Exp. Eye Res. (1990)

50, 751-757

S-Antigen: TOSHIMICHI

SHINOHARA”,

From VIJAY TOHRU

Gene

to Autoimmune

Uveitis

K. SINGH, MASAHIKO TSUDA, ABE AND SHUJI SUZUKI

KUNIHIKO

YAMAKI,

Molecular Biology Section, Laboratory of Retinal Cell and Molecular Biology, National Eye Institute, National Institutes of Health, Bethesda, MD 20892, U.S.A. Retinal S-antigen(S-Ag) is capableof inducing experimentalautoimmuneuveitis (EAU) in laboratory animals.FAU may serveas an animal modelfor studying human uveitis. As a first stepwe have determinedthe nucleotidesequenceof an S-Ag geneand its cDNAs.The amino acidsequences werededucedfrom the cDNAsof various animalsand human. Four uveitopathogenicsites in bovine S-Ag were characterized.One of the sites(peptideM) has sequencehomology with non-self proteinsfrom baker’syeast, potato, E. coIi, hepatitisB virus, moloneymurine leukemiavirus, Moloney murine sarcomavirus, AKR murine leukemiavirus and baboonendogenousvirus. Mononuclear cells from animalsimmunizedwith peptideM showedsignificantproliferationwhen incubatedwith synthetic peptidescorrespondingto the aminoacid sequences of the above-mentionedforeign proteins.In addition. all the peptidesinducedEAUin Lewisrats with a doseof 10-2000 pg. Moreover,native histoneH3 from baker’s yeast histone H3 induced EAU in Lewis rats. Thus, we found severalexamplesof antigenic mimicry between self and non-self proteins. These findings establisha base to study further the mechanismof autoimmuneinflammation. Keywords: autoimmunediseases ; experimentalautoimmuneuveitis (EALJ): S-antigen; gene; molecular mimicry.

1. Introduction Uveitis comprises a diverse group of numerous intraocular inflammations and is a major cause of visual impairment, affecting approximately 10% of visually handicapped patients in the U.S.A. Although the etiology of most uveitis is unknown, T-cell mediated autoimmune processesare thought to play a major role in the pathogenesis of certain types of uveitis in humans (seeFaure, 1980: Gery, Mochizuki and Nussenblatt, 1986). S-Ag, a potent uveitopathogenic antigen was purified from bovine retina (Wacker et al., 19 77 ; Dorey and Faure, 1977). It is capable of inducing experimental autoimmune uveitis (EAU) and experimental autoimmune pinealitis (EAP). S-Ag is also known as the ’ 4%kDa-protein ’ or ‘Arrestin’. an abundant photoreceptor, (Kuhn, Hall and Wilden, 1984 ; Pfister et al., 198 5) and pineal gland (Kalsow and Wacker, 1977, 1978) protein involved in visual transduction. In this report, we summarize our recent work on EAU induced by S-Ag and discussthe hypothesis of molecular mimicry between self and non-self antigens in ocular uveitis. 2. Hypothesis of Molecular Mimicry Although many theories have been proposedfor the induction of autoimmunity, the etiologies of these conditions are unknown. One mechanism for the development of autoimmune uveitis may involve ‘ molecular mimicry ‘. whereby selected antigenic * For correspondence. 00144835/90/060751+07

%03.00/O

determinants (epitope) from non-self, such as viruses and bacteria, have similar structures with selfepitopes. Non-self antigens can elicit the formation of antibodies or effector lymphocytes, which in turn may react with similar epitopes of a self antigen. These epitopes are similar enough to stimulate the immune system but different enough to escapefrom immunological tolerance. This processdoes not require either an ongoing replicating agent or the continuous presence of the immunogen. An ongoing cycle could be initiated and causing tissue injury by an autoimmune responsethat, in turn, could releasemore self antigen inducing additional lymphocyte responses (Fujinami and Oldstone, 198 5 ; Oldstone and Notkins, 1986: Oldstone, 1987; Cohen, 1988). 3. Experimental Animal Models for Human Uveitis Animal models offer the best hope of understanding the mechanisms of autoimmunity. EAU is such an animal model for studying certain types of human uveitis. EAU is induced in someanimals by an injection of microgram amounts of S-Ag with complete Freund’s adjuvant (CFA) (Wacker et al., 1977). The pathology of EAU is characterized by an inflammation in the retina with loss of photoreceptor cells (Faure, 1980; Gery et al., 1986). Although humoral antibodies (B-cells) may play a role in the development of EAU (De Kozak et al., 19 76, 1979), it has been reported that helper T cells are primary mediators (Mochizuki et al., 1984, 1985; Chan et al., 1985 ; Caspiet al., 1986). Cytotoxic T-cells are believed to mediate the delayed type hypersensitivity (DTH) (Mochizuki et al., 1985). 0 1990 AcademicPressLimited

