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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 University of 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 of Biochemistry) We accept t h i s thesis as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA June 1990 Gregory James Connell, 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 Y/^/tffi DE-6 (2/88) - i i -Abstract Peripherin, a membrane protein with an apparent molecular weight of 34 kDa, has been previously l o c a l i z e d to the rim region of the vertebrate rod photoreceptor disk membrane using monoclonal antibodies and immunocytochemical l a b e l l i n g techniques. As an i n i t i a l step i n determining the structure and function of t h i s protein, cDNA containing i t s coding sequence has been cloned and sequenced. A bovine r e t i n a l / g t l l expression l i b r a r y was screened with antiperipherin monoclonal antibodies, and a 583 base p a i r clone was i n i t i a l l y i s o l a t e d . The remaining part of the coding sequence was obtained from subsequent rescreenings of the same l i b r a r y and an independent XgtlO l i b r a r y . A C-terminal CNBr fragment of peripherin was p u r i f i e d by immunoaffinity chromatography and reverse phase high-performance l i q u i d chromatography. The amino acid sequence of the i s o l a t e d C-terminal peptide and the N-terminal sequence analysis of immunoaffinity p u r i f i e d peripherin are i n agreement with the cDNA sequence. At the amino acid l e v e l , the sequence of peripherin has 92.5% sequence i d e n t i t y to the gene proposed to be responsible for the r e t i n a l degeneration slow defect i n mice (Travis et al.(1989) Nature 338, 70-73) The differences between the two sequences can be attributed to species differences. The i d e n t i t y of the r e t i n a l degeneration slow - i i i -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 previously unknown. The cDNA sequence of peripherin predicts that there are possibly four transmembrane domains. On the basis of immunocytochemical studies and sequence analysis, the hydrophilic C-terminal segment containing the antigenic s i t e s f o r the antiperipherin monoclonal antibodies has been l o c a l i z e d on the cytoplasmic side of the disk membrane. There are three consensus sequences for asparagine linked glycosylation. Deglycosylation studies have indicated that at l e a s t one of these s i t e s i s u t i l i z e d . The complete coding sequence of peripherin was expressed i n COS-1 c e l l s . Western bl o t analysis of the expressed peripherin suggest that i t e x i s t s as a homodimer i n the absence of a reducing agent. The possible function of peripherin i n r e l a t i o n to i t s primary structure i s discussed. - i v -TABLE OF CONTENTS page I. ABSTRACT i i II . TABLE OF CONTENTS — : i v II I . LIST OF FIGURES • v i i IV. LIST OF ABBREVIATIONS v i i i V. ACKNOWLEDGEMENT — ix VI. INTRODUCTION A. The Vertebrate Retina 1 B. The Outer Segment 3 C. Vis u a l E x c i t a t i o n • 5 D. Rod Outer Segment Proteins 10 E. Photoreceptor C e l l Peripherin 11 F. Retinal Degeneration Slow 14 G. Thesis Investigation 17 VII. MATERIALS AND METHODS A. Materials : 18 B. Screening of the Retinal L i b r a r i e s 19 C. I s o l a t i o n of Phage DNA and Sequencing 21 D. Northern Blot Analysis 22 E. Immunoaffinity Column Preparation 2 3 F. Rod Outer Segment Preparation 2 3 G. P u r i f i c a t i o n for N-terminal Sequence Analysis 24 H. Is o l a t i o n of a CNBr Peripherin Peptide for Amino Acid Sequencing 24 I. Deglycosylation of Peripherin 2 5 -v-J . SDS - Polyacrylamide Gel Electrophoresis and Gel Transfer 2 6 K. Subcloning of the Complete Coding Sequence of Peripherin into pAX 111 -a COS C e l l Expression Vector 26 L. COS C e l l Maintenance 27 M. Transfection of COS-1 C e l l s with the pAX 111 Vector 29 N. Harvesting of the Transfected COS C e l l s 3 0 VIII. RESULTS A. P u r i f i c a t i o n of Peripherin for N-terminal Sequence Analysis 31 B. I s o l a t i o n of Peripherin cDNA Clones 3 3 C. I s o l a t i o n of a C-terminal CNBr Fragment of Peripherin for Amino Acid Sequence Analysis -3 5 D. Amino Acid Sequence of Peripherin 35 E. Blot Hybridization Analysis 43 F. Protein Homologies • 43 G. Synthesis of a Construct Containing the Complete Open Reading Frame of Peripherin 4 3 G. Expression of Peripherin i n COS C e l l s 49 IX. DISCUSSION 52 X. CONCLUSION 63 - v i -XI REFERENCES 65 - v i i -L I S T OF FIGURES Page F i g u r e 1. N e u r a l C e l l s o f t h e R e t i n a 2 F i g u r e 2. S t r u c t u r e o f t h e Rod C e l l 4 F i g u r e 3. M o d e l f o r Rod O u t e r Segment F o r m a t i o n 6 F i g u r e 4. 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 9 F i g u r e 5. L o c a l i z a t i o n o f P e r i p h e r i n 12 F i g u r e 6. I d e n t i f i c a t i o n o f P e r i p h e r i n o n a n SDS - p o l y a c r y l a m i d e g e l 15 F i g u r e 7. S c h e m a t i c o f t h e pAX 111 V e c t o r 28 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 32 F i g u r e 9. N - t e r m i n a l S e q u e n c e A n a l y s i s o f P e r i p h e r i n a n d D e s i g n o f a D e g e n e r a t e O l i g o n u c l e o t i d e P r o b e 3 4 F i g u r e 10. 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 3 6 F i g u r e 1 1 . P u r i f i c a t i o n o f a C - t e r m i n a l F r a g m e n t o f P e r i p h e r i n 37 F i g u r e 12. Amino A c i d S e q u e n c e o f P e r i p h e r i n 40 F i g u r e 13. 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 42 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 o f P e r i p h e r i n 44 M e s s a g e s F i g u r e 15. A n 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 P e r i p h e r i n w i t h t h e P r o p o s e d r d s S e q u e n c e 46 F i g u r e 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 C o d i n g S e q u e n c e o f P e r i p h e r i n 47 F i g u r e 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 51 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 F i g u r e 18. A S t r u c t u r a l M o d e l o f P e r i p h e r i n 57 - v i i i -LIST OF ABBREVIATIONS bp base p a i r BSA bovine serum albumin DEAE diethylaminoethyl EDTA ethylenediamine tetraacetate Endo H endo -6-N-acetylglucosaminidase H. HPLC high-performance l i q u i d chromatography Ig immunoglobin kb kilobase Mr apparent molecular weight PTH penylthiohydantoin rd r e t i n a l degeneration rds r e t i n a l degeneration slow ROS rod outer segment SDS sodium dodecyl s u l f a t e TEMED — : N,N,N',N'-Tetramethylethylenediamine UV —• u l t r a v i o l e t T r i s — • — tris(hydroxymethyl)aminomethane - i x -ACKNOWLEDGEMENT I would l i k e to thank Dr. Robert Molday f o r h i s excellent supervision, encouragement and support of t h i s project. I would l i k e to extend my gratitude and thanks to Dr. Ross MacGillivray and the many members of hi s lab who have given me assistance over the course of these studies. I would also l i k e to thank Dr. Rob Kay for the use of h i s pAX 111 expression vector and for h e l p f u l discussions 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 to thank Laurie Molday, Drs. Simon and Delyth Wong, Dr. J e f f Leung, Dr. Robert McMaster, Dr. Dale Laird, Shu-Chan Hsu and Dr. Rod Mclnnes for many he l p f u l discussions. - 1 -INTRODUCTION The Vertebrate Retina The r e t i n a , located at the posterior end of the eyeball, transduces l i g h t into neural signals, integrates these signals and then transmits them through the optic nerve to the brain. The photoreceptor c e l l s , i n which the transduction of l i g h t into neural signals occurs, are the layer of r e t i n a l neural c e l l s furthest removed from the eye lens (Figure 1). These c e l l s are c l a s s i f i e d as rod or cone c e l l s on the basis of t h e i r p h o tosensitivity and morphology. The photosensitive pigment of the photoreceptor c e l l consists of a protein component, opsin, that i s covalently linked to 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 of the d i f f e r e n t opsin molecules with the r e t i n a l that determines the absorption maxima (Nathans et a l . , 1986). The human blue, green, and red sensi t i v e cone pigments have absorption maxima at 420, 530 and 560 nm respectively. Rhodopsin, the rod photosensitive molecule has an absorption maxima at 500 nm. In the human ret i n a there are approximately 120 m i l l i o n rod c e l l s and 6.5 m i l l i o n cone c e l l s (Pirenne, 1967). The cone c e l l s are located primarily i n the fovea, the centre of the v i s u a l f i e l d , whereas the rod c e l l s are located 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 some 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 . The 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 . The 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 ( m o d i f i e d a f t e r D o w l i n g , 1 9 7 0 ) . - 3 -more peripheral regions of the r e t i n a . Both types of c e l l s consist of a synaptic terminal, an inner segment and an outer segment (Figure 2). The synaptic terminal makes chemical synaptic connections with the bip o l a r and horizontal neural c e l l s (Figure 1). The inner segment contains the nucleus and mitochondria of the c e l l . A l l protein synthesis takes place i n the inner segment. The outer segments of cone and rod c e l l s are s p e c i a l i z e d organelles which contain the opsin molecules and the other proteins required for the transduction of l i g h t into e l e c t r i c a l signals as part of the v i s u a l e x c i t a t i o n process. The outer segment i s attached to the inner segment by a connecting c i l i u m . The Outer Segment The outer segment of the rod c e l l contain hundreds of c l o s e l y stacked membrane disks that are discontinuous with a surrounding plasma membrane throughout most of the rod outer segment. These disks are organized into a highly ordered array (Figure 2). In vertebrates the disks also have infoldings or incisures. The cone outer segment d i f f e r s from the rod outer segment i n that the disks are continuous with the plasma membrane throughout the ent i r e structure. I t i s not yet c l e a r how the rod outer segment disk membranes are formed. Steinberg et al.(1980) have proposed a model for the formation of rod outer segments that 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 s e g m e n t . The c o n e p h o t o r e c e p t o r c e l l d i f f e r s f r o m 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 S z u t s , 1 9 8 2 ) . - 5 -on electron microscopic observations. In t h e i r model the disks r e s u l t from an outgrowth of the c i l i a r y plasma membrane. There are components i n the c i l i a r y membrane that are proposed to intera c t with binding s i t e s e i t h e r d i r e c t l y on the c i l i u m or with a cytoskeletal element c l o s e l y associated with the ci l i u m (Figure 3). Act i n has been found to be associated with the d i s t a l region of the ci l i u m (Chaitin et a l . 1984 and Chaitin and Bok 1986). I t has been suggested that an a c t i n mediated c o n t r a c t i l e mechanism could be the cause for the outward growing membrane foldi n g back on i t s e l f . Treatment of rabbit r e t i n a i n vivo with cytbchalasin D, an i n h i b i t o r of a c t i n polymerization, r e s u l t s i n the formation of large membrane outgrowths instead of normal outer segment disks (Vaughan & Fisher, 1989). Rim formation, which seals the folded membrane into an in t a c t disk i s proposed to s t a r t at the attachment s i t e near the c i l i u m and move outwards towards the plasma membrane. Upon formation of the completed rim, the disk becomes detached from the plasma membrane. Visu a l E x c i t a t i o n A model for the v i s u a l e x c i t a t i o n process i n the rod outer segment i s outlined i n figure 4. In the dark Na + and to a le s s e r degree C a + 2 are able to enter the c e l l through a cGMP dependent channel located i n the outer segment (Hodgkin - 6 -o o O O membrane vesicle;; INNER SEGMENT rim ^formation mature rl disk oluter -^ segment \ F i g u r e 3 . A h y p o t h e t i c a l m o d e l 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 s egment 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 n e a r t h e b a s e 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 on t h e c i l i u m (#.) . The o u t g r o w t h o f t h e membrane i s h y p o t h e s i z e d t o b e l i m i t e d b y an i n w a r d d i r e c t e d a c t i n m e d i a t e d c o n t r a c t i l e m e c h a n i s m ^ ^_ ) . b) R i m f o r m a t i o n t a k e s p l a c e b e t w e e n 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 i s removed from the outer segment through a Na +\ C a + 2 exchanger. For every four Na + ions that are taken into the outer segment a K + ion and a C a + 2 ion are extruded (Cervetto et a l . , 1989). The Na + i s removed from the c e l l by a Na +\K + ATPase that i s located i n the inner segment (Stahl & Baskin, 1984). The rod inner segment Na +\K + ATPase has not been that well characterized, but the ATPase of the human erythrocyte pumps three Na + ions out of the c e l l for every two K + ions that are taken i n (Sen & Post, 1964). This ion flow into the outer segment and out through the inner segment i s known as the dark current. Absorption of a photon by the 11-cis r e t i n a l group of the opsin causes a series of conformational changes that eventually leads to i t s conversion to the a l l trans form and to i t s release from the opsin. During the process the conformation of the opsin i s altered so that i t i s able to inte r a c t and activate transducin or G-protein. Transducin consists of an oc subunit(39 kDa), a 6 subunit(36 kDa), and a 2f subunit(8 kDa) (Fung, 1983). The a c t i v a t i o n involves the exchange of GDP on the inactive form of the oc subunit for GTP. The only known function of the R and .jr'subunits i s to po s i t i o n the oc subunit so that i t can int e r a c t with rhodopsin. The activated =X subunit i n turn can activate phosphodiesterase. The phospodiesterase consists of a n ° ^ subunit(88 kDa), a 6 subunit(84 kDa) and an i n h i b i t o r y & subunit (11 kDa) (Baehr et a l . , 1979). A c t i v a t i o n i s thought - 8 -to involve the displacement of the i n h i b i t o r y subunit (Hurley & Stryer, 1982). Upon ac t i v a t i o n , phosphodiesterase i s able to hydrolyze cGMP. The decreased l e v e l s of cGMP i n the outer segment r e s u l t s i n the c l o s i n g of some of the sodium channels (Fesenko et a l . , 1985). Since the Na +\K + ATPase i s s t i l l operating i n the inner segment, the c l o s i n g of the channels r e s u l t i n the hyperpolarization of the photoreceptor membrane. This hyperpolarization modifies the synaptic output to the connecting neurons. The enti r e e x c i t a t i o n process occurs within milliseconds of the i l l u m i n a t i o n of the photoreceptor c e l l . There are several mechanisms used to deactivate the v i s u a l cascade. Shortly a f t e r a c t i v a t i o n , rhodopsin kinase(68 kDa) binds to the cytoplasmic face of rhodopsin and phosphorylates up to nine serine and threonine residues (Wilden & Kuhn, 1982). This phosphorylation decreases the a f f i n i t y of rhodopsin for transducin and increases the a f f i n i t y f o r a r r e s t i n . Arrestin(48 kDa) competes f o r the same binding s i t e on rhodopsin as transducin, and by binding to rhodopsin i t prevents a c t i v a t i o n of further transducin molecules. In addition, the subunit of transducin has a GTPase a c t i v i t y that hydrolyzes i t s bound GTP to GDP which returns any activated protein to i t s inactive state. A r r e s t i n i s also thought to be able to d i r e c t l y i n h i b i t the activated phosphodiesterase (Zuckerman et a l . 1985). - 9 -P l a s t n a P0£ Membrane Disc Memtxane F i g u r e 4: A mo d e l f o r v i s u a l t r a n s d u c t i o n . The cGMP g a t e d c a t i o n c h a n n e l i s o p e n i n t h e d a r k . U pon i l l u m i n a t i o n , r h o d o p s i n u n d e r g o e s a c o n f o r m a t i o n a l c h a n g e t h a t p e r m i t s i t t o 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 . T he a c t i v a t e d t r a n s d u c i n i n t u r n a c t i v a t e s a p h o s p h o d i e s t e r a s e t h a t 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 Proteins The photoactive protein, rhodopsin, i s the major constituent of both the plasma membrane and the disk membrane of the rod outer segment. The C-terminus of rhodopsin and the F ^ I ^ loop which joins the f i f t h and si x t h membrane spanning hel i c e s are oriented on the cytoplasmic face of the membranes where they are able to i n t e r a c t with other components of the v i s u a l cascade such as rhodopsin kinase and transducin (Kuhn, 1981; Hargrave, 1982). The N-terminal segment which contains two asparagine linked carbohydrate chains i s orientated on the e x t r a c e l l u l a r surface of the plasma membrane and i n the lumen of the disk (Rohlich, 1976; Hargrave, 1977; Clark & Molday, 1979). In addition to rhodopsin, the plasma membrane contains a unique set of proteins that are not present i n the disk membrane. Several r i c i n binding glycoproteins were i d e n t i f i e d that are s p e c i f i c for the plasma membrane (Molday & Molday, 1987a). These proteins were used as markers i n the p u r i f i c a t i o n of the plasma membrane from the disk membrane using a density perturbation method (Molday & Molday 1987b). The 63 kDa cGMP - gated cation channel and the 220 kDa sodium - calcium exchanger were subsequently shown to be r e s t r i c t e d to the plasma membrane (Bauer, 1988; Cook et a l . , 1989, Reid et a l . , 1990). The disk membrane, i t s e l f , can be divided into two domains with unique protein compositions. Falk and Fatt - 1 1 -(1969) have shown that an osmium tetroxide s o l u t i o n dissolved the lamellae of the disks but not the rims. Papermaster et al.(1978) used a polyclonal antibody to l o c a l i z e a 290 kDa protein to the disk incisures and rims of frog ROS. This polyclonal serum does not, however, cross react with any component present i n the mammalian photoreceptor. Molday et a l . (1987) l o c a l i z e d a 33 kDa protein, peripherin, s p e c i f i c a l l y to the rim region of the bovine disk membrane using monoclonal antibodies and immunocytochemical l a b e l i n g techniques (Figure 5). A 22 0 kDa concanavalin A - binding glycoprotein and several minor proteins are also present i n the mammalian disk membrane, but they have not yet been l o c a l i z e d to e i t h e r domain (Molday & Molday 1987b). Photoreceptor C e l l Peripherin Peripherin cannot be extracted from the disk membrane with chelating and chaotropic agents, but i t requires a detergent for s o l u b i l i z a t i o n (Molday et a l . , 1987). The protein migrates as a dimer on an SDS - polyacrylamide gel i n the absence of 2 - mercaptoethanol suggesting that i t may e x i s t as two subunits held together by one or more d i s u l f i d e bonds (Molday et 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 of 2 -mercaptoethanol, peripherin 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 at a molecular weight of 3 3 -12-Figure 5. The l o c a l i z a t i o n of peripherin within the rod outer segment, a) A longitudinal section through a rod outer segment that had been l a b e l l e d with 3B6, an antiperipherin monoclonal antibody, followed by goat antimouse Ig-gold dextran p a r t i c l e s . Labelling i s shown to be concentrated along the peripheral region of the outer segment where the disk membranes come into close contact with the plasma membrane. Bar=0.25um. b) An is o l a t e d disk showing the binding of 3B6 p r e f e r e n t i a l l y along the rim region. Bar=.05um (Molday et a l . , 1987). -13-- 1 4 -kDa (Figure 6). The determination of the number of peripherin molecules present i n the rod outer 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 . Peripherin appears, though, to constitute l e s s than 5% of the t o t a l disk protein. This could s t i l l make i t , a f t e r rhodopsin, one of the more abundant membrane proteins of the photoreceptor c e l l . Retinal Degeneration Slow Retinal degeneration slow (rds) i s a congenital r e t i n a l disorder that has been i d e n t i f i e d i n a s t r a i n of mice (van Nie et a l . , 1978). Those mice that are homozygous for the defect are i n i t i a l l y characterized by an absence of photoreceptor c e l l outer segments (Sanyal & Jansen, 1981). The inner segments of the photoreceptor c e l l s and the neurons that synapse with them i n i t i a l l y appear to be normal (Jansen & Sanyal, 1984). Membrane v e s i c l e s have been shown to accumulate close to the region where outer segment formation would normally occur. At 2 to 3 weeks a f t e r b i r t h , there i s a gradual loss of the remaining portion of the photoreceptor c e l l s , and a f t e r one year, the photoreceptors are completely degenerated. The other r e t i n a l c e l l types remain unaffected. The defective gene i s c l o s e l y linked to the H2 gene complex, and i t maps to chromosome 17 (Demant et a l . , 1979). -15-F i g u r e 6. Rod o u t e r segment 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) o r 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 t h e p r e s e n c e o f 2 - m e r c a p t o e t h a n o l r u n s u n d e r n e a t h t h e b r o a d r h o d o p s i n b a n d a t 34 kDa. I n t h e a b s e n c e o f 2-m e r c a p t o e t h a n o l t h e 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 l a b e l a b a n d c l o s e r t o 68 kDa. The 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 kDa 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 kDa 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 b y t h e 1D4 a n t i b o d y . The 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 o f t h e p r e s e n c e o f 2 - m e r c a p t o e t h a n o l ( M o l d a y e t a l . , 1 9 8 7 ) . - 1 6 -Travis et a l . (1989) have recently i d e n t i f i e d a candidate gene for the rds defect. By carrying out subtractive and d i f f e r e n t i a l colony screening of wild-type mice minus a s t r a i n homozygous for r e t i n a l degeneration (rd), photoreceptor s p e c i f i c cDNA clones were obtained. The rd defect, which i s unrelated to rds and maps to chromosome 5, i s characterized by a rapid degeneration of the photoreceptor c e l l s that i s complete within four weeks a f t e r b i r t h (Carter-Dawson et a l . , 1978). A l l other r e t i n a l c e l l types remain unaffected. A panel of mouse x hamster hybrid c e l l - l i n e DNAs was used to determine the chromosome assignments of the d i f f e r e n t photoreceptor s p e c i f i c cDNA clones. One clone that mapped to chromosome 17, the same loca t i o n as the rds defect, was i d e n t i f i e d . Genomic clones were i s o l a t e d from l i b r a r i e s made from both wild type and rds mice using t h i s cDNA clone as a probe. The genomic clone from the rds s t r a i n was found to contain 10 kb of DNA inserted into i t that are not present i n the wild-type gene. Northern b l o t analysis revealed that normal sized 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 basis, the photoreceptor s p e c i f i c cDNA clone mapping to chromosome 17 was i d e n t i f i e d as being the gene responsible f o r the rds defect. Neither the gene product nor i t s l o c a l i z a t i o n within the photoreceptor c e l l had been determined. -17-Thesis Investigation The l o c a l i z a t i o n of photoreceptor c e l l peripherin s p e c i f i c a l l y to the rim region of the disk membrane introduced several questions. How does t h i s l o c a t i o n i n the disk membrane r e l a t e to i t s function? Is i t a s t r u c t u r a l protein that i s somehow involved i n the formation of the ordered array of disk membranes within the outer segment? Does i t have anything to do with rim formation on the disk membranes? The d i f f u s i o n of rhodopsin throughout the disk membrane i s necessary for i t s function (Leibman & Pugh, 1981). Is peripherin sequestered into the rim region of the disk membrane so that i t w i l l not i n t e r f e r e with rhodopsin? Does peripherin have a ro l e i n the v i s u a l e x c i t a t i o n process? How does protein sorting take place among the rim and lamellar regions of the disk membrane and the plasma . membrane of the outer segment? This study i s an i n i t i a l attempt to p a r t i a l l y answer some of these questions. The work involves the molecular cloning, expression and characterization of peripherin. -18-MATERIALS and METHODS Materials General laboratory reagents were obtained from BDH Chemicals (Toronto, Ontario), Fisher S c i e n t i f i c (Ottawa, Ontario) or Sigma Chemical Company (St. Louis, MO). The r e s t r i c t i o n enzymes and the other DNA modifying enzymes were purchased from Bethesda Research Laboratories (Burlington, Ontario), Boehringer Mannheim (Dorval, Quebec), Pharmacia (Baie d'Urfe, Quebec) or The United States Biochemical Corporation (Cleveland, 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 of Dr. Jeremy Nathans (John Hopkins) and Dr. Dan Oprian (Brandeis) respectively. The BA85 Schleicher & Schuell n i t r o c e l l u l o s e f i l t e r s that were used for the plaque l i f t s were obtained from Chemonics S c i e n t i f i c Ltd. (Richmond, B.C.), and the nylon f i l t e r s (Hybond) were obtained from Amersham (Oakville, Ontario). The Gene Clean k i t was purchased from Bio 101 Inc. (La J o l l a , CA). Oligo(dT) - c e l l u l o s e (type 3) was purchased from Collaborative Research (Bedford, MA). A l l oligonucleotides were synthesized by Dr. Tom Atkinson (U.B.C). The radioactive isotopes and the Quanta III i n t e n s i f y i n g screens were obtained from Dupont (Markham, Ontario). Bovine r e t i n a were eithe r purchased frozen from Hormel (Austin, MN) or obtained fresh 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 - 1 9 -purchased from Fisher S c i e n t i f i c . The HPLC grade t r i f l u o r o a c e t i c acid was obtained from Pierce (Rockford, IL). Acrylamide, ammonium persulfate, N-N' methylene bisacrylamide and TEMED were obtained from Bio - Rad (Richmond, CA). The Immobilon paper and the reverse phase C18 HPLC column were obtained from M i l l i p o r e (Mississauga, Ontario). N - Glycanase F was obtained from Genzyme (Boston, MA) and Endo H from Seikagaku Kogyo (Tokyo, Japan). C e l l culture media, f e t a l c a l f sera, t r y p s i n , p e n i c i l l i n and streptomycin were obtained from Gibco (Burlington, Ontario). DEAE - dextran (MW 500,000) was obtained from Pharmacia and chloroquine 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 that was prepared i n the phage expression vector jLgt 11 (Barrett et a l . , 1985) was screened using monoclonal antibodies (Young & Davis, 1983). This l i b r a r y was amplified from 3.5 x 10 5 independent clones. The average si z e of the o l i g o dT primed cDNA ins e r t s that were used i n the construction of the l i b r a r y was lkb. One complete l i b r a r y was plated on Escherichia c o l i Y1090 [ lacU169 Ion proA + araD139 strA supF (pMC9)] at a density of 180 phage/cm2 and incubated at 42°C for 3 hours. N i t r o c e l l u l o s e f i l t e r s that had been saturated with isopropyl-l-thio-B-D-galactopyranoside were ov e r l a i d on the plaques, and the plates were incubated at 37°C f o r 5 to 8 hours. The f i l t e r s were quenched i n 3% BSA and then - 2 0 -incubated with a mixture of the antiperipherin monoclonal antibodies 2B6 and 3B6 (Molday et a l . , 1987). The f i l t e r s were reacted with goat antimouse Ig that had been l a b e l l e d with 1 2 5 i to a s p e c i f i c a c t i v i t y of 4xl 0 9 dpm/mg using the chloramine T method (Hunter & Greenwood, 1962). After both the primary and secondary antibody incubations, the f i l t e r s were washed 5 times with a solution containing 150 mM NaCl, 10 mM Na 2HP0 4 and 0.05% Tween 20. Autoradiography was ca r r i e d out for approximately 18 hours. Po s i t i v e signals were plaque p u r i f i e d by four additional rounds of screenings at successively lower phage d i l u t i o n s . cDNA that was used as probes i n l a t e r screenings of the same Agt l l l i b r a r y and a ^ g t l O l i b r a r y (Nathans & Hogness, 1983) was l a b e l l e d usingc<- 3 2p-ATP and random hexamer primers according to the method of Feinberg and Vogelstein (1983). The s p e c i f i c a c t i v i t y of these probes was approximately 5 x 10 7 cpm/pmol. The ^ g t l O l i b r a r y had been amplified from 2.5 x 10 5 independent clones. Oligo dT primed cDNAs greater than 500 bp were used i n i t s construction. The degenerate oligonucleotide mixture that was used to screen the yCg t lO l i b r a r y was 5' end l a b e l l e d using 2f- 3 2P-ATP and T4 polynucleotide kinase (Maniatis et a l . , 1982). On average, the end-labelled probes had a s p e c i f i c a c t i v i t y of 1-2 x 10 6 cpm/pmol. Hybond nylon membrane f l t e r s (Amersham) were used to make the plaque l i f t s f or the DNA hybridizations. A f t e r denaturation, the DNA was UV cross - linked to the f i l t e r s . -21-The f i l t e r s were prehybridized for 2 hours at the hybridization temperature i n a solution containing 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% polvinylpyrrolidone 360, 0.02% BSA), 0.5% SDS and 1 mM EDTA. The prehybridization solution was removed and replaced with fresh solution containing 0.5 - 1 x 10 6 cpm per ml of la b e l l e d probe. The hybridizations with the l a b e l l e d cDNA probes were c a r r i e d out at 68° C for 12 hours, and the hybridizations with the l a b e l l e d degenerate oligonucleotide mixture were ca r r i e d out at 40° C for 18 hours. The f i l t e r s were washed at the hybridization temperature f o r several hours with several changes of a solution containing 6 x SSC, .5% SDS and 1 mM EDTA. Autoradiography was done at -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 is o l a t e d by the procedure of Maniatis et a l (1982). EcoRl digested DNA was fractionated on an agarose gel, and the in s e r t DNA was p u r i f i e d with the Gene Clean k i t which uses the glass powder e l u t i o n method of Vogelstein 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 li g a t e d into M13mpl8 or M13mpl9 f o r sequencing by the dideoxynucleotide chain termination method (Sanger et a l . , 1977) using eit h e r the Klenow fragment of DNA polymerase I or modified T7 DNA polymerase. Both strands of the region -22-c o d i n g f o r the open r e a d i n g frame were sequenced t o completion. Northern blot analysis T o t a l RNA was i s o l a t e d from bovine r e t i n a s a c c o r d i n g t o the method of Chirgwin e t a l . (1979). The r e t i n a s were d i s s e c t e d from the eyes a t the s l a u g h t e r house and immediately f r o z e n i n l i q u i d n i t r o g e n . The f r o z e n r e t i n a s (10 g) were p l a c e d i n 100 ml of homogenizing s o l u t i o n (7.5 M g u a n i d i n e - HC1, 0.025M sodium c i t r a t e and 10 mM d i t h i o t h r e i t o l ) and d i s r u p t e d w i t h t h r e e 15 second b u r s t s of a P o l y t r o n (Kinematica) s e t a t medium speed. The sample was e n r i c h e d f o r RNA through s e v e r a l rounds of p r e c i p i t a t i o n w i t h one t h i r d volume of 95% e t h a n o l f o l l o w e d by e x t r a c t i o n i n s u c c e s s i v e l y s m a l l e r volumes of homogenization s o l u t i o n . P o l y a d e n y l a t e d r e t i n a l RNA was then p u r i f i e d from the mixture by passage through an o l i g o ( d T ) - c e l l u l o s e column (Aviv & Leder, 1972). Approximately 700 ug o f p o l y a d e n y l a t e d RNA was o b t a i n e d from 10 g of t i s s u e . Bovine l i v e r RNA was the generous g i f t o f Walter Funk. RNA was separated on a 1.2% agarose g e l c o n t a i n i n g 2.2M formaldehyde and t r a n s f e r r e d t o Hybond n y l o n membrane. The t r a n s f e r s were h y b r i d i z e d i n 5x Denhardts, 9% dextran, 43% formamide, 0.1% SDS, 100 ug/ml salmon sperm DNA, and 5x SSPE ( l x SSPE = 180 mM NaCl, 10 mM NaH 2P0 4, ImM EDTA, pH 7.4) a t 42°C w i t h 1 0 6 dpm/ml of 3 2 P l a b e l l e d cDNA probes. The membranes were then washed a t 70°C i n .lxSSPE and .1% SDS. - 2 3 -Immunoaffinity Column Preparation The antiperipherin monoclonal antibody 2B6 was purified from ascites f l u i d by precipitation with 50% ammonium sulfate followed by DEAE-Sepharose chromatography. SDS -polyacrylamide gel electrophoresis indicated that the antibody was essentially pure. The 2B6 monoclonal antibody was covalently coupled to Sepharose 2BC1 using a CNBr activation 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 light from 80 frozen retinas as previously described (Molday & Molday, 1987b). Briefly, 80 frozen retinas were placed in 30 ml of a homogenization solution (2 0% sucrose, 10 mM glucose, 2 mM MgCl2 and 2 0 mM Tris acetate pH 7.2) and gently shaken for 1 minute. This treatment breaks off the outer segments from the connecting ciliums. The solution was passed through a Teflon screen and then layered onto six 24 ml 25 - 60 % (wt/vol) sucrose gradients which also contained 10 mM glucose, 2 mM MgCl2, and 20 mM Tris - acetate pH 7.2. The gradients were spun at 25,000 rpm in a SW - 27 rotor (Beckman Instruments) for one hour. The pink rod outer segment band was collected and washed several times with homogenizing solution. Approximately 60 mg of protein were obtained from the 80 retinas. -24-Purification of peripherin for N-terminal sequence analysis The rod outer segments were resuspended at a protein concentration of 2mg/ml i n a buffer containing 20 mM T r i s -acetate pH 7.4, lOOmM NaCl and 0.4mM phenylmethylsulfonylfluoride. Approximately 8 mg of the resuspended ROS were s o l u b i l i z e d with an equal volume of 50 mM o c t y l glucoside or 18 mM CHAPS and passed through 1ml of the 2B6 antibody - Sepharose 2BC1 column. The column was washed with 20 volumes of the resuspension buffer and then eluted with e i t h e r 0.1 M acetic acid or 0.05 M formic acid. During both the washing and elu t i o n of the column, the detergent was kept at one ha l f the concentration that was used during the i n i t i a l s o l u b i l i z a t i o n . The eluted protein was then dialyzed against 0.1% SDS and sequenced by the Protein Microchemistry Centre at the University of V i c t o r i a . Protein quantitation was done with the BCA protein assay reagent(Pierce) using bovine serum albumin as the standard. 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 H20, lOmg of the ROS preparation were resuspended i n 0.7ml of 98% formic acid, and the volume was adjusted to 1ml with d i s t i l l e d H 20. CNBr was added to a f i n a l concentration of 0.05g/ml, and the digest was l e f t f o r 14 hours at room temperature i n the dark. The mixture was dried under vacuum, and the p e l l e t was extracted several times with 0.05M NH4HC03 pH 8.0. The extracted CNBr cleaved peptides were passed through 2ml of the 2B6 antibody - Sepharose 2BC1 column, and the - 2 5 -column was washed with 15 volumes of 0.05M NH4HC03 pH 7.0. The column was then eluted with 5 volumes of 0.05M formic acid, and the eluate was dried under vacuum. The sample was resuspended i n lOOul of d i s t i l l e d H 20 containing 0.05% t r i f l u o r o a c e t i c acid and loaded onto a reverse phase C18 HPLC column. The HPLC column was eluted with a d i s t i l l e d water - a c e t o n i t r i l e gradient that contained 0.05% t r i f l u o r o a c e t i c acid. The gradient ran from 10% to 80% a c e t o n i t r i l e over a period of 100 minutes. Peptide e l u t i o n was detected by monitoring the column e f f l u e n t at 215 nm. Peak fractio n s were assayed for the presence of the peripherin peptide by dot b l o t t i n g onto Immobilon paper and screening with the 2B6 monoclonal antibody as previously described (Molday et a l . , 1987). The peak f r a c t i o n assaying p o s i t i v e f o r 2B6 antibody binding 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 at a protein concentration of 3mg/ml i n 2% SDS i n the presence and absence of 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 with eithe r N-Glycanase F buffer (0.2M sodium phosphate pH 8.6, 1.5% Nonidet P-40, 0.4mM phenylmethylsulfonylfluoride and lOmM phenanthroline) or Endo H buffer (0.05M sodium phosphate pH 5.5, 1.5% Nonidet P-40 and 0.4mM phenylmethylsulfonylfluoride). The di l u t e d ROS were incubated at 37° for 24 hours with either 9 units/ml N-Glycanase F or 0.06 units/ml Endo H. The reaction products - 2 6 -were separated on an 8% polyacrylamide g e l , and peripherin was detected by western b l o t t i n g as previously described (Molday et a l . , 1987). SDS - Polyacrylamide Gel Electrophoresis and Gel Transfer The protein samples were combined with an equal volume of gel loading buffer (5% SDS, 40% sucrose, lOmM T r i s pH 6.8 and 10% 2-mercaptoethanol), and upto a maximum of 20 ug of protein was applied to the gel wells i n a maximum volume of 2 0 u l . The r a t i o of acrylamide to N-N' methylene bisacrylamide i n the gels (0.75mm thickness X 3.0 cm length) was 30 to 0.8. Electrophoresis was c a r r i e d out by using the buffer system of Laemmli (1970). The proteins were e l e c t r o p h o r e t i c a l l y transferred from the polyacrylamide gels to Immobilon paper using a Hoefer transblot apparatus (model TE22). The transfers were c a r r i e d out for 30 minutes at 0.30 amps i n a buffer containing 20 mM T r i s pH 7.4, 2 mM EDTA, 0.01% SDS and 10% methanol. Subcloninq of the Complete Coding Sequence of Peripherin  into pAX 111 - a COS C e l l Expression Vector A schematic diagram showing the s i g n i f i c a n t features of the pAX 111 vector (R. Kay & R.K. Humphries - personal communication) i s shown on figure 7. The vector contains both the Col E l and SV40 orig i n s of r e p l i c a t i o n . The DNA coding for the complete open reading frame of peripherin was li g a t e d into the BamHI cloning s i t e s which put the -27-expression of peripherin under the control of the cytomegalo v i r u s promoter. The vector containing the peripherin i n s e r t was transformed into E . c o l i DH5»<p3, a d e r i v a t i v e of E. c o l i DH5«*. containing the p 3 plasmid. The p 3 plasmid contains c o n s t i t u t i v e kanamycin resistance, and i t also 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 resistance. These amber mutants become active following transformation with pAX 1 1 1 which contains the Sup F gene. Approximately 2 0 0 colonies were obtained from the transformation of competent DH5o<p3 c e l l s with 4 . 5 ng of the l i g a t e d vector. The transformation mixture was plated i n the presence of 40 ug/ml a m p i c i l l i n , 2 5 ug/ml kanamycin and 8 ug/ml t e t r a c y c l i n e . The orientation of the inserted peripherin sequence r e l a t i v e to the cytomegalo promoter was determined by r e s t r i c t i o n mapping using a B g l l l digest and an EcoRI - Kpnl double digest. Transformants containing both orientations of the i n s e r t were iso l a t e d , and the plasmid DNA was CsCl p u r i f i e d according to the method described by Maniatis et a l . ( 1 9 8 2 ) . Approximately 1 0 0 ug of p u r i f i e d plasmid DNA was obtained from a 2 0 0 ml culture. COS C e l l Maintenance COS -1 c e l l s (Gluzman, 1 9 8 1 ) were grown on 10 cm diameter p e t r i plates i n medium A (Dulbecco's modified Eagles medium that was supplemented with 10 % heat inactivated ( 5 6 ° for 30 minutes) f e t a l c a l f serum, 2 mM glutamine, 10 mM Hepes p H 7 . 2 , streptomycin ( 1 0 0 units/ml), and p e n i c i l l i n ( 1 0 0 - 2 8 -/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 111 v e c t o r . The BamHI and S m a l c l o n i n g s i t e s a r e a d j a c e n t t o t h e 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 a n d SV40 o r i g i n s o f r e p l i c a t i o n ( O R I ) . - 2 9 -units/ml)) . Upon reaching 60 to 80% confluency, the c e l l s were rinsed once with PBS (137 mM NaCl, 2.7 mM KCl, 1.5 mM KH 2P0 4, 8 mM Na 2HP0 4 pH 7.3) and then incubated with 2 ml of 0.05% t r y p s i n for 3 minutes. The t r y p s i n was quenched with 8 ml of medium A, and the c e l l s that were loosened from the p e t r i plate were then spun at approximately 400g i n a IEC table-top centrifuge. The c e l l p e l l e t was e i t h e r resuspended i n fresh medium A for r e p l a t i n g or i n 2 to 3 ml of c e l l freezing media (medium A containing 8.3% dimethyl sulfoxide and 10% heat inactivated f e t a l c a l f serum). The c e l l s that were to be stored frozen were aliquoted into 1 ml cryogenic tubes, placed i n an insulated box at -60° overnight and then transferred to l i q u i d N 2. Transfection of COS-1 C e l l s with the pAX 111 Vector The tr a n s f e c t i o n procedure that was used was taken from Hammarskjold et a l . (1986). The COS c e l l s were divided into 10 cm diameter p e t r i plates one or two days p r i o r to the tra n s f e c t i o n so that they would be about 60% confluent when transfected. About 7.5 ug of the pAX 111 plasmid DNA was added to 2 ml TS (140 mM NaCl, 25 mM T r i s , 5 mM KCl, 0.5 mM Na 2HP0 4, 1 mM MgCl 2 pH 7.5) and t h i s was then added to 2ml of TS containing 1.0 mg/ml DEAE-dextran. The COS c e l l s were washed once with TS and then once with TD (TS without any Mg + 2 or C a + 2 ) . The c e l l s were then incubated with the DNA -DEAE-dextran solut i o n at room temperature f o r 10 minutes and then at 37°C for 40 minutes. The DNA - DEAE-dextran solution - 3 0 -was removed and 4 ml of 20% g l y c e r o l i n TS was added. The soluti o n was gently swirled o f f and on the c e l l s f or 2 minutes. The gl y c e r o l solution was aspirated o f f , and the c e l l s were washed successively with TS followed by medium A. The plates were then incubated for 5 hours at 37°C with 100 uM chloroquine diphosphate i n medium A. The solut i o n was aspirated o f f and replaced with medium A. The c e l l s were l e f t to grow for 60 to 70 hours with fresh medium A being added to the c e l l s d a i l y . Harvesting of the Transfected COS C e l l s The plates were washed once with PBS, and then 1 ml of l y s i s buffer (1% CHAPS and 0.2 mg/ml phenylmethanesulfonylfluoride i n PBS) was added. The plates were scraped with a rubber policeman and the l y s i s solution was spun i n a microfuge for 15 minutes at 4°C. The post nuclear supernatant (about 1.2 ml) was transferred to a fresh tube and frozen at -60°C. Analysis of the expressed peripherin product was done by western b l o t t i n g . -31-RESULTS P u r i f i c a t i o n of Peripherin for N-terminal Sequence Analysis Peripherin was p u r i f i e d from rod outer segments by immunoaffinity chromatography. Western b l o t t i n g and radioimmune assays both indicated that there was s t i l l some rhodopsin contamination within the p u r i f i e d 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 to make up between 10% to 50% of the t o t a l protein i n the preparation. The sample shown i n figure 8 i s the best preparation that was obtained, and the rhodopsin content i s approximately 10%. Since rhodopsin has an acetylated N - terminus (Hargrave, 1977), i t was reasoned that t h i s impurity would have no e f f e c t on the sequencing. The r e s u l t from the N-terminal sequence analysis i s shown i n figure 9. The y i e l d of PTH-amino acids from each cycle of sequencing was approximately 10 to 20 pmol. I n i t i a l l y , there was not a high degree of confidence i n the sequence analysis. The y i e l d of amino acids calculated from each cycle of sequencing was approximately a factor of 100 le s s than what was expected. Peripherin forms very large aggregates under non reducing conditions, and i t i s possible that a large aggregate of peripherin may not have been accessible to the analysis. There i n i t i a l l y was concern, though, that peripherin may have a blocked N-terminus, and the sequence that was obtained could have been from a minor - 3 2 -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 b y 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 Hepes pH 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 CHAPS. The s o l u b i l i z e d 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 2ml 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 2BC1 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 CHAPS. The c o l u m n was 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 CHAPS. 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 2-m e r c a p t o e t h a n o l . 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 s e g m e n t 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 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 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, a n 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 noise of the analysis was also high. I s o l a t i o n of peripherin cDNA clones A mixture of two antiperipherin monoclonal antibodies 2B6 and 3B6 was used to screen the >*gtll bovine r e t i n a l cDNA l i b r a r y . Plaque screening of 350 000 recombinants led to the i s o l a t i o n of 3 clones which reacted to both antibodies. Clone JL.5, with the largest i n s e r t , has a 105 bp open reading frame (Figure 10) that i s i n frame with the £-galactosidase gene i n the / g t l l vector. The l i b r a r y was rescreened with the complete i n s e r t from JK.5. Over 300 p o s i t i v e signals were obtained from the primary screen. Twenty of these p o s i t i v e s were chosen at random and plaque p u r i f i e d . Clones A.4 and J..8, shown i n figure 10, contain the largest i n s e r t s . The two clones have complete sequence overlap with X.5 but diverge from each other at the 3' untranslated end. The open reading frame continues to the 5' end of clone J-.8. The 249 bp EcoRI-Hpall fragment at the 5' end ofX.8 was used to rescreen the same l i b r a r y . Two additional clones X.11 and / .17 were obtained. These clones have complete sequence overlap with the EcoRl-Hpall probe but diverge from each other at the 5' ends (Figure 10). An independent/gt10 bovine r e t i n a l l i b r a r y was screened with the 499 bp EcoRI - PstI fragment f r o m / . 17 and with a degenerate oligonucleotide derived from the putative N-terminal sequence of peripherin (Figure 9). From a screen - 3 4 -a) 1 5 10 15 Ala Leu Leu Lys Val Lys Phe Asp Gin Lys Lys Arg Val Lys Leu 20 25 30 Ala Gin Gly Leu ### Leu Met Asn Trp Phe Ser Val Leu Ala Gly H e H e b) 5' CCA ATT CAT CAG CCA CAG CCC CTG 3' G T T T A A A G G G Figure 9. a)The N-terminal amino acid sequence was determined from immunoaffinity p u r i f i e d peripherin. One of the tryptophan residues, as determined from the cDNA sequence, was destroyed during the protein sequencing (###) and was not detected, b) The degenerate oligonucleotide mixture that was used to screen a XgtlO l i b r a r y was derived from amino acids 17 to 24. -35-of 500 000 recombinants, only one p o s i t i v e s i g n a l , clone X-18 (Figure 10), was obtained which reacted to both probes. Clone J^. 18 has complete sequence overlap with X. 17 f o r 234 bp before diverging from i t at i t s 5' end. I s o l a t i o n of a C-terminal CNBr fragment of peripherin f o r  amino acid sequencing Total rod outer segment proteins were digested with CNBr, and the digested products were separated on a reverse phase HPLC column (11a). Individual column fr a c t i o n s were assayed for the presence of the C-terminal CNBr fragment of peripherin by dot b l o t t i n g onto Immobilon paper and screening with the 2B6 monoclonal antibody. A f t e r the digest was passed through a 2B6 immunoaffinity column, only 3 major peaks were v i s i b l e on the chromatogram (Figure l i b ) . One of these chromatogram peaks was i d e n t i f i e d as the C-terminal CNBr fragment of peripherin, one of the peaks was i d e n t i f i e d as the N-terminal CNBr fragment of rhodopsin, and the other remaining peak was l e f t u n i d e n t i f i e d . Predicted Amino acid sequence of peripherin The sequencing strategy used for the d i f f e r e n t cDNA clones i s outlined i n figure 10. The complete amino acid sequence of peripherin i s shown i n figure 12A. The sequence of the f i r s t 32 amino acids of peripherin was obtained from the N-terminal sequence analysis (Figure 12A). The i n i t i a l amino acid i n the mature protein i s alanine i n d i c a t i n g that eith e r a leader sequence or the i n i t i a t o r methionine was -36-E J I E E _J L. Clones X.S J I X.4 J 1.8 TQA P K _ i i H J..17 100 bp X.18 Figure 10. R e s t r i c t i o n map and sequencing strategy of cloned cDNA encoding peripherin. The r e s t r i c t i o n map shows only the relevant s i t e s for EcoRI (E) , Hpall (H) , Kpnl (K) , and PstI (P) . The i n s e r t from clone X.5, obtained from screening the cDNA r e t i n a l l i b r a r y with the antiperipherin monoclonal antibodies, was used to rescreen the l i b r a r y . Clones J. . 4 and X.8 were is o l a t e d . The 5' EcoRI - Hpall fragment from X.8 was used to rescreen the same l i b r a r y . Two additional clones jL.11 and X.17 were obtained. The 499 bp EcoRI - PstI fragment from X.17 and a degenerate oligonucleotide probe derived from the N-terminal amino acid sequence of peripherin were used to screen an independent A g t l O l i b r a r y . Clone X-18 hybridized to both probes. Identical sequence overlap among the clones i s represented by the bold lines and sequence divergence by the narrow lines. The dotted segment above clone X.1% represents the sequence that i s i n agreement with the N-terminal amino acid sequence. The sequence coding for the f i r s t 5 N-terminal amino acids of the mature protein i s not present on clone X-18. The arrows represent the d i r e c t i o n and extent of sequence determination. The sequence was determined by the dideoxynucleotide method using the universal M13 sequencing primer (solid arrow) or synthesized oligonucleotides (dashed arrow) . The position of the termination codon (TGA) on clone A. 11 and the position of the s t a r t of the open reading frame (^r) on clone X'18 are indicated. -37-Figure 11. P u r i f i c a t i o n of a C-terminal CNBr fragment of peripherin. a) Total rod outer segment proteins were digested with CNBr, and 10 mg of the digested products were passed through a reverse phase C18 HPLC column. The HPLC column was eluted with a d i s t i l l e d water - a c e t o n i t r i l e gradient that contained 0.05% t r i f l u o r o a c e t i c acid. The gradient ran from 10% to 80% a c e t o n i t r i l e over a period of 100 minutes. Peptide e l u t i o n was detected by monitering the column e f f l u e n t at 215 nm. b) Afte r digestion with CNBr, 10 mg of the rod outer segment preparation were passed through a 2B6 antibody - Sepharose 2BC1 column. The eluted material was then run through the HPLC column under the same conditions described i n a. Peak fracti o n s were assayed for the presence of the peripherin peptide by dot b l o t t i n g onto Immobilon paper and screening with the 2B6 monoclonal antibody as previously described (Molday et a l . , 1987). The fracti o n s were also screened with 4D2, a monoclonal antibody directed to the N-terminus of rhodopsin. The peak f r a c t i o n assaying p o s i t i v e for 2B6 binding was sequenced. - 3 8 -b 2 B 6 4 D 2 r-cj> CM*'. c— ^  -c- c-£ o r- t/-. \ o « r 1 <-j «- -o a? V <^  CJ <D CD <X- "O °~ - 3 9 -removed. Except for the f i r s t five N-terminal amino acids, the coding region of clone ,£.18 i s in 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 clones^.11 and /.17 . As shown in figure 12A, the amino acid sequence that was obtained directly from the C-terminal CNBr fragment of peripherin i s also in complete agreement with the cDNA sequence. The predicted size of the mature protein i s 345 amino acids (Figure 12A). There are three potential asparagine linked glycosylation sites (Neuberger & Marshall, 1968) within the sequence (Figure 12A). Treatment of peripherin with Endo H or N-Glycanase F causes a small decrease in i t s mobility (Figure 13) indicating the presence of N-linked carbohydrate on at least one of these sites. The hydrophobicity of the predicted peripherin sequence was examined. The Kyte Doolittle plot predicts that there are four hydrophobic regions that are long enough to be potential membrane spanning domains (Figure 12B). This i s in agreement with the solubility properties of the protein which suggest that peripherin is an integral 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 in the original paper (Rao & Argos, 1986). Figure 12. Amino acid sequence of peripherin. a) The amino aci d sequence of an iso l a t e d C-terminal peptide of peripherin and the N-terminal amino ac i d sequence of immunoaffinity p u r i f i e d peripherin (parentheses) i s i n agreement with a conceptual t r a n s l a t i o n of the cDNA sequence. Some of the cysteine and tryptophan residues were destroyed during the protein sequencing and were not detected. The segments that are predicted by the hydrophobicity plo t (b) to be possible membrane spanning domains are underlined. There are three p o t e n t i a l asparagine linked glycosylation s i t e s within the sequence(•) . The part of the sequence containing the antigenic s i t e s f o r the antiperipherin monoclonal antibodies i s bracketed, b) A Kyte-Doolittle hydrophobicity p l o t was done on the translated sequence. Hydrophobic amino acids are p l o t t e d above the dashed l i n e and hydrophilic residues below the l i n e . - 4 1 -(Ala Leu Leu 10 Lys Val Lys Phe Asp Gin Lys Lys Arg Val Lys Leu Ala 20 Gln,Gly Leu Trp Leu Met Asn Trp Phe Ser Val Leu Ala 30 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 l i e 40 l i e ) He Phe Gly Leu Gly Leu Phe Leu. Lys He Glu Leu Arg Lys 50 Arg Ser Asp Val Met T Asn Asn Ser Glu Ser His Phe Val 60 Pro 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 TTT GTG CCC Asn Ser Leu I l e Gly Val Gly Val Leu 70 Ser Cys Val Phe Asn Ser Leu Ala Gly,Lys 80 He Cys Tyr Asp Ala Leu Asp Pro Ala Lys 90 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 Ala Lys Trp Lys Pro Trp Leu Lys Pro 100 Tyr,Leu Ala Val Cys Val Leu Phe Asn Val 110 Vat Leu Phe Leu Val Ala Leu Cys Cys Fhe 120 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 Leu.Arg Gly Ser Leu Glu Ser Thr Leu 130 Ala His Gly Leu Lys Asn Gly Met Lys Phe 140 Tyr Arg Asp Thr Asp Thr Pro Gly -rg Cys 150 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 Met Lys Lys Thr He Asp Met Leu Gin 160 He Gtu Phe Lys Cys Cys Gly Asn Asn Gly 170 Phe Arg Asp Trp Phe Glu H e Glr. Trp • le 180 Ser 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 Asn Arg Tyr Leu Asp Phe Ser Ser Lys 190 Glu Val Lys Asp Arg He Lys Ser Asn Val 200 Asp Gly Arg Tyr Leu Val Asp Gly Val Fro 210 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 Ser Cys Cys Asn Pro Asn Ser Pro Arg 220 Pro Cys He Gin Tyr Gin Leu Thr ATT> Asn 230 Ser Ala His Tyr Ser Tyr Asp His Gin Thr 240 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 Glu Leu Asn Leu Trp Leu Arg Gly Cys 250 Arg Ala Ala Leu Leu Ser Tyr Tyr Ser Asn 260 Leu Met ksn Thr (Thr Gly Ala Val Thr Leu 270 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 Val Trp Leu Phe Glu Val Thr He Thr 280 Val Gly leu,Arg Tyr Leu His Thr Ala Leu 290 Glu Gly Met [Ala Asn Pro Glu Asp Pro 300 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 Glu Ser Glu Gly 310r-Trp Leu)Leu Glu Lys Ser[Val Pro Glu Thr Trp Lys Ala Phe Leu 320 Glu Ser Val Lys Lys Leu Gly Lys Gly Asn 330 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 Glu Ala Glu Gly Glu Asp Ala Gly Gin Ala Pro Ala Ala 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 Z48 388 A m i n o acid number -42-DTT DTT + 33 kDa -- + - + Endo H Endo H F i g u r e 13. 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 . The 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 37°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 Endo H. S a m p l e s w e r e s e p a r a t e d on a n 8 % 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 t i c a l l y t r a n s f e r r e d 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 f o l l o w e d b y 1 2 5 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 r e d u c i n g a g e n t , p e r i p h e r i n m i g r a t e s a s a d i m e r . 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 when 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. -43-Blot Hybridization analysis Northern blot analysis of retinal poly-A RNA revealed two major transcripts that hybridized to peripherin cDNA probes (Figure 14). Two predominant bands of 6.5kb and 2.9kb are present. Both the number and large size of the peripherin transcripts were unexpected from the western blot analysis of ROS which had indicated that there i s only one protein of apparent molecular weight 33 kDa (Molday et a l . , 1987). The large 3'- untranslated region on some of the cDNA clones (Figure 10), however, is in agreement with the hybridization results. No hybridization was seen to bovine l i v e r RNA. Protein Homologies At the amino acid level, peripherin has 92.5% sequence identity to the gene identified by Travis et a l . (1989) as being responsible for the retinal degeneration slow defect in mice (Figure 15). No significant homology to any other protein was detected in 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 is 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-bp 9416 6682 4361-2322 2027 B 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 -a g a r o s e g e l e l e c t r o p h o r e s i s , t r a n s f e r r e d t o a n y l o n f i l t e r a n d 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 d e s c r i b e d i n " E x p e r i m e n t a l P r o c e d u r e s " . The s e v e n E c o R I f r a g m e n t s f r o m c l o n e s x ( . 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 , a n d t h e y g a v e s i m i l a r r e s u l t s . S i z e m a r k e r s w e r e d e n a t u r e d 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. -45-sequence with the peripherin sequence, however, revealed that the i n i t i a t o r methionine was the only amino acid missing from the mature protein sequence (Figure 15). A construct containing the complete open reading frame of peripherin, as based on the N-terminal amino acid sequence analysis of peripherin and the alignment with the rds sequence, was made from clones j L . l l , J..17,/. 18 and from a synthetic cassette (Figure 16). The synthetic cassette was designed to contain: BamHI and EcoRI cloning s i t e s , the Kozak consensus sequence for 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 (Kozak, 1989), the coding sequence for the f i r s t s i x amino acids that are not encoded by the cDNA sequence, and the sequence overlapping with clone A 1 8 up to the unique S t y l cloning s i t e . Three l i g a t i o n s were required to make the construct. F i r s t , the cassette was l i g a t e d with the 612bp S t y l - H i n d l l l fragment from ^ .18 into BamHI-Hindlll cut pUC19. Second, the 852 bp EcoRI - BstEII fragment from /..17 was l i g a t e d to the 492 bp BstEII - EcoRI fragment from/. 11 into EcoRI cut pUC19. The 341 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 were confirmed i n the f i n a l construct through DNA sequencing and r e s t r i c t i o n mapping (Figure 16). - 4 6 -PERIPHERIN- ALLKVKFDQKKRVKLAQGLWLMNWFSVLAGIIIFGLGLFLKIELRKRSD -49 RDS x^LLKVKFDQKKRVKLAQGLWLMNWLSVLAGIVLFSLGLFLKIELRKRSE -50 PERIPHERIN- VMNNSESHFVPNSLIGVGVLSCVFNSLAGKICYDALDPAKYAKWKPWLKP -99 RDS VMNNSESHFVPNSLIGVGVLSCVFNSLAGKICYDALDPAKYAKWKPWLKP -100 PERIPHERIN- YLAVCVLFNWLFLVALCCFLLRGSLESTLAHGLKNGMKFYRDTDTPGRC -149 RDS YLAVCIFFNVILFLVALCCFLLRGSLESTLAYGLKNGMKYYRDTDTPGRC -150 PERIPHERIN- FMKKTIDMLQIEFKCCGNNGFRDWFEIQWISNRYLDFSSKEVKDRIKSNV -199 RDS FMKKTIDMLQIE FKCCGNNGFRDWFEIQWISNRYLDFS S KEVKDRIKS NV -200 PERIPHERIN- DGRYLVDGVPFSCCNPNSPRPCIQYQLTNNSAHYSYDHQTEELNLWLRGC -249 RDS DGRYLVDGVPFSCCNPSSPRPCIQYQLTNNSAHYSYDHQTEEIiNLWLRGC -250 PERIPHERIN- RAALI^YYSNIJWTTGAVTLLVWLFEVTITVGLRYLHTALEGMANPEDPE -299 RDS PJ^LI^YYSSLJWSMGWTLLVWLFEVSITAGLRYLHTALESVSNPEDPE -300 PERIPHERIN- CESEGWLLEKSVPETWKAFLESVKKLGKGNQVEAEGEDAGQAPAAG -34 5 RDS CESEGWLLEKSVPETWKAFLESFKKLGKSNQVEAEGADAGPAPEAG -34 6 F i g u r e 15. An a l i g n m e n t o f t h e a m i n o a c i d s e q u e n c e o f t h e m a t u r e p e r i p h e r i n ( b o v i n e ) w i t h t h e p r o p o s e d r d s s e q u e n c e ( m o u s e ) . The s e q u e n c e s 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 f r o m t h e m a t u r e 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 two s e q u e n c e s may b e a t t r i b u t e d t o s p e c i e s p o l y m o r p h i s m . -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 restriction sites are shown for BamHI (Ba), Bst EII (Bs), EcoRI (E), Hin d l l l (H), Maell (M), and Styl (S). Identical sequence overlap among the cDNA clones i s represented by the bold lines and the sequence divergence by the narrow lines. The open boxed region of clone /.18 i s J^gtlO sequence that was not removed from the cDNA insert because one of the original EcoRI cloning sites was missing. The translational 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 for the efficient i n i t i a t i o n of translation (underlined) and the sequence for the f i r s t six amino acids that are not encoded by the cDNA sequence of clone X.18. Three ligations were required to make the construct. First, the 612 bp Styl - Hindlll fragment from clone A-18 was ligated with the cassette into BamHI -Hindlll cut pUC19. Second, the 852 bp EcoRI - BstEII fragment from clone X.17 was ligated to the 492 bp BstEII -EcoRI fragment from clone/.-H into EcoRI cut pUC19. The 341 bp BamHI - Maell fragment from the f i r s t ligation was then ligated to the 994 bp Maell - EcoRI fragment from the second ligation into BamHI - EcoRI cut pUC19. The ligations in the fina l construct were confirmed through restriction mapping and DNA sequencing (dashed arrows). -48-T G A „ B s I B a E A . 1 1 M B s A . 1 7 M H X . 1 8 100 bp GATCC^GAATTcfcACCATGGCGCTGCTCAAAGTCAAATTTGACCAGAAGAAGCGGGTCAAGTTGG'cC SYNTHETIC GCTTAAGGTGGTACCGCGACGAGTTTCAGTTTAAACTGGTCTTCTTCGCCCAGTTCAACCGGGTTC C ASS E TT E J — i CONSTRUCT - 4 9 -Expression of Peripherin in COS ce l l s Because of the unique position of peripherin in the disk membrane, the primary structural characteristics obtained from the cDNA sequence and i t s relationship to the rds defect, i t seems possible that peripherin could be the component in the growing c i l i a r y membrane that interacts with binding sites at or near the cilium (Figure 3). It 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 participate in the formation of the rim. To test this second possibility, work was directed towards expressing the complete coding sequence of peripherin in a COS c e l l line. If the aggregation of peripherin causes rim formation in the disk membrane, the same aggregation of peripherin within the COS c e l l plasma membrane could result in the formation of membrane infoldings. The expression of peripherin in COS cells should also c l a r i f y whether in 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 ligated into the pAX 111 vector. Western blot analysis was used to detect the peripherin produced by the transfected COS-1 cells (Figure 17). In the presence of a reducing agent there appears to be two bands produced by the COS ce l l s which comigrate on an SDS - polyacrylamide gel with the peripherin purified from rod outer segments. The relative intensity of the two bands produced by the COS - 5 0 -c e l l s , however, i s d i f f e r e n t from the rod outer segment peripherin. This may r e f l e c t a difference i n the glycosyl a t i o n of peripherin or some other type of post t r a n s l a t i o n a l modification. Rhodopsin when i t i s expressed i n COS c e l l s migrates at a s l i g h t l y higher molecular weight from the rod outer segment protein (Oprian et a l . 1987). In the absence of a reducing agent the expressed peripherin migrates as a dimer s i m i l a r to the peripherin from the rod outer segments. In both samples there i s some remaining monomer present. -51-+ 2 - M E - 2 - M E Figure 17. Western blot analysis of the peripherin expressed within the COS-1 c e l l s . COS-1 cells were transfected with the pAX 111 vector containing the peripherin coding sequence in either the correct (c) or wrong (d) orientation for expression. Approximately 7 ul of the COS c e l l postnuclear fractions along with 10 ug of total rod outer segment proteins (a) and 0.8 ug of the 2B6 antibody - Sepharose 2BC1 column purified peripherin (b) were loaded onto 8% SDS - polyacrylamide gels. The gels were run in 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 1 2 5 i goat antimouse Ig for autoradiography. -52-Discussion A cDNA sequence for peripherin, a membrane protein l o c a l i z e d i n the rim of rod outer segment disks, has been determined. The i n i t i a l cDNA clones were obtained by screening a bovine r e t i n a l expression l i b r a r y with antiperipherin monoclonal antibodies. These antibodies have been shown to bind to cone photoreceptor membrane as well as to the rod disk membranes (Hicks & Molday - unpublished r e s u l t s ) . Many of the major photoreceptor proteins are d i f f e r e n t i n the two c e l l types (Hurwitz et a l . , 1 9 8 5 ; Nathans et a l . , 1 9 8 6 ; Lerea et a l . , 1 9 8 6 ) , and i t i s possible that there could be more than one form of peripherin. The northern b l o t analysis i s i n agreement with t h i s p o s s i b i l i t y . Since over 90% of the photoreceptor c e l l s i n the bovine r e t i n a are rod c e l l s , i t i s more l i k e l y that the cDNA sequence presented here encodes the rod form of the protein rather than the cone form. One CNBr cleavage peptide that bound to the antiperipherin monoclonal antibody 2B6 was i s o l a t e d . The antigenic s i t e for t h i s antibody has been determined to be somewhere within the C-terminal 3 5 amino acids of peripherin. The amino acid sequence obtained from the i s o l a t e d C-terminal peptide i s i n agreement with that predicted from the cDNA sequence. I f there i s a d i f f e r e n t form of peripherin i n the cones, i t must eithe r be i n such a low abundance i n the outer segment preparation as not to be detected, or a l t e r n a t i v e l y i t could diverge from the rod -53-protein at a location further removed from the antigenic s i t e . Several divergent cDNA clones were i s o l a t e d that do not contain the 5' end of the coding sequence (Figure 10). If these clones were derived from true mRNA molecules, they would give r i s e to protein products that are truncated at the N-terminal end. The truncated protein products produced from these clones would s t i l l contain the antigenic s i t e s for the 2B6 and 3B6 antiperipherin monoclonal antibodies. No evidence was obtained for the existence of these truncated products from eit h e r the N-terminal sequence analysis of immunoaffinity p u r i f i e d peripherin or from the western b l o t t i n g of t o t a l outer segment proteins. The p o s s i b i l i t y e x i s t s , however, that these truncated forms could be present i n the r e t i n a at locations other then the photoreceptor outer segments. There i s no evidence from the immunocytochemical studies to support t h i s p o s s i b i l i t y . Other labs have had problems obtaining complete unscrambled clones from the two l i b r a r i e s that were used i n t h i s study (G. Khorana and D. Oprian - personal communications). I t , therefore, seems l i k e l y that these 5' divergent regions are cloning a r t i f a c t s . There are no consensus sequences for intron s p l i c e s i t e s at the points of sequence divergence which would have indicated that the cDNA clones had been derived 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 to cDNA in s e r t was not i n excess, several -54-= unrelated blunt ended cDNA fragments could have been l i g a t e d together within the vector. 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 the peripherin cDNA probes (Figure 14). I t i s possible that these two bands could 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 blot analysis of BALB/c mouse genomic DNA suggests that there could p o t e n t i a l l y be two genes that code fo r peripherin or possibly for peripherin and a related protein (Travis et a l . , 1989). A protein that has 36% amino acid sequence i d e n t i t y to peripherin has been i d e n t i f i e d i n the human ret i n a (R. Mclnnes - personal communication). This protein and any other peripherin related protein would not be detected on western blo t s i f the antigenic s i t e s f or the antiperipherin monoclonal antibodies, which are located on the C-terminal 35 amino acids, are not conserved. A l t e r n a t i v e l y , i t i s possible that the two t r a n s c r i p t s could have been the r e s u l t of the alternate s p l i c i n g of a single gene product. Several t r a n s c r i p t s that are the r e s u l t of the u t i l i z a t i o n of d i f f e r e n t 3' s p l i c e s i t e s are produced for rhodopsin and many of the neural,proteins. The purpose for 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 fo r the organization of peripherin i n the disk membrane i s outlined i n figure 18. The protein i s shown as having four transmembrane domains as predicted from the Kyte-Doolittle hydrophobicity p l o t . As with rhodopsin each -55-of the hydrophobic segments i s bordered on i t s carboxy -terminal side by a positi v e l y s c h a r g e d residue that i s capable of int e r a c t i n g with the phospholipid head groups (Nathans & Hogness, 1983). I t was possible to orientate the C-terminus of the protein r e l a t i v e to the membrane because the two antiperipherin monoclonal antibodies had been previously shown to bind on the cytoplasmic face of the protein (Molday et a l . , 1987). The antigenic s i t e s f or the same antibodies were also l o c a l i z e d to the C-terminal portion of the protein by the cDNA clones obtained from the immunological screening of the l i b r a r y . There are three p o t e n t i a l asparagine linked glycosylation s i t e s present on the protein, and i n the model presented i n figure 18, a l l of these s i t e s are located within the lumen of the disk. The use of glycosidases has indicated that there i s N - linked carbohydrate on peripherin which i s i n agreement with the placement of at lea s t one of these s i t e s within the lumen. The or i e n t a t i o n of the other features presented i n the model i s highly speculative since there i s only a small database on which to base the prediction of the presence of a transmembrane segment. In the model (Figure 18), there are seven cysteine residues placed within the lumen of the disk that would have the p o t e n t i a l to form intramolecular or intermolecular d i s u l f i d e bonds. The greater reducing p o t e n t i a l of the cytoplasm i s thought to i n h i b i t such interactions on the cytoplasmic side of the membrane. Molday et a l . (1987) -56-demonstrated that i n the absence of a reducing agent, peripherin migrates as a dimer. I t was not cl e a r at the time whether peripherin i s forming a heterodimer or a homodimer. The peripherin that i s expressed i n the COS c e l l s forms a dimer i n the absence of a reducing agent. I t , therefore, seems l i k e l y that the rod outer segment peripherin i s also forming a homodimer. The p o s s i b i l i t y of a mixture of heterodimers and homodimers i n the rod outer segment, however, cannot be excluded. The protein encoded by the cDNA sequence of peripherin would have a molecular weight close to 39 kDa. This value i s higher than the 33 kDa estimated from SDS - polyacrylamide gel electrophoresis. Peripherin migrates with an apparent molecular weight closer to 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) or when the rhodopsin i n the rod outer segment preparations i s removed with a mild Staphylococcus, aureus V-8 protease digestion (Molday et al.,1987). I t appears that rhodopsin, which makes up approximately 70% of the t o t a l ROS protein, could be e f f e c t i n g the e l e c t r o b l o t t i n g of peripherin to the Immobilon paper. The large quantity of rhodopsin could be saturating most of the s i t e s on the Immobilon paper so that only the leading edge of the peripherin band i s tansferred. As a r e s u l t , the peripherin i n the t o t a l ROS protein appears as a narrower band and appears to migrate faster on an SDS -polyacrylamide gel then the 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 of peripherin. The model shows the orientation of the protein within the disk membrane. The loc a t i o n of the antigenic s i t e s of the antiperipherin monoclonal antibodies (Mab) i s indicated. The t h i r t e e n cysteine residues of the sequence are highlighted. The negatively (•) and p o s i t i v e l y (0) charged amino acids and the three p o t e n t i a l s i t e s for asparagine linked glycosylation (^ ) are also shown. Intradisk (Lumen) side -59-(Figure 8). This e f f e c t i s also observed i n a comparison of lane a of figure 17 i n both the presence and absence of a sul f h y d r y l reducing agent. In the presence of a reducing agent, peripherin comigrates with rhodopsin and the e f f i c i e n c y of transfer i s si g n i c a n t l y less then when i n the absence of a reducing agent, peripherin, unlike rhodopsin, migrates as a dimer. During the immunoaffinity p u r i f i c a t i o n of peripherin, several higher molecular weight bands are v i s i b l e on the western blo t s (Figure 8). These bands are the r e s u l t of an aggregation of peripherin that occurs during the p u r i f i c a t i o n and are not usually observed i n western blots of t o t a l ROS protein obtained from f r e s h l y dissected r e t i n a . Since the presence of some of the higher molecular weight bands are not observed i n the presence of a su l f h y d r y l reducing agent (Figure 17), some of these aggregates may have formed as a r e s u l t of the formation of d i s u l f i d e bonds between adjacent peripherin molecules. Peripherin, l i k e rhodopsin does not have any obvious sign a l peptide. Very l i t t l e i s known about how protein sor t i n g takes place among the rod outer segment plasma membrane, the lamellar region of the disk membrane and the rim region of the disk membrane. Protein sorting among some of the c e l l u l a r organelles has been shown to be directed by s p e c i f i c amino acid sequences. Membrane targeting sequences have been i d e n t i f i e d for the transportation of proteins into the endoplasmic reticulum (Munro & Pelham, 1987), the -60-mitochondria (Hartl et a l . , 1989), and the nucleus (Gomez-Marquez & Segade, 1988). The disk membranes of the rod outer segments could be viewed as cellular organelles. If the peripherin that i s being expressed in the COS cel l s i s found to be localized to the plasma membrane, i t would suggest that there i s not a disk specific signal sequence and that protein sorting among the 3 membrane domains occurs after the i n i t i a l insertion into the plasma membrane. Photoreceptor c e l l peripherin at the amino acid level was shown to have 92.5% sequence identity to the gene proposed to be responsible for the rds defect. Most of the differences between the two sequences represent f a i r l y conservative changes, and these changes are scattered throughout the sequence. One of the consensus sequences for asparagine linked glycosylation i s not conserved in the rds sequence. This suggests that this site may not be ut i l i z e d in 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 substitution of an alanine for a glutamic acid residue at amino acid position 336 i s compensated by the opposite substitution at position 345. This i s suggestive that the conservation of the net negative charge on this part of the protein may be important for the function of peripherin. The rds gene product and i t s intracellular localization had not previously been identified. - 6 1 -The function of peripherin i s not known. The l o c a l i z a t i o n of peripherin to the rim region of the disk membrane and the phenotype associated with the rds defect i s suggestive that i t may play a ro l e i n anchoring the disks to the cytoskeletal system of the rod c e l l . The C-terminal cytoplasmic domain of peripherin i s highly charged, and i t i s possible that i t may be int e r a c t i n g with a cytoskeletal component. Electron microscopic studies have indicated that there are filamentous structures extending from the rims of the disks (Roof & Heuser, 1982), and a 240 kDa spectrin -l i k e protein has been suggested to play a r o l e i n disk -membrane interactions (Wong & Molday, 1986). A l t e r n a t i v e l y , charge repulsion among adjacent peripherin molecules on the cytoplasmic surface may cause curvature i n the disk membrane, and t h i s could a i d i n the formation of the rim region. Interactions of the sulfhydryl groups or carbohydrate chains that are present on peripherin within the lumen of the disk may also help to form the disk rim. Disk morphogenesis i n Xenopus and frog r e t i n a has been shown to be disrupted by tunicamycin, an i n h i b i t o r of N-1inked glycosylation ( F l i e s l e r et a l . , 1984; F l i e s l e r et a l . , 1985a). In tunicamycin - treated r e t i n a , membrane v e s i c l e s are formed i n the space between the rod inner and outer segments where disk formation would normally occur ( F l i e s l e r et a l . , 1985a). F l i e s l e r et al.,(1985b) suggested that the carbohydrate chains of rhodopsin on the opposing disk -62-membrane faces may int e r a c t and cause membrane adhesion. The in t e r a c t i o n of the opposing membrane faces appears to be greatest near the rim of the disk. Although rhodopsin and other disk proteins may be involved i n membrane adhesion, the l o c a l i z a t i o n of peripherin to the rim makes i t a prime candidate as the major adhesive molecule i n the membrane. - 6 3 -Conclusion In this study the cDNA sequence of peripherin was determined. This sequence 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 integral 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 localized to the cytoplasmic 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 in the absence of a reducing agent, and i t was also shown to be the defective protein responsible for the retinal degeneration slow defect. On the basis of the localization of peripherin to the rim region of the disk membrane, the primary structural characteristics of peripherin and the phenotype associated with the retinal degeneration slow defect, i t was proposed that peripherin may play a role in disk morphogenesis. It was suggested that peripherin could serve to anchor the disks to the cytoskeletal system of the outer segment. 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