f p e r i p h e r i n . ROS p r o t e i n s w e r e s o l u b i l i z e d i n 2% SDS i n t h e p r e s e n c e (+DTT) o r i n t h e a b s e n c e (-DTT) o f d i t h i o t h r e i t o l . T h e s o l u b i l i z e d ROS w e r e i n c u b a t e d a t 3 7 ° C f o r 24 h o u r s i n t h e p r e s e n c e (+) o r i n t h e a b s e n c e (-) o f E n d o H. S a m p l e s w e r e s e p a r a t e d o n a n 8 % polyacrylamide gel, electrophoretically transferred t o I m m o b i l o n p a p e r a n d l a b e l e d w i t h 2B6 a n t i p e r i p h e r i n a n t i b o d y followed by I g o a t a n t i m o u s e I g f o r a u t o r a d i o g r a p h y . The m o b i l i t y s h i f t i s more e v i d e n t when, i n t h e a b s e n c e o f a reducing agent, p e r i p h e r i n migrates as a dimer. S i m i l a r r e s u l t s w e r e o b t a i n e d w h e n ROS w e r e t r e a t e d w i t h N - G l y c a n a s e F. 1  2  5  -43-  Blot Hybridization analysis Northern b l o t analysis of r e t i n a l poly-A RNA  revealed  two major t r a n s c r i p t s that hybridized to peripherin cDNA probes (Figure 14). Two  predominant bands of 6.5kb and 2.9kb  are present. Both the number and large s i z e of the peripherin t r a n s c r i p t s were unexpected from the western b l o t analysis of ROS  which had indicated that there i s only  protein of apparent molecular weight 33 kDa  one  (Molday et a l . ,  1987). The large 3'- untranslated region on some of the cDNA clones  (Figure 10), however, i s i n agreement with the  hybridization r e s u l t s . No hybridization was liver  seen to bovine  RNA.  Protein Homologies At the amino acid l e v e l , peripherin has 92.5%  sequence  i d e n t i t y to the gene i d e n t i f i e d by Travis et a l . (1989) as being responsible for the r e t i n a l degeneration slow defect i n mice (Figure 15). protein was  No s i g n i f i c a n t homology to any  other  detected i n a search of the Swiss-PROT 13 data  bank using the FASTP algorithm  (Lipman & Pearson, 1985).  Synthesis of a Construct Containing the Complete Open Reading Frame of Peripherin Since the N-terminal amino acid of the mature peripherin i s not methionine, and since the cDNA clones do not contain the complete 5' end of the gene, i t was possible that there could have been a signal peptide that had been cleaved from the nascent protein. A comparison of the complete rds  -44-  B  bp  9416 6682 4361-  2322 2027  564 -  F i g u r e 14. N o r t h e r n b l o t a n a l y s i s . RNA i s o l a t e d f r o m b o v i n e r e t i n a (A) a n d l i v e r (B) w e r e s e p a r a t e d b y f o r m a l d e h y d e agarose g e l electrophoresis, t r a n s f e r r e d t oa nylon f i l t e r and h y b r i d i z e d w i t h P l a b e l e d p e r i p h e r i n cDNA p r o b e s a s described i n "Experimental Procedures". Theseven EcoRI f r a g m e n t s f r o m c l o n e s ( . 4 a n d J-.8 ( f i g u r e 10) w e r e u s e d i n d i v i d u a l l y a s t h e h y b r i d i z a t i o n p r o b e s , and t h e y gave s i m i l a r r e s u l t s . S i z e markers were denatured H i n d l l l f r a g m e n t s o b t a i n e d f r o m X DNA. x  -45-  sequence w i t h t h e p e r i p h e r i n sequence, however, r e v e a l e d t h a t t h e i n i t i a t o r methionine was t h e o n l y amino a c i d m i s s i n g from t h e mature p r o t e i n sequence  ( F i g u r e 15). A  c o n s t r u c t c o n t a i n i n g t h e complete open r e a d i n g frame o f p e r i p h e r i n , as based on t h e N - t e r m i n a l amino a c i d  sequence  a n a l y s i s o f p e r i p h e r i n and t h e alignment w i t h t h e r d s sequence, was made from c l o n e s j L . l l , J..17,/. 18 and from a synthetic cassette  (Figure 16).  The s y n t h e t i c c a s s e t t e was designed t o c o n t a i n : BamHI and EcoRI c l o n i n g s i t e s , t h e Kozak consensus sequence f o r e f f i c i e n t i n i t i a t i o n of translation  (Kozak, 1989), t h e  c o d i n g sequence f o r t h e f i r s t s i x amino a c i d s t h a t a r e not encoded by t h e cDNA sequence, and t h e sequence  overlapping  w i t h c l o n e A 1 8 up t o t h e unique S t y l c l o n i n g s i t e .  Three  l i g a t i o n s were r e q u i r e d t o make t h e c o n s t r u c t . F i r s t , t h e c a s s e t t e was l i g a t e d w i t h t h e 612bp S t y l - H i n d l l l from ^.18  fragment  i n t o BamHI-Hindlll c u t pUC19. Second, t h e 852 bp  EcoRI - B s t E I I fragment from /..17 was l i g a t e d t o t h e 492 bp B s t E I I - EcoRI fragment f r o m / . 11 i n t o EcoRI c u t pUC19. The 341 bp BamHI - M a e l l fragment from t h e f i r s t  l i g a t i o n was  then l i g a t e d t o t h e 994 bp M a e l l - EcoRI fragment from t h e second l i g a t i o n i n t o BamHI - EcoRI c u t pUC19. The l i g a t i o n s were confirmed i n t h e f i n a l c o n s t r u c t through DNA sequencing and r e s t r i c t i o n mapping (Figure 16).  -46-  ALLKVKFDQKKRVKLAQGLWLMNWFSVLAGIIIFGLGLFLKIELRKRSD  -49  x^LLKVKFDQKKRVKLAQGLWLMNWLSVLAGIVLFSLGLFLKIELRKRSE  -50  P E R I P H E R I N - VMNNSESHFVPNSLIGVGVLSCVFNSLAGKICYDALDPAKYAKWKPWLKP  -99  PERIPHERINRDS  VMNNSESHFVPNSLIGVGVLSCVFNSLAGKICYDALDPAKYAKWKPWLKP  -100  PERIPHERIN- YLAVCVLFNWLFLVALCCFLLRGSLESTLAHGLKNGMKFYRDTDTPGRC  -149  RDS  YLAVCIFFNVILFLVALCCFLLRGSLESTLAYGLKNGMKYYRDTDTPGRC  -150  PERIPHERIN-  FMKKTIDMLQIEFKCCGNNGFRDWFEIQWISNRYLDFSSKEVKDRIKSNV  -199  RDS  FMKKTIDMLQIE FKCCGNNGFRDWFEIQWISNRYLDFS S KEVKDRIKS NV -200  PERIPHERIN-  DGRYLVDGVPFSCCNPNSPRPCIQYQLTNNSAHYSYDHQTEELNLWLRGC -249  RDS  DGRYLVDGVPFSCCNPSSPRPCIQYQLTNNSAHYSYDHQTEEIiNLWLRGC  RDS  -250  P E R I P H E R I N - RAALI^YYSNIJWTTGAVTLLVWLFEVTITVGLRYLHTALEGMANPEDPE  -299  RDS  -300  PJ^LI^YYSSLJWSMGWTLLVWLFEVSITAGLRYLHTALESVSNPEDPE  P E R I P H E R I N - CESEGWLLEKSVPETWKAFLESVKKLGKGNQVEAEGEDAGQAPAAG  -34 5  RDS  -34 6  CESEGWLLEKSVPETWKAFLESFKKLGKSNQVEAEGADAGPAPEAG  F i g u r e 15. An a l i g n m e n t o f t h e amino a c i d s e q u e n c e o f t h e mature p e r i p h e r i n (bovine) w i t h t h e proposed r d s sequence (mouse). The sequences a r e 9 2 . 5 % i d e n t i c a l . The i n i t i a t o r m e t h i o n i n e i s m i s s i n g from t h e mature p r o t e i n . The r e m a i n i n g d i f f e r e n c e s b e t w e e n t h e t w o s e q u e n c e s may b e a t t r i b u t e d t o species polymorphism.  -47-  Figure 16. A construct containing the complete open reading frame of peripherin was made from clones J..11, A 17, A.IS, and from a synthetic cassette. The relevant r e s t r i c t i o n s i t e s are shown f o r BamHI (Ba), Bst EII (Bs), EcoRI (E), H i n d l l l (H), Maell (M), and S t y l (S). Identical sequence overlap among the cDNA clones i s represented by the bold l i n e s and the sequence divergence by the narrow l i n e s . The open boxed region of clone /.18 i s J^gtlO sequence that was not removed from the cDNA insert because one of the o r i g i n a l EcoRI cloning s i t e s was missing. The t r a n s l a t i o n a l stop codon (TGA) on clone / - . l l and the sequence of clone A. 18 that overlaps with the N-terminal amino acid sequence (dashed line) are indicated. The cassette contains the Kozak consensus sequence f o r the e f f i c i e n t i n i t i a t i o n of t r a n s l a t i o n (underlined) and the sequence f o r the f i r s t s i x amino acids that are not encoded by the cDNA sequence of clone X.18. Three l i g a t i o n s were required to make the construct. F i r s t , the 612 bp S t y l - H i n d l l l fragment from clone A-18 was l i g a t e d with the cassette into BamHI H i n d l l l cut pUC19. Second, the 852 bp EcoRI - BstEII fragment from clone X.17 was l i g a t e d to the 492 bp BstEII EcoRI fragment from c l o n e / . - H into EcoRI cut pUC19. The 3 4 1 bp BamHI - Maell fragment from the f i r s t l i g a t i o n was then l i g a t e d to the 994 bp Maell - EcoRI fragment from the second l i g a t i o n into BamHI - EcoRI cut pUC19. The l i g a t i o n s i n the f i n a l construct were confirmed through r e s t r i c t i o n mapping and DNA sequencing (dashed arrows).  -48-  Bs  I  „ Ba  E  A.11  Bs  M  M  TGA  A.17  H  X.18  100 b p  SYNTHETIC  GATCC^GAATTcfcACCATGGCGCTGCTCAAAGTCAAATTTGACCAGAAGAAGCGGGTCAAGTTGG'cC GCTTAAGGTGGTACCGCGACGAGTTTCAGTTTAAACTGGTCTTCTTCGCCCAGTTCAACCGGGTTC  J—i  C ASS  E TT  E  CONSTRUCT  -49-  Expression of Peripherin i n COS c e l l s Because of the unique p o s i t i o n of peripherin i n the disk membrane, the primary s t r u c t u r a l c h a r a c t e r i s t i c s obtained from the cDNA sequence and i t s r e l a t i o n s h i p t o the rds defect, i t seems possible that peripherin could be the component i n the growing c i l i a r y membrane that i n t e r a c t s with binding s i t e s at or near the c i l i u m (Figure 3). I t i s also possible that an aggregation of the peripherin molecules that are i n i t i a l l y concentrated  at the c i l i a r y end  of the disk p a r t i c i p a t e i n the formation of the rim. To t e s t t h i s second p o s s i b i l i t y , work was directed towards expressing the complete coding sequence of peripherin i n a COS c e l l l i n e . I f the aggregation of peripherin causes rim formation  i n the disk membrane, the same aggregation of  peripherin within the COS c e l l plasma membrane could r e s u l t i n the formation of membrane i n f o l d i n g s . The expression of peripherin i n COS c e l l s should also c l a r i f y whether i n the absence of a reducing agent peripherin forms a heterodimer or a homodimer. The 1130 bp BamHI fragment from the peripherin construct (Figure 16) was l i g a t e d into the pAX 111 vector. Western blot analysis was used to detect the peripherin produced by the transfected COS-1 c e l l s (Figure 17). In the presence of a reducing agent there appears to be two bands produced by the COS c e l l s which comigrate on an SDS - polyacrylamide g e l with the peripherin p u r i f i e d from rod outer segments. The r e l a t i v e i n t e n s i t y of the two bands produced by the COS  -50-  c e l l s , however, i s d i f f e r e n t from t h e r o d o u t e r segment p e r i p h e r i n . T h i s may r e f l e c t a d i f f e r e n c e i n t h e g l y c o s y l a t i o n o f p e r i p h e r i n o r some o t h e r type o f 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 . Rhodopsin when i t i s expressed  i n COS c e l l s migrates a t a s l i g h t l y  higher  m o l e c u l a r weight from t h e r o d o u t e r segment p r o t e i n (Oprian et a l . 1987). In t h e absence o f a r e d u c i n g agent t h e expressed p e r i p h e r i n migrates as a dimer s i m i l a r t o t h e p e r i p h e r i n from t h e r o d o u t e r segments. In both t h e r e i s some remaining monomer p r e s e n t .  samples  -51-  + 2-ME  -2-ME  Figure 17. Western blot analysis of the peripherin expressed within the COS-1 c e l l s . COS-1 c e l l s were transfected with the pAX 111 vector containing the peripherin coding sequence i n either the correct (c) or wrong (d) orientation for expression. Approximately 7 u l of the COS c e l l postnuclear fractions along with 10 ug of t o t a l rod outer segment proteins (a) and 0.8 ug of the 2B6 antibody - Sepharose 2BC1 column p u r i f i e d peripherin (b) were loaded onto 8% SDS - polyacrylamide gels. The gels were run i n the presence (+2-ME) or absence (-2-ME) of 2-mercaptoethanol. The gels were then electroblotted onto Immobilon paper and screened with the 2B6 antibody followed by i goat antimouse Ig f o r autoradiography. 1  2  5  -52-  Discussion A cDNA sequence f o r p e r i p h e r i n , a membrane p r o t e i n l o c a l i z e d i n the rim o f rod o u t e r segment d i s k s , has been determined.  The  i n i t i a l cDNA c l o n e s were o b t a i n e d by  s c r e e n i n g a bovine r e t i n a l e x p r e s s i o n l i b r a r y w i t h a n t i p e r i p h e r i n monoclonal  a n t i b o d i e s . These a n t i b o d i e s have  been shown t o b i n d t o cone p h o t o r e c e p t o r membrane as w e l l  as  t o the rod d i s k membranes (Hicks & Molday - u n p u b l i s h e d r e s u l t s ) . Many o f the major p h o t o r e c e p t o r p r o t e i n s are d i f f e r e n t i n the two c e l l types  (Hurwitz e t a l . ,  1985;  Nathans e t a l . , 1 9 8 6 ; Lerea e t a l . , 1 9 8 6 ) , and i t i s p o s s i b l e t h a t t h e r e c o u l d be more than one  form o f  p e r i p h e r i n . The n o r t h e r n b l o t a n a l y s i s i s i n agreement w i t h t h i s p o s s i b i l i t y . S i n c e over 90% o f the p h o t o r e c e p t o r  cells  i n the bovine r e t i n a are rod c e l l s ,  that  i t i s more l i k e l y  the cDNA sequence p r e s e n t e d here encodes the rod form o f the p r o t e i n r a t h e r than the cone form. One  CNBr c l e a v a g e p e p t i d e  t h a t bound t o the a n t i p e r i p h e r i n monoclonal  a n t i b o d y 2B6  was  i s o l a t e d . The a n t i g e n i c s i t e f o r t h i s antibody has been determined  t o be somewhere w i t h i n the C - t e r m i n a l 3 5 amino  a c i d s o f p e r i p h e r i n . The amino a c i d sequence o b t a i n e d from the i s o l a t e d C - t e r m i n a l p e p t i d e i s i n agreement w i t h t h a t p r e d i c t e d from the cDNA sequence. I f t h e r e i s a d i f f e r e n t form o f p e r i p h e r i n i n the cones,  i t must e i t h e r be i n such a  low abundance i n the o u t e r segment p r e p a r a t i o n as not t o be d e t e c t e d , o r a l t e r n a t i v e l y i t c o u l d d i v e r g e from the rod  -53-  p r o t e i n a t a l o c a t i o n f u r t h e r removed from t h e a n t i g e n i c site. S e v e r a l d i v e r g e n t cDNA c l o n e s were i s o l a t e d t h a t do not c o n t a i n t h e 5' end o f t h e coding sequence ( F i g u r e 10). I f these c l o n e s were d e r i v e d from t r u e mRNA molecules, would g i v e r i s e t o p r o t e i n products the N-terminal  that are truncated a t  end. The t r u n c a t e d p r o t e i n products  from these c l o n e s would s t i l l  they  produced  contain the antigenic s i t e s  f o r t h e 2B6 and 3B6 a n t i p e r i p h e r i n monoclonal a n t i b o d i e s . No evidence  was obtained  products  from e i t h e r t h e N-terminal  immunoaffinity  f o r t h e e x i s t e n c e o f these  truncated  sequence a n a l y s i s o f  p u r i f i e d p e r i p h e r i n o r from t h e western  b l o t t i n g o f t o t a l outer segment p r o t e i n s . The p o s s i b i l i t y e x i s t s , however, t h a t these t r u n c a t e d forms c o u l d be present i n t h e r e t i n a a t l o c a t i o n s other then t h e p h o t o r e c e p t o r o u t e r segments. There i s no evidence immunocytochemical s t u d i e s t o support  from t h e this  possibility.  Other l a b s have had problems o b t a i n i n g complete unscrambled c l o n e s from t h e two l i b r a r i e s t h a t were used i n t h i s (G. Khorana and D. Oprian  study  - p e r s o n a l communications). I t ,  t h e r e f o r e , seems l i k e l y t h a t these 5' d i v e r g e n t r e g i o n s a r e c l o n i n g a r t i f a c t s . There a r e no consensus sequences f o r i n t r o n s p l i c e s i t e s a t t h e p o i n t s o f sequence  divergence  which would have i n d i c a t e d t h a t t h e cDNA c l o n e s had been d e r i v e d from unprocessed mRNA molecules.  I t i s possible that  i f during the construction of the l i b r a r i e s the r a t i o of EcoRI l i n k e r s t o cDNA i n s e r t was n o t i n excess,  several  -54-=  u n r e l a t e d b l u n t ended cDNA fragments c o u l d have been l i g a t e d together  within the vector.  Northern b l o t a n a l y s i s o f r e t i n a l poly-A RNA two  revealed  major t r a n s c r i p t s t h a t h y b r i d i z e d t o t h e p e r i p h e r i n cDNA  probes (Figure 14). I t i s p o s s i b l e t h a t t h e s e two bands c o u l d be t r a n s c r i p t s from two d i f f e r e n t genes. Southern b l o t a n a l y s i s o f BALB/c mouse genomic DNA suggests t h a t  there  c o u l d p o t e n t i a l l y be two genes t h a t code f o r p e r i p h e r i n o r p o s s i b l y f o r p e r i p h e r i n and a r e l a t e d p r o t e i n al.,  (Travis et  1989). A p r o t e i n t h a t has 36% amino a c i d sequence  i d e n t i t y t o p e r i p h e r i n has been i d e n t i f i e d i n t h e human retina and  (R. Mclnnes - p e r s o n a l  communication). T h i s p r o t e i n  any other p e r i p h e r i n r e l a t e d p r o t e i n would n o t be  detected  on western b l o t s i f t h e a n t i g e n i c s i t e s f o r t h e  a n t i p e r i p h e r i n monoclonal a n t i b o d i e s , which a r e l o c a t e d on the C-terminal  35 amino a c i d s , a r e not conserved.  Alternatively,  i t i s p o s s i b l e t h a t t h e two t r a n s c r i p t s c o u l d  have been t h e r e s u l t o f t h e a l t e r n a t e s p l i c i n g o f a s i n g l e gene p r o d u c t . S e v e r a l t r a n s c r i p t s t h a t a r e t h e r e s u l t o f the u t i l i z a t i o n o f d i f f e r e n t 3' s p l i c e s i t e s a r e produced f o r rhodopsin and many o f t h e n e u r a l , p r o t e i n s . The purpose f o r these d i f f e r e n t t r a n s c r i p t s i s not understood  ( J . McGinnis,  1990). A model f o r t h e o r g a n i z a t i o n o f p e r i p h e r i n i n t h e d i s k membrane i s o u t l i n e d i n f i g u r e 18. The p r o t e i n i s shown as h a v i n g f o u r transmembrane domains as p r e d i c t e d from t h e K y t e - D o o l i t t l e hydrophobicity  p l o t . As w i t h r h o d o p s i n each  -55-  of t h e hydrophobic segments i s bordered on i t s carboxy terminal  s i d e by a p o s i t i v e l y c h a r g e d r e s i d u e t h a t i s s  capable o f i n t e r a c t i n g w i t h t h e p h o s p h o l i p i d  head groups  (Nathans & Hogness, 1983). I t was p o s s i b l e t o o r i e n t a t e t h e C-terminus o f t h e p r o t e i n r e l a t i v e t o t h e membrane because the two a n t i p e r i p h e r i n monoclonal a n t i b o d i e s had been p r e v i o u s l y shown t o b i n d on t h e c y t o p l a s m i c protein  face o f the  (Molday e t a l . , 1987). The a n t i g e n i c s i t e s f o r t h e  same a n t i b o d i e s were a l s o l o c a l i z e d t o t h e C-terminal p o r t i o n o f t h e p r o t e i n by t h e cDNA c l o n e s o b t a i n e d immunological s c r e e n i n g  from t h e  o f t h e l i b r a r y . There a r e t h r e e  p o t e n t i a l asparagine l i n k e d g l y c o s y l a t i o n s i t e s p r e s e n t  on  the p r o t e i n , and i n t h e model presented i n f i g u r e 18, a l l o f t h e s e s i t e s a r e l o c a t e d w i t h i n t h e lumen o f t h e d i s k . The use  o f g l y c o s i d a s e s has i n d i c a t e d t h a t t h e r e  i s N - linked  carbohydrate on p e r i p h e r i n which i s i n agreement w i t h t h e placement o f a t l e a s t one o f these s i t e s w i t h i n t h e lumen. The  o r i e n t a t i o n o f the other  f e a t u r e s p r e s e n t e d i n t h e model  i s h i g h l y speculative since there i s only a small  database  on which t o base t h e p r e d i c t i o n o f t h e presence o f a transmembrane segment. In t h e model (Figure 18), t h e r e a r e seven c y s t e i n e r e s i d u e s p l a c e d w i t h i n t h e lumen o f t h e d i s k t h a t would have the p o t e n t i a l t o form i n t r a m o l e c u l a r  or intermolecular  d i s u l f i d e bonds. The g r e a t e r r e d u c i n g  p o t e n t i a l of the  cytoplasm i s thought t o i n h i b i t such i n t e r a c t i o n s on t h e cytoplasmic  s i d e o f t h e membrane. Molday e t a l . (1987)  -56-  demonstrated t h a t i n t h e absence o f a r e d u c i n g  agent,  p e r i p h e r i n migrates as a dimer. I t was not c l e a r a t the time whether p e r i p h e r i n i s forming a heterodimer o r a homodimer. The  p e r i p h e r i n t h a t i s expressed i n t h e COS c e l l s forms a  dimer i n t h e absence o f a reducing  agent. I t , t h e r e f o r e ,  seems l i k e l y t h a t t h e r o d o u t e r segment p e r i p h e r i n i s a l s o forming a homodimer. The p o s s i b i l i t y o f a mixture o f heterodimers and homodimers i n t h e r o d o u t e r segment, however, cannot be excluded. The  p r o t e i n encoded by t h e cDNA sequence o f p e r i p h e r i n  would have a m o l e c u l a r weight c l o s e t o 39 kDa. T h i s v a l u e i s h i g h e r than t h e 33 kDa estimated from SDS - p o l y a c r y l a m i d e g e l e l e c t r o p h o r e s i s . P e r i p h e r i n m i g r a t e s w i t h an apparent m o l e c u l a r weight c l o s e r t o 35 kDa  when i t i s p u r i f i e d by  a f f i n i t y chromatography (Figure 8 ) , when i t i s expressed i n COS c e l l s  (Figure 17) o r when t h e rhodopsin i n t h e r o d o u t e r  segment p r e p a r a t i o n s  i s removed w i t h a m i l d  aureus V-8 p r o t e a s e d i g e s t i o n  Staphylococcus,  (Molday e t a l . , 1 9 8 7 ) . I t  appears t h a t rhodopsin, which makes up approximately 70% o f the t o t a l ROS p r o t e i n , c o u l d be e f f e c t i n g t h e e l e c t r o b l o t t i n g o f p e r i p h e r i n t o t h e Immobilon paper. The large quantity  o f rhodopsin c o u l d be s a t u r a t i n g most o f the  s i t e s on t h e Immobilon paper so t h a t only t h e l e a d i n g edge of t h e p e r i p h e r i n band i s t a n s f e r r e d . As a r e s u l t , t h e p e r i p h e r i n i n t h e t o t a l ROS p r o t e i n appears as a narrower band and appears t o migrate f a s t e r on an SDS p o l y a c r y l a m i d e g e l then t h e immunoaffinity p u r i f i e d  protein  -57-  F i g . 18. A s t r u c t u r a l model o f p e r i p h e r i n .  The model shows  the o r i e n t a t i o n o f the p r o t e i n w i t h i n the d i s k membrane. The 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 o f t h e a n t i p e r i p h e r i n monoclonal a n t i b o d i e s  (Mab) i s i n d i c a t e d .  The t h i r t e e n  c y s t e i n e r e s i d u e s o f the sequence a r e h i g h l i g h t e d . The negatively  (•) and p o s i t i v e l y (0)  charged amino a c i d s and  the t h r e e p o t e n t i a l s i t e s f o r asparagine l i n k e d glycosylation  (^) a r e a l s o  shown.  Intradisk (Lumen) side  -59-  ( F i g u r e 8 ) . T h i s e f f e c t i s a l s o observed i n a comparison o f lane a o f f i g u r e 17 i n both t h e presence and absence o f a s u l f h y d r y l reducing  agent. In t h e presence o f a  reducing  agent, p e r i p h e r i n comigrates w i t h rhodopsin and t h e e f f i c i e n c y o f t r a n s f e r i s s i g n i c a n t l y l e s s then when i n the absence o f a reducing  agent, p e r i p h e r i n , u n l i k e  rhodopsin,  m i g r a t e s as a dimer. During t h e immunoaffinity p u r i f i c a t i o n o f p e r i p h e r i n , s e v e r a l h i g h e r m o l e c u l a r weight bands a r e v i s i b l e on t h e western b l o t s  (Figure 8).  These bands a r e t h e r e s u l t o f an  a g g r e g a t i o n o f p e r i p h e r i n t h a t occurs d u r i n g t h e p u r i f i c a t i o n and a r e not u s u a l l y observed i n western b l o t s o f t o t a l ROS p r o t e i n o b t a i n e d from f r e s h l y d i s s e c t e d r e t i n a . S i n c e t h e presence o f some o f t h e h i g h e r m o l e c u l a r weight bands a r e not observed i n t h e presence o f a s u l f h y d r y l reducing  agent (Figure 17), some o f these aggregates may  have formed as a r e s u l t o f t h e formation o f d i s u l f i d e bonds between a d j a c e n t p e r i p h e r i n molecules. Peripherin, s i g n a l peptide.  l i k e rhodopsin does not have any obvious Very l i t t l e  i s known about how p r o t e i n  s o r t i n g takes p l a c e among t h e r o d o u t e r segment plasma membrane, t h e l a m e l l a r r e g i o n o f t h e d i s k membrane and t h e rim r e g i o n o f t h e d i s k membrane. P r o t e i n s o r t i n g among some of t h e c e l l u l a r o r g a n e l l e s has been shown t o be d i r e c t e d by s p e c i f i c amino a c i d sequences. Membrane t a r g e t i n g sequences have been i d e n t i f i e d f o r t h e t r a n s p o r t a t i o n o f p r o t e i n s the endoplasmic r e t i c u l u m  (Munro & Pelham, 1987), t h e  into  -60-  mitochondria (Hartl et a l . , 1989), and the nucleus (GomezMarquez & Segade, 1988). The disk membranes of the rod outer segments could be viewed as c e l l u l a r organelles. I f the peripherin that i s being expressed i n the COS c e l l s i s found to be l o c a l i z e d to the plasma membrane, i t would suggest that there i s not a disk s p e c i f i c signal sequence and that protein sorting among the 3 membrane domains occurs a f t e r the i n i t i a l i n s e r t i o n into the plasma membrane. Photoreceptor c e l l peripherin at the amino acid l e v e l was shown to have 92.5% sequence i d e n t i t y to the gene proposed to be responsible for the rds defect. Most of the differences between the two sequences represent  fairly  conservative changes, and these changes are scattered throughout the sequence. One of the consensus sequences f o r asparagine linked glycosylation i s not conserved i n the rds sequence. This suggests that t h i s s i t e may not be u t i l i z e d i n peripherin. In some locations where there i s a major amino acid change there also tends to be a nearby compensatory change. In the cytoplasmic  C-terminal  domain,  the s u b s t i t u t i o n of an alanine f o r a glutamic acid residue at amino acid p o s i t i o n 336 i s compensated by the opposite substitution at p o s i t i o n 345. This i s suggestive that the conservation of the net negative charge on t h i s part of the protein may be important f o r the function of peripherin. The rds gene product and i t s i n t r a c e l l u l a r l o c a l i z a t i o n had not previously been i d e n t i f i e d .  -61-  The  f u n c t i o n o f p e r i p h e r i n i s n o t known. The  l o c a l i z a t i o n of peripherin t o the rim region of the disk membrane and t h e phenotype a s s o c i a t e d w i t h t h e r d s d e f e c t i s suggestive  t h a t i t may p l a y a r o l e i n a n c h o r i n g t h e d i s k s t o  the c y t o s k e l e t a l system o f t h e r o d c e l l . The cytoplasmic  C-terminal  domain o f p e r i p h e r i n i s h i g h l y charged, and i t  i s p o s s i b l e t h a t i t may be i n t e r a c t i n g w i t h a c y t o s k e l e t a l component. E l e c t r o n m i c r o s c o p i c t h e r e a r e filamentous the d i s k s  s t u d i e s have i n d i c a t e d t h a t  s t r u c t u r e s extending from t h e rims o f  (Roof & Heuser, 1982), and a 240 kDa s p e c t r i n -  l i k e p r o t e i n has been suggested t o p l a y a r o l e i n d i s k membrane i n t e r a c t i o n s (Wong & Molday, 1986). A l t e r n a t i v e l y , charge r e p u l s i o n among adjacent cytoplasmic  p e r i p h e r i n molecules on the  s u r f a c e may cause c u r v a t u r e  i n the disk  membrane, and t h i s c o u l d a i d i n t h e formation  of the rim  region. I n t e r a c t i o n s o f t h e s u l f h y d r y l groups o r carbohydrate chains t h a t a r e p r e s e n t  on p e r i p h e r i n w i t h i n t h e lumen o f  the d i s k may a l s o h e l p t o form t h e d i s k rim.  Disk  morphogenesis i n Xenopus and f r o g r e t i n a has been shown t o be d i s r u p t e d by tunicamycin, an i n h i b i t o r o f N-1inked glycosylation  ( F l i e s l e r e t a l . , 1984; F l i e s l e r e t a l . ,  1985a). In tunicamycin - t r e a t e d r e t i n a , membrane v e s i c l e s are formed i n t h e space between the r o d i n n e r and o u t e r segments where d i s k formation  would normally o c c u r  (Fliesler  e t a l . , 1985a). F l i e s l e r e t al.,(1985b) suggested t h a t t h e carbohydrate chains  o f rhodopsin on t h e opposing d i s k  -62-  membrane f a c e s may i n t e r a c t and cause membrane adhesion. The i n t e r a c t i o n o f t h e opposing membrane f a c e s appears t o be g r e a t e s t near t h e r i m o f t h e d i s k . Although r h o d o p s i n and o t h e r d i s k p r o t e i n s may be i n v o l v e d i n membrane adhesion, the l o c a l i z a t i o n o f p e r i p h e r i n t o t h e r i m makes i t a prime c a n d i d a t e as t h e major adhesive molecule i n t h e membrane.  -63-  Conclusion  In t h i s study the cDNA sequence of peripherin determined. This sequence was  was  confirmed from both the amino  acid sequence of an isolated C-terminal  CNBr fragment of  peripherin and from the N-terminal amino acid sequence analysis. Peripherin was  shown to be an i n t e g r a l membrane  protein that has at least one membrane spanning domain and possibly as many as four. Peripherin was  demonstrated to be  a glycoprotein. The C-terminus of peripherin was to the cytoplasmic  localized  side of the disk membrane, and a model  for the organization of peripherin within the disk membrane was  proposed. Peripherin was  shown to be able to form a  homodimer i n the absence of a reducing agent, and i t was also shown to be the defective protein responsible for the r e t i n a l degeneration slow defect. On the basis of the l o c a l i z a t i o n of peripherin to the rim region of the disk membrane, the primary s t r u c t u r a l c h a r a c t e r i s t i c s of peripherin and the phenotype associated with the r e t i n a l degeneration slow defect, i t was that peripherin may was  proposed  play a role i n disk morphogenesis. I t  suggested that peripherin could serve to anchor the  disks to the cytoskeletal system of the outer segment. Future studies might be directed towards i d e n t i f y i n g t h i s hypothetical cytoskeletal element. I t was  also suggested  that an aggregation of peripherin within the disk membrane  -64-  could a i d i n the formation of the disk rim. To t e s t t h i s p o s s i b i l i t y peripherin has been expressed i n a COS c e l l l i n e . I f the aggregation of peripherin causes rim formation i n the disk membrane, the same aggregation of peripherin within the COS c e l l plasma membrane could r e s u l t i n the formation of membrane i n f o l d i n g s . Work i n the future could be directed towards investigating t h i s p o s s i b i l i t y . I f the expressed peripherin i s l o c a l i z e d to the plasma membrane of the COS c e l l , i t would also have implications f o r protein sorting within the outer segment.  -65-  REFERENCES A v i v , H., and Leder, P. (1972) Proc. N a t l . Acad. S c i . U.S.A. 6 9 , 1408-1412. B a r r e t t , D. J . , Redmond, T. M., Wiggert, B., Oprian, D. D., Chader, G. J . , and Nickerson, J . M. (1985) Biochem.Biophys. Res. Commun. 1 3 1 , 1086-1093. 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