752

T. SHINOHARA

1 Bo. Hu. MO.

Ra.

10

20

30

40

ET AL

50

MKANKPAPNHVIFKKISRDKSVTIYLGKRDYIDHVERVEPVDGWLVDPE MAASG:AS:SEPN::::::I::::::::I::N:::I:::SQ:Q::::::::::D MAACG:TN:S---::::::V::::::::Y::K:::V:::SQ:E::::::::::E MAACV:TN:S---::::::V::::::::Y::K:::I:::SQ:E::::::::::E

60

70

80

90

LVKGKRVYVSLTCAFRYGQEDIDVMGLSFRRDLYSFRRDLYSFQVQVFPPVGASGAT .. .. .. .. .*K:::T:::::::::-:V:WI::T::::::::R:::Y:::::ASTP .. .. .. .. .. K:::T:::::::::E:I:VM::T::::::::R:::Y:::::MSVL .. .. .. .. .. K:::T:::::::::E:I:VI::T::::::::R:::Y:::::MSAP 110 120 130 140 TRLQESLIKKLGANTYPFLLTFPDYLPCSVMLQPAPQDVGKSCGVDFEIK :K::E::L::::S:::::::::::::::::::::::::S:::::::::V: :Q::E::L::::D:::::::::::::::::::::::::V:::::::::V: :Q::L::R::::D:::::::::::::::::::::::::V:::: :::::V: 160 170 180 190 AFATHSTDVEEDKIPKKSSVRLLIRKVQHAPRDMGPQPRAEASWQFFMSD :::TDS::A::::::::::::S:::S::RW:LEL::::R::::::::::E :::SDI::P::::::::::::L:::K::HA:PEM::::S::::::::::D :::TDI::A::::::::::::L:::K::HA:PEM::::C::::::::::D 210 220 240 230 KPLRLAVSLSKEIYYHGEPIPVTVAVTNSTEKTVKKIKVLVEQVTNWLY :::H:A:C:TR:TNF:::::::::D:::N:E:T:::::ASW::VA::::: :::N:S:S:SK:IYF:::::::::T:::N:D:V:::::VSV::IA::::: . ...H.S:S:SK:IYF:::::::::T:::N:E:V:::::VSV::IA::::: .

260

270

280

290

100

150

200

250

300

SSDYYIKTVAAEEAQEKVPPNSSLTKTLTLVPLLANNRERRGIALDGKIK .. .. .. .**V:P::M::A::::P:::T:::::T:L::::::::::::::::::: . .. .. .. .* .*V:P ::S::T::::Q:::T:::::V:V::::::::::::::::::: .. .. .. .* .*V:P ::S::T::::Q:::T:::::V:V:::::::::::::::::::

310

320

330

340

350

390

400

HEDTNLASSTIIKEGIDKTVGILVSYQIKVKLTVSGLLGELTSSEVATE .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .*:R::L::::::Q:::::::::F:::::::::::: .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .*R::M::::::H:::::::::F:::::::::::: .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. ...R::M::::::H:::::::::F:::::::::::: .

360

370

380

VPFRLMHPQPEDPDTAKESFQDENFVFEEFARQNLKDAGEYKEEKTDQE~DE .. .. .. .. .. .. .. .. .. .. .. .. .__. . . .. .. .*Y::A:L:::::::H::::A::AE:G:R:KNDA-:: .. .. .. .. .. .. .. .. .. ....*. ..__... . . . ..V::E:L:::::::Q:: : :T: :NT:G:K:EDAGQ: .*............ . . . . . . . . . . . .__. . .. .. ..V::E:L:::::::Q:: : :T: :NT:G:K:EDAGQ::

:

(404) (405) (403) (403)

FIG. 1. Comparison of the amino acidsequences of four different speciesof S-Ag.Alignment of the amino acid sequences was achievedby the methodof Kyte and Doolittle (1982). Colons( : ) indicatethe identicalamino acid residues.Gaps(-) have been introducedin the sequences to optimizethe alignment.Numbersabovethe sequenceare basedupon the bovine S-Ag sequence. Numbers in parentheses at the end of the sequences indicate the total amino acid residues in S-Ag. Boxes are uveitopathogenic sites. Bo = S-Ag from bovine retina. Hu = S-Ag from human retina. MO = S-Ag from mouse retina. Ra = S-Ag from rat pineal gland. The one-letter code has been used in order to conserve space.

4. Amino Acid Sequences We have determined by DNA sequencing and partial amino acid sequencing the entire amino acid sequence of S-Ag from human (Yamaki, Tsuda and Shinohara, 1988), bovine (Shinohara et al., 1987: Yamaki et al., 1987) and mouse (Tsuda et al., 1988) retina and rat pineal gland (Abe et al., 1989) (Fig. 1). Comparison of the amino acid sequencesreveals a high degree of sequence similarity, among these species. The molecular weight of S-Ag is approximately 45 kDa and the

number of residues is 403. 404 or 405, as shown in Fig. 1. Some internal sequence repeat was detected, perhaps reflecting gene duplication for S-Ag (Wistow et al., 1986). Searching the data bank revealed no extensive sequencesimilarity between S-Ag and other proteins. However, there were regional sequence similarities with a-transducin (Wistow et al., 1986: Shinohara et al., 1988). Comparison of the amino acid sequencesof S-Ag from the rat pineal gland and mouse retina showed that they are virtually identical (Abe et al., 1989).

753

S-ANTIGEN 2

S-Ag Protein 48 kDa

34

5

a

67

9

10

DVMG

NH,

11

12 13 14 15

GIADLGK STI KEG

APQDVGK

16 NLKD GENTEGK

C00t-1 403

1 H 20AA UGA I

AUG

mRNA 1.6 kbp

5

34

a

67

9

10

11

12 131415

16

-

AAA

H 1OObp

Gene >60 kbp

/? A

hMGS

H 2kbp

I3 C

//

D+/

E F G

Frc. 2. Diagrammaticpresentationof the protein, mRNA and gene.Top: The protein structure. The aminoacid sequences of the putative binding sitesto rhodopsin are shown by single-letterabbreviation. Middle: The mRNA structure. The AUG initiation and UGA termination codonsare shownabove boxes.Bottom: The structure of S-Ag gene.Closedboxesdenotethe codingexonsand open boxesdenotethe 5’ and 3’-non-codingregions.hMGS(A-G) indicatesthe partial genomicfragments insertedinto bacteriophageA. Secondary structure prediction and circular dichroism spectroscopy analysis showed that S-Ag has predominantly a P-sheet conformation (Shinohara et al., 1987). 5. Gene Organization The mouse S-Ag gene was isolated from four genomic libraries with mouse cDNA probes and its partial sequencewas determined (Fig. 2). Total length of the S-Ag gene is approximately 50 kbp and it consistsof 16 exons and 15 introns. Most of the exons are less than 100 bp in length: the smallest one is exon 15. which is only 10 bp in length. In contrast to the exons, the size of the introns is relatively large, with most of them being more than 5 kbp in length. Overall the introns constitute approximately 9 7 % and the exons only 3% of the gene. All of the donor and accepter splice sites are in good agreement with the GT/AG rule. Approximately 1.5 kbp upstream of the 5’-flanking region of S-Ag gene sequence was also determined and a typical TATA box or CAAT box was not revealed (Tsuda et al., unpubl. res.). The S-Ag gene is assignedto chromosome 2Q33-34 in the human (Ngo et al., in press) and to the centromeric portion of chromosome 1 near the IDH-1 locus in the mouse (Danciger et al., 1989). 6. Messenger RNA The northern blot analysis indicated that S-Ag mRNA is approximately 1600 nucleotides in length

and has one long open reading frame. In addition, it has a small open reading frame of 150-160 nucleotides in the S/-non-coding region. By analogy with yeast GCN4 mRNA, this small open reading frame may be involved in controlling translation (Mueller and Hinnebusch. 1986). 7. Immunogenic Sites The knowledge of S-Ag amino acid sequences allowed us to identify the uveitopathogenic (epitope) sites. In general T-cells do not recognize the intact protein molecule but rather a small peptide (epitope) (Watt et al., 1984). The recognition of the epitope by T-cells requires a trimolecular complex consisting of a class I or class II MHC molecule. the T-cell antigen receptor and the antigen (see Schwartz, 1985). Initially a seriesof 23 oligopeptides corresponding to the entire amino acid sequence of the bovine Santigen were tested for their ability to induce EAU in Lewis rats and guinea-pigs (Donoso et al.. 1986, 1987a. b; Singh et al., 1988a. b, 1989a). In these studies. four synthetic peptides, designated peptide M and N in bovine S-Ag consistently induced EAU when immunized at a SO-100 pugdose. Peptide K and 3 induced mild EAU with higher doses (100-500 pg) (Donoso et al.. 1987a; Singh et al., 1989a). In general, the various epitopes from a single protein are not recognized with equal efficiency and there appears to be a hierarchy in the ability of any of these epitopes to be recognized by T-cells (seeGammon et al., 198 7). The sequence of uveitopathogenic peptides M and

754

T SHINOHARA TABLE

ET AL.

1

Major uveitopathogenic sites in S-Ag M Bo Hu MO Ra

DTNLASSTIIKEGIDKIT : : : : : : : : : : : : :: :R:: : : :::::::::::: :R: : : : : : : : : : : : : : : : : R: :

Amino acid sequence similarities indicated by a colon (: ). The bovine amino acid residues.

Mimicry

VPLLANNRERRGIALDGKIKHE : : : : :::::::::::::::::: : : : : : : : : :::::::::::::: : : : : : : ::::::::::::::::

of pathogenic sites in bovine (Bo). human (Hu). pathogenic peptides N (282-302) and M (303-317)

N. which behave similarly in various animals, are virtually identical in all species tested (Table I). The peptides K and 3 have different sequences among the species. The EAU induced by peptide K and 3 of bovine S-Ag always shows a mild disease in Lewis rats (Donoso et al., 1987a; Singh et al., 1988a), which may be explained by the differences in antigen sequence. Although not in every case, two type of immunodominant oligopeptides are predicted when whole proteins are used for induction of immune response (Rothbard and Taylor, 1988). One type is an amphipathic a-helix conformation (DeLisi and Berzofsky, 1985 ; Margalit et al., 1987), while the other type is glycine or another charged amino acid followed by two to three hydrophobic residues and a hydrophilic residue (Sette et al., 1987). The peptides M, K and 3 have neither amphipathic a-helix conformation (Shinohara et al., 198 7) nor the latter case. Peptide N, however, has the latter case, i.e. glycine followed by isoleucine-alanine-leucine (hydrophobic) and aspartic acid (hydrophilic). Peptide M has an interesting conformational structure. At physiological pH, it has a strong tendency to form macromolecular assemblies adopting an intermolecular P-sheet structure. The intermolecular ,& sheets are stabilized by ionic interactions between the carboxylate groups and basic residues of the neighboring peptide molecules (Muga et al., in press). Such an antigenic structure may contribute for MHC restricted antigen recognition by T-cells. 8. Molecular

N

in EAU

Knowledge of the uveitopathogenic sites of S-Ag enabled us to search for similar sequences in foreign antigens. We compared amino acid sequences of a variety of proteins listed in the National Biomedical Research Foundation data base for sequence homology to peptide M and found sequence similarity to a variety of proteins including viral, fungus, bacteria and others as shown in Table II (Donoso et al., 1987a; Singh et al., 1989b, c, d). We did not find any protein having sequence similarity with peptide N. The best match with peptide M was with baker’s yeast (Saccharomyces cerevisiae) histone H3 (Singh et al., 1989c) and E. coli hypothetical protein, where six or five amino acids are

mouse (MO) and rat (Ra) S-Ag. Identical residues are are indicated. The one-letter code is used to designate

identical (Singh et al., 1989b). Potato proteinase inhibitor and proteins in E. coli elongation factor also have a four or five consecutive amino acid sequence identity. Among viral proteins we found several homologous proteins in hepatitis B virus, baboon virus, AKR murine leukemia virus, Moloney murine leukemia virus and Moloney murine sarcoma virus. Oligopeptides corresponding to these proteins were synthesized by conventional solid-phase chemistries and purified. The incidence of EAU after immunization of Lewis rats with different doses of these peptides is summarized in Table II. At 100 and 200 rug of histone H3, 50% of the animals developed EAU, while at the 400 pg dose, all animals developed EAU by day 21 (Singh et al., 1989c). E. coli hypothetical peptide and other viral peptides also induced EAU at higher doses (500-2000 pug) (Singh et al., 1989b). The first clinical signs of an inflammatory response were hyperemia of the conjunctiva, iris vasodilation and the appearance of exudate and cells in the anterior chamber and vitreous cavity. Four to six days following the onset of EAU the inflammatory activity gradually subsided, and the cells in the anterior chamber disappeared (Singh et al., 1989b). Histophathologically, a more severe inflammatory response was present in the rats immunized with the higher doses of the peptide (400 ,ug or more) in contrast to the lower doses (100-200 pg), where the disease was mild. In the severely inflamed eyes the retina was totally detached, showing a complete loss of the rod outer segments of the photoreceptor cells. A subretinal exudate was present containing mononuclear lymphocytes. In addition, rats with EAU showed an associated EAP characterized by a lymphocytic infiltration of the subcapsular area of the pineal glands (Singh et al., 1989b, c), The other peptides from E. coli protein, potato inhibitor and four viral proteins also induced EAU with the histology of retina and pineal gland being indistinguishable from EAU induced by peptide M (Singh et al., 1989b). To extend our results using the synthetic polypeptides, we immunized Eve Lewis rats with a histone preparation purified from S. cerevisiae. This contained highly purified histones (more than 95 %) although histone H3 only consisted of 10-l 5 % of the prep-

755

S-ANTIGEN

TABLE II

Non-self protein or peptideswhich inducedexperimental autoimmune inflammation Experimental autoimmune inflammation

Non-selfprotein/ Self protein Raker’syeast histone H3 Baker’syeast native histoneH3* E. roli hypothetical protein Potato inhibitor Hepatitisvirus Baboonvirus Moloney murine sarcomavirus Moloney murine leukemiavirus AKR murine leukemiavirus Hepatitisvirus * Indicates native protein. The rest of them indicate et al. (1990~ ; 4. Fujkami and Oldstone (1985).

synthetic

peptides.

aration. Five rats were immunized: all developed an EAU 20-24 days postimmunization with 500 pg of native histone and CFA (Singh et al., 1989c). There was cellular infiltration in the anterior chamber as well as in the vitreous cavity and posterior iris synechiae. Histological examination of the eye showed a severe inflammatory response with destruction of the photoreceptor cell layer of the retina, similar to that by native S-Ag, peptide M or histone H3. Rats with EAU showed an associated pinealitis characterized by lymphocytic infiltration of the pineal gland (Singh et al., 1989c). No histopathological change was observed in the eye or pineal gland of control animals injected with PBS and CFA emulsion. We also injected 18 other oligopeptides of S-Ag into Lewis rats and none developed EAU (Donoso et al., 19 8 7a : Singh et al., 1989~). These results illustrate that native histone as well as synthetic preparations are effective agents for the induction of EAU in Lewis rats. 9. Lymphocyte Cross-reaction and Adaptive Transfer In order to confirm the lymphocyte cross-reaction, the responsiveness of lymph-node cells from rats immunized with peptide M. histone H3 and native histone was tested. Lymph-node cells from rats immunized with antigens reacted well against immunizing antigen. In addition, lymph-node cells from rats immunized with peptide M also responded well to histone H3 or native histone (Singh et al., 1989~ d). Similarly, cells from rats immunized with either histone H3 or native histone showed significant proliferation when cultured with peptide M. None of the lymphocytes reacted with calf thymus histone. There was a significant response to purified protein derivative (PPD) and Con A (Singh et al., 1989~). These results indicate that peptide M and histone H3 peptide are similar enough to cross-react. Lymph-node cells of peptide M and histone H3 immunized animals were prepared for adaptive transil

EAIJ EAU EAU EAU EAU EAU EAU EAIJ EAU EAE References:

Keference 1 1

2 3 3 3 3 3 3 4

1. Singh et al. (1989~):

2, Singh et al. (1989b):

3. Singh

fer studies. The cells were stimulated with either peptide M or histone H3 in vitro culture and injected i.p. in naive syngeneic rats. The rats induced EAU at 6-12 days. Histopathology showed severe inflammation in the uveal tract and a complete loss of photoreceptor cells in EAU positive animals which were indistinguishable from rats induced by peptide M, or native S-Ag (Singh et al., 1989d). Thus EAU is adaptively transferred to the naive rats by lymph-node cells stimulated with either peptide M or histone H3. 10. Molecular Mimicry in Other Systems In experimental allergic encephalomyelitis (EAE), an animal model of multiple sclerosis, caused by immunization with myelin basic protein (MBP), Fujinami and Oldstone (1985) showed that a six amino acid sequence of hepatitis B virus polymerase (HBVP) was identical to the encephalitogenic site of MBP. Rabbits injected with HBVP synthetic peptide showed antibody responses that cross-reacted with MBP. The peripheral blood mononuclear cells proliferated when incubated with either MBP or HBVP. This finding is the first and only example of molecular mimicry reported in which EAE is induced by T-cell mediated autoimmune inflammation (Table II). 11. Conclusions and Remarks Studies by us and others provide important clues in understanding autoimmune diseasesof the eye and pineal gland. Autoimmune response provoked by molecular mimicry occurs when the non-self and self determinants are similar enough to cross-react. Our findings that selected bacterial, fungal. viral and other proteins with sequencesimilarity to retinal S-Ag can induce EAU add to the evidence supporting molecular mimicry as an etiology of autoimmune uveitis. Synthetic oligopeptides offer useful probes for studying molecular mimicry. The finding that native histone H3 induces EAU in EEK ill

756

T SHINOHARA

Lewis rats is, to the best of our knowledge, the first clear example of molecular mimicry at the experimental level. Our results implicate that microbial infection may activate the immune system. In addition to proteins of infectious microorganisms. proteins of yeast and potato, both common food, are also possibleinitiators of autoimmune inflammation. Although genetic predisposition, multiple lymphatic factors and lymphocyte interactions may play major roles in the development of autoimmune diseases. molecular mimicry between self and non-self proteins may be an underlying primary initiator of autoimmune inflammation. Studies of cell-mediated immune responsesand of antibodies in animal models and patients with uveitis should help test further the role of molecular mimicry in the pathogenesis of autoimmune inflammatory uveitis as well of autoimmune diseasesin general.

References Abe, T., Yamaki, K.. Tsuda, M.. Singh, V. K.. Suzuki, S., McKinnon, R., Klein, D. C., Donoso, L. A. and Shinohara. T. (1989). Rat pineal S-antigen: sequence analysis reveals presence of a-transducin homologous sequence.FEBS Lett. 247, 307-l 1. Caspi, R. R., Roberge, F. G., McAllister. C. G., El-Saied, M.. Kuwabara, T., Gery, I.. Hanna, H. and Nussenblatt, R. B. (1986). T cell lines mediating experimental autoimmune uveoretinitis (EAU) in the rat. 1. Immunol. 136, 928-33. Chan. C.-C., Mochizuki. M., Nussenblatt. R. B., Palestine, A. G., McAllister, C., Gery. I., and BenEzra, D. (1985). T Lymphocyte subsets in experimental autoimmune uveitis. Clin. Immunol. Immunopathol. 35. 103-10. Cohen, I. R.. (1988). The self, the world and autoimmunity. Sci. Am. 258, 52-60. Danciger, M., Kozak. C. A., Tsuda. M., Shinohara, T. and Farber, D. B. (1989). The gene for retinal S-antigen (48 kDa protein) maps to the centromeric portion of mouse chromosome 1 near IDH- 1. Genomics 5 q 3 78-8 1. De Kozak. Y., Audibert, F., Thillaