RS1, a Discoidin Domain-containing Retinal Cell Adhesion Protein Associated with X-linked Retinoschisis, Exists as a Novel Disulfide-linked Octamer (2005)

RS1, also known as retinoschisin, is an extracellular protein that plays a crucial role in the cellular organization of the retina. Mutations in RS1 are responsible for X-linked retinoschisis, a common, early-onset macular degeneration in males that results in a splitting of the inner layers of the retina and severe loss in vision. RS1 is assembled and secreted from photoreceptors and bipolar cells as a homo-oligomeric protein complex. Each subunit consists of a 157-amino acid discoidin domain flanked by two small segments of 39 and 5 amino acids. To begin to understand how the structure of RS1 relates to its role in retinal cell adhesion and X-linked retinoschisis, we have determined the subunit organization and disulfide bonding pattern of RS1 by SDS gel electrophoresis, velocity sedimentation, and mass spectrometry. Our results indicate that RS1 exists as a novel octamer in which the eight subunits are joined together by Cys59-Cys223 intermolecular disulfide bonds. Subunits within the octamer are further organized into dimers mediated by Cys40-Cys40 bonds. These cysteines lie just outside the discoidin domain indicating that these flanking segments primarily function in the octamerization of RS1. Within the discoidin domain, two cysteine pairs (Cys63-Cys219 and Cys110-Cys142) form intramolecular disulfide bonds that are important in protein folding, and one cysteine (Cys83) exists in its reduced state. Because mutations that disrupt subunit assembly cause X-linked retinoschisis, the assembly of RS1 into a disulfide-linked homo-octamer appears to be critical for its function as a retinal cell adhesion protein. RS1, also known as retinoschisin, is an extracellular protein that plays a crucial role in the cellular organization of the retina. Mutations in RS1 are responsible for X-linked retinoschisis, a common, early-onset macular degeneration in males that results in a splitting of the inner layers of the retina and severe loss in vision. RS1 is assembled and secreted from photoreceptors and bipolar cells as a homo-oligomeric protein complex. Each subunit consists of a 157-amino acid discoidin domain flanked by two small segments of 39 and 5 amino acids. To begin to understand how the structure of RS1 relates to its role in retinal cell adhesion and X-linked retinoschisis, we have determined the subunit organization and disulfide bonding pattern of RS1 by SDS gel electrophoresis, velocity sedimentation, and mass spectrometry. Our results indicate that RS1 exists as a novel octamer in which the eight subunits are joined together by Cys59-Cys223 intermolecular disulfide bonds. Subunits within the octamer are further organized into dimers mediated by Cys40-Cys40 bonds. These cysteines lie just outside the discoidin domain indicating that these flanking segments primarily function in the octamerization of RS1. Within the discoidin domain, two cysteine pairs (Cys63-Cys219 and Cys110-Cys142) form intramolecular disulfide bonds that are important in protein folding, and one cysteine (Cys83) exists in its reduced state. Because mutations that disrupt subunit assembly cause X-linked retinoschisis, the assembly of RS1 into a disulfide-linked homo-octamer appears to be critical for its function as a retinal cell adhesion protein. X-linked retinoschisis (XLRS) 1The abbreviations used are: XLRS, X-linked retinoschisis; CHAPS, 3-[(3-cholamidopropyl)dimethylammoniol]-1-propanesulfonate; WT, wild-type; rWT, recombinant WT; PBS, phosphate-buffered saline; ER, endoplasmic reticulum; NEM, N-ethylmaleimide; DTT, dithiothreitol; IAM, iodoacetamide; MS, mass spectrometry; LC, liquid chromatography; MALDI-TOF, matrix-assisted laser desorption/ionization time-of-flight; MOPS, 4-morpholinepropanesulfonic acid; bicine, N,N-bis(2-hydroxyethyl)glycine; Bis-Tris, 2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)propane-1,3-diol.1The abbreviations used are: XLRS, X-linked retinoschisis; CHAPS, 3-[(3-cholamidopropyl)dimethylammoniol]-1-propanesulfonate; WT, wild-type; rWT, recombinant WT; PBS, phosphate-buffered saline; ER, endoplasmic reticulum; NEM, N-ethylmaleimide; DTT, dithiothreitol; IAM, iodoacetamide; MS, mass spectrometry; LC, liquid chromatography; MALDI-TOF, matrix-assisted laser desorption/ionization time-of-flight; MOPS, 4-morpholinepropanesulfonic acid; bicine, N,N-bis(2-hydroxyethyl)glycine; Bis-Tris, 2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)propane-1,3-diol. is a common, inherited macular degeneration that affects males early in life (1George N.D. Yates J.R. Moore A.T. Br. J. Ophthalmol. 1995; 79: 697-702Crossref PubMed Scopus (170) Google Scholar, 2Sieving P.A. Traboulsi E.I. Genetic Diseases of the Eye. Oxford University Press, New York1998Google Scholar, 3Tantri A. Vrabec T.R. Cu-Unjieng A. Frost A. Annesley Jr., W.H. Donoso L.A. Surv. Ophthalmol. 2004; 49: 214-230Abstract Full Text Full Text PDF PubMed Scopus (152) Google Scholar). Affected individuals show a significant loss in central and in some cases peripheral vision, a splitting of the inner layers of the retina, and a loss in the b-wave of the electroretinogram. The gene responsible for XLRS was identified by positional cloning and shown to encode a retinal-specific 224-amino acid protein, known as RS1 or retinoschisin, containing a discoidin domain (4Sauer C.G. Gehrig A. Warneke-Wittstock R. Marquardt A. Ewing C.C. Gibson A. Lorenz B. Jurklies B. Weber B.H. Nat. Genet. 1997; 17: 164-170Crossref PubMed Scopus (402) Google Scholar). RS1 is expressed and secreted from photoreceptor cells of the outer retina and bipolar cells of the inner retina as a multisubunit protein (5Reid S.N. Akhmedov N.B. Piriev N.I. Kozak C.A. Danciger M. Farber D.B. Gene (Amst.). 1999; 227: 257-266Crossref PubMed Scopus (65) Google Scholar, 6Molday L.L. Hicks D. Sauer C.G. Weber B.H. Molday R.S. Invest. Ophthalmol. Vis. Sci. 2001; 42: 816-825PubMed Google Scholar, 7Reid S.N. Yamashita C. Farber D.B. J. Neurosci. 2003; 23: 6030-6040Crossref PubMed Google Scholar, 8Grayson C. Reid S.N. Ellis J.A. Rutherford A. Sowden J.C. Yates J.R. Farber D.B. Trump D. Hum. Mol. Genet. 2000; 9: 1873-1879Crossref PubMed Scopus (121) Google Scholar). The secreted protein associates with the surface of rod and cone photoreceptors at the level of the inner segment, outer nuclear, and outer plexiform layers and the surface of bipolar cells within the inner nuclear and inner plexiform layers of the retina. Biochemical studies further show that RS1 is tightly associated with the membrane fraction of retinal cell homogenates (6Molday L.L. Hicks D. Sauer C.G. Weber B.H. Molday R.S. Invest. Ophthalmol. Vis. Sci. 2001; 42: 816-825PubMed Google Scholar). RS1 is generally believed to function as a retinal cell adhesion protein, because mice deficient in RS1 have a highly disorganized retina with displacement of bipolar cells into the outer retinal layer, gaps between bipolar cells within the inner retina, disruption of the photoreceptor-bipolar synapse, and progressive degeneration of rod and cone photoreceptors (9Weber B.H. Schrewe H. Molday L.L. Gehrig A. White K.L. Seeliger M.W. Jaissle G.B. Friedburg C. Tamm E. Molday R.S. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 6222-6227Crossref PubMed Scopus (175) Google Scholar). The dominant structural feature of the RS1 polypeptide is the 157-amino acid discoidin domain, also known as an F5/8 type C domain, which comprises over 75% of the processed polypeptide chain (4Sauer C.G. Gehrig A. Warneke-Wittstock R. Marquardt A. Ewing C.C. Gibson A. Lorenz B. Jurklies B. Weber B.H. Nat. Genet. 1997; 17: 164-170Crossref PubMed Scopus (402) Google Scholar). Discoidin domains are present in a wide range of membrane and extracellular proteins where they mediate a variety of cell adhesion and cell signaling processes (10Vogel W. FASEB J. 1999; 13: S77-S82PubMed Google Scholar, 11Baumgartner S. Hofmann K. Chiquet-Ehrismann R. Bucher P. Protein Sci. 1998; 7: 1626-1631Crossref PubMed Scopus (164) Google Scholar). Some proteins that contain discoidin domains are Factors V and VIII involved in blood coagulation, neuropilins 1 and 2, which mediate nervous system regeneration and degeneration, discoidin domain receptors implicated in cancer metastasis, and discoidin I involved in cellular adhesion during slime mold differentiation and development (10Vogel W. FASEB J. 1999; 13: S77-S82PubMed Google Scholar, 11Baumgartner S. Hofmann K. Chiquet-Ehrismann R. Bucher P. Protein Sci. 1998; 7: 1626-1631Crossref PubMed Scopus (164) Google Scholar, 12Fuentes-Prior P. Fujikawa K. Pratt K.P. Curr. Protein Pept. Sci. 2002; 3: 313-339Crossref PubMed Scopus (65) Google Scholar, 13Lee C.C. Kreusch A. McMullan D. Ng K. Spraggon G. Structure. (Camb.). 2003; 11: 99-108Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar). Still other members of the discoidin family play a role in key physiological processes ranging from heart development, milk lactation, and sperm-egg adhesion in vertebrates, to immunity and metamorphosis in the silkworm, and to post-fertilization events in the sea urchin zygote (14Yamakawa M. Tanaka H. Dev. Comp. Immunol. 1999; 23: 281-289Crossref PubMed Scopus (71) Google Scholar, 15Matese J.C. Black S. McClay D.R. Dev. Biol. 1997; 186: 16-26Crossref PubMed Scopus (48) Google Scholar, 16Shur B.D. Ensslin M.A. Rodeheffer C. Curr. Opin. Cell Biol. 2004; 16: 477-485Crossref PubMed Scopus (30) Google Scholar, 17Hvarregaard J. Andersen M.H. Berglund L. Rasmussen J.T. Petersen T.E. Eur. J. Biochem. 1996; 240: 628-636Crossref PubMed Scopus (134) Google Scholar). Bioinformatic studies have also identified a number of discoidin domain-containing proteins of unknown function (11Baumgartner S. Hofmann K. Chiquet-Ehrismann R. Bucher P. Protein Sci. 1998; 7: 1626-1631Crossref PubMed Scopus (164) Google Scholar). The high resolution structures of the C2 discoidin domain of Factors V and VIII and the b1 discoidin domain of neuropilin 1 have been determined (13Lee C.C. Kreusch A. McMullan D. Ng K. Spraggon G. Structure. (Camb.). 2003; 11: 99-108Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar, 18Macedo-Ribeiro S. Bode W. Huber R. Quinn-Allen M.A. Kim S.W. Ortel T.L. Bourenkov G.P. Bartunik H.D. Stubbs M.T. Kane W.H. Fuentes-Prior P. Nature. 1999; 402: 434-439Crossref PubMed Scopus (220) Google Scholar, 19Pratt K.P. Shen B.W. Takeshima K. Davie E.W. Fujikawa K. Stoddard B.L. Nature. 1999; 402: 439-442Crossref PubMed Scopus (283) Google Scholar). These domains consist of eight antiparallel β-strands arranged in a barrel-like structure with two or three loops, or “spikes,” projecting from one end of the core structure. In the case of Factors V and the are involved in the of these proteins to the surface of as a key in the blood P. Fujikawa K. Pratt K.P. Curr. Protein Pept. Sci. 2002; 3: 313-339Crossref PubMed Scopus (65) Google Scholar). The discoidin domain of RS1 been from the C2 discoidin domain of Factors V and VIII Molday R.S. J. Biol. 2003; Full Text Full Text PDF PubMed Scopus Google Scholar, L. G. 2003; PubMed Scopus Google these the structure consists of eight core and three In to its discoidin domain, RS1 a acid or by a acid known as the The plays an role in the of the RS1 polypeptide chain into the is in the of the by a as a key in the of RS1 from cells (4Sauer C.G. Gehrig A. Warneke-Wittstock R. Marquardt A. Ewing C.C. Gibson A. Lorenz B. Jurklies B. Weber B.H. Nat. Genet. 1997; 17: 164-170Crossref PubMed Scopus (402) Google Scholar, Molday R.S. J. Biol. 2003; Full Text Full Text PDF PubMed Scopus Google Scholar). The domain cysteine of these an intermolecular disulfide with of subunit to form a disulfide-linked homo-oligomeric Molday R.S. J. Biol. 2003; Full Text Full Text PDF PubMed Scopus Google Scholar). The or function of the three other cysteines in the domain been To mutations in the RS1 gene have been associated with XLRS at or cells RS1 have been used to understand how mutations cause Molday R.S. J. Biol. 2003; Full Text Full Text PDF PubMed Scopus Google Scholar, K. Trump D. Hum. Mol. Genet. 2002; 11: PubMed Scopus Google Scholar). mutations in the RS1 discoidin domain in severe of the protein and in the Mutations in the of RS1 into the membrane in of these to the and K. Trump D. Hum. Mol. Genet. 2002; 11: PubMed Scopus Google Scholar). two cysteine mutations and outside the discoidin domain protein or from cells Molday R.S. J. Biol. 2003; Full Text Full Text PDF PubMed Scopus Google Scholar). these to into a multisubunit that is crucial for the of RS1 as an extracellular adhesion protein. studies have shown that RS1 exists as a disulfide-linked protein, the number and of these subunits in the and the role of intramolecular and intermolecular disulfide bonds and in the assembly and of the RS1 have been In we have used and together with velocity and by mass to into the structure of retinal and recombinant RS1 and we show that RS1 an structure of disulfide-linked dimers within a disulfide-linked complex. involved in the of key intramolecular and intermolecular disulfide are RS1 and the RS1 was into the the and The was used to the cysteine the the and and the cysteine to the of the mutations and of The was and to as (9Weber B.H. Schrewe H. Molday L.L. Gehrig A. White K.L. Seeliger M.W. Jaissle G.B. Friedburg C. Tamm E. Molday R.S. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 6222-6227Crossref PubMed Scopus (175) Google Scholar). Cell and Protein of cells known as cells with and RS1 as Molday R.S. J. Biol. 2003; Full Text Full Text PDF PubMed Scopus Google Scholar). The cellular fraction of the protein was by the cells in by The was in of containing and to an of containing NEM, and for at the was at in a for and the was for SDS gel The secreted fraction of the protein was by of the at in a at for to membrane The for with the with and protein was with SDS in and in a in the or of and or SDS or SDS as or SDS a of and was the was used SDS was with and was also to for in containing of the a gel was and the protein was for in containing with milk in and with the containing milk in and for 1 The and with to containing milk in and SDS for for by the octamer the a gel MOPS, and 1 as the containing bicine, Bis-Tris, 1 and The form of retinal RS1 and the form of the RS1 of RS1 from Cell RS1 and the as in The containing was the with was with containing The containing was a for or The secreted RS1 protein was with at and in the of the cell The protein was at of RS1 from in containing by at in an The cells as the was in of NEM, and and for by a to a To the membrane the cells to a and for at in a The at the of the and with by at in a The with and at for The with was and retinal RS1 was with containing CHAPS, and The was at and the was and to the and of RS1 from was from retina an as and with as The cells in containing CHAPS, NEM, and for and at for The was with of the for 1 and in of and with containing the was in SDS and by SDS gel of retinal RS1, recombinant RS1, and RS1 containing the a containing at for and in a retinal RS1 was by and as The secreted fraction of and RS1 was by as to velocity a mass of was as a for at in a to the to and with the The of the was with the in fraction was determined by to the of protein the of of RS1 for was by the RS1 with a The gel with to in a of and and the with the with for or by for with and from The gel containing the with the at for The was and the gel with and 5 also The for at The was in a and the gel with and with The in a and in of mass RS1 was to an The was and with two of acid in a with and a laser of was used for of the RS1 as and and in with a and from was used for and in and in at with a to over to at and for to B. The used and of gel also to to and other RS1 as a RS1 was shown to SDS as a and a high Molday R.S. J. Biol. 2003; Full Text Full Text PDF PubMed Scopus Google Scholar). To the of the RS1 a disruption of the was secreted from and shown in the secreted a of eight ranging from the to the protein. The secreted fraction of cells RS1 was also by SDS gel shown in a of was also for protein for with the the the for RS1, to the of the by the dominant complex. was by the of the mass for the the of the at which these G. T.E. Protein Press, Scholar). the the number of subunits in the of the was to be that RS1, the exists as a disulfide-linked of in we that the and of RS1 are responsible for disulfide-linked assembly Molday R.S. J. Biol. 2003; Full Text Full Text PDF PubMed Scopus Google Scholar). of these cysteine with in a loss of the disulfide-linked and the of a disulfide-linked SDS To which of the one or cysteine in RS1 are responsible for we of cysteine and with a in a and the cellular and secreted protein from These cysteines because studies that the cysteines and are involved in intramolecular disulfide bonds within the discoidin domain Molday R.S. J. Biol. 2003; Full Text Full Text PDF PubMed Scopus Google Scholar). of cellular and SDS and as the from the secreted fraction as a the three containing an or as a of dimers and the containing a as the in the cellular and secreted These studies that of subunits are involved in loss in present in the and and to a the from structural which the of intermolecular disulfide bonds. of these cysteine an protein just the in the cellular fraction of 2, in the secreted to a that SDS to the of intramolecular disulfide bonds. is by the system of the as a protein and is secreted from the RS1 within the that intermolecular disulfide bonds are been and and between of two In one the Cys59-Cys223 disulfide bonds are responsible for octamer and disulfide bonds form between two or subunits within the In the Cys59-Cys223 intermolecular disulfide bonds in the of further together in a disulfide bonds to form the the one that RS1 containing a form an for the in To between these we expressed a which is in disulfide-linked for and SDS that the secreted RS1, as a and a octamer results for the These results the in which an octamer is Cys59-Cys223 disulfide bonds in the of mediated RS1 from RS1 from retina a disulfide-linked structure as recombinant protein, we the of these with the and shown in a fraction of RS1 from the retina as a and a octamer to recombinant RS1. a significant fraction of retinal RS1 a of RS1 as a and fraction as a between the and at or for 5 in the of and SDS the in the retinal RS1 a was the was by Reid S.N. Yamashita C. Farber D.B. J. Neurosci. 2003; 23: 6030-6040Crossref PubMed Google for retinal These for the recombinant or protein expressed in cells they in RS1 from cells The pattern of RS1 from retina was also for and retina indicating that the in the retina are to from or from the of These that a of RS1 expressed in photoreceptor bipolar cells a type of of RS1 studies to RS1 exists as an octamer or a 5 for retinal and recombinant RS1 from of from a The recombinant protein just of retinal RS1 and used as a that retinal and RS1 as a disulfide-linked octamer as as and form for the small in and for retinal RS1 to the recombinant protein The with its a as that play a significant role in octamer or of RS1 and of the by further the disulfide bonding pattern of RS1, the reduced and octamer of RS1 from retina with and the by and in I and in and identified from of RS1. of for RS1 and of for RS1 to the of the processed RS1 that at as of the protein The from the RS1 discoidin domain In a to a mass of was was in with the mass of the from to The of the in the of retinal of reduced RS1 retinal RS1 was with in the of and an reduced with DTT, with IAM, and with MS, reduced with and with for and the mass is the mass with for the of the was from a of and was from a of The are for the reduced and the was from a of and was from a of The are for the reduced and the was from a of and was from a of The are for the reduced and the in a of from reduced and RS1. retina was with in the of and a with RS1 was The RS1 was and with with was also by mass in the and in the reduced form with and with to the the and the are The mass of from RS1 is shown in In to identified in reduced RS1, a at of was to a containing disulfide to a containing the mass of the a mass of is from the of the of these two to of two disulfide at of to the disulfide-linked with the the in an state. To that these contain a disulfide we reduced with and the cysteines with these the at of and and a at The mass of is with the containing an The containing the was by mass was by of RS1 in the of RS1 was with and with or The a with an of and the a with an of and These to containing that have been with that the within the discoidin domain of RS1 exists in its reduced state. that have to to the containing one or amino acids. In we have the structure of RS1 as an important in how extracellular protein in retinal cell adhesion and how mutations in RS1 cause RS1 is of eight subunits that are together intermolecular disulfide bonds between and These disulfide bonds are for octamer because of and with octamerization of RS1 as by and as by velocity In to the Cys59-Cys223 intermolecular disulfide RS1 a Cys40-Cys40 disulfide responsible for is of and octamerization is of because in the of the other RS1 is of disulfide-linked dimers within a disulfide-linked structure. The of the of the high resolution structure of RS1. The cysteine involved in these intermolecular disulfide bonds are in segments that the discoidin domain, the acid domain and a acid of the discoidin domain a function of these flanking is to RS1 into a disulfide-linked appears to be for the function of RS1 in retinal cell because the and which the and of RS1, are known to cause XLRS Molday R.S. J. Biol. 2003; Full Text Full Text PDF PubMed Scopus Google Scholar, R. G. S. P.A. Hum. 1999; PubMed Scopus Google Scholar, Hum. Mol. Genet. 1998; 7: PubMed Scopus Google Scholar). is is critical for RS1 because of with folding, or octamerization of RS1 Molday R.S. J. Biol. 2003; Full Text Full Text PDF PubMed Scopus Google and to mutations at have been is that to the of the be critical for its for retinal cell adhesion to In to cysteine at the RS1 domain two cysteine at and of these with octamerization Molday R.S. J. Biol. 2003; Full Text Full Text PDF PubMed Scopus Google and affects as shown to the of and by mass spectrometry. is that these form an intramolecular disulfide or intermolecular disulfide bonds that the and cysteine in reduced state. is highly in RS1 proteins that have been to The discoidin domain is the of the RS1 protein. the discoidin domains of other RS1 cysteine at the and the end of its discoidin resolution structures of the discoidin domains of and neuropilin 1 indicate that these cysteines the and end of the discoidin domain form an intramolecular disulfide Because and have been shown to in to other in studies of RS1 and are for protein Molday R.S. J. Biol. 2003; Full Text Full Text PDF PubMed Scopus Google is that these cysteines form an intramolecular disulfide in the RS1 In to and the RS1 discoidin domain cysteine The and in and been to form intramolecular disulfide and Molday R.S. J. Biol. 2003; Full Text Full Text PDF PubMed Scopus Google Scholar). been in the present the mass of a containing from RS1. The function of the in the RS1 discoidin domain to be in the C2 discoidin domain of Factors V and VIII are known to into the of as of the blood of RS1 with the C2 domain of Factors V and VIII in the of the P. Fujikawa K. Pratt K.P. Curr. Protein Pept. Sci. 2002; 3: 313-339Crossref PubMed Scopus (65) Google together with have to the that RS1 with in a L. G. 2003; PubMed Scopus Google Scholar). the disulfide be important in the of RS1 for into the cysteine in the discoidin domain of other is present at of the RS1 discoidin of and that cysteine is in its reduced state. is with which that is within the protein structure S. Bode W. Huber R. Quinn-Allen M.A. Kim S.W. Ortel T.L. Bourenkov G.P. Bartunik H.D. Stubbs M.T. Kane W.H. Fuentes-Prior P. Nature. 1999; 402: 434-439Crossref PubMed Scopus (220) Google Scholar). of with the folding, or assembly of RS1, that cysteine be for the structure or function of RS1. to mutations at have been the role of cysteines in disulfide bonding is shown in Our results a of events to the of RS1 from cells in the case of secreted the the and of the RS1 polypeptide chain the in the the at to the processed RS1 The of the discoidin domain with the of the and intramolecular disulfide bonds. intermolecular disulfide bonds Cys40-Cys40 and Cys59-Cys223 within the segments flanking the discoidin domain form to the RS1 octamer for from Our studies that a of RS1 in retina an the of which to be studies indicate that the and of RS1 the system for that the of the RS1 discoidin domain of disulfide-linked subunit the function of RS1 as a cell adhesion protein The of the RS1 homo-octamer adhesion its into the surface of the multisubunit of RS1 its to the surface of a cell by of In RS1 with other extracellular proteins or to cell adhesion and the cellular of the retina H. Weber for the RS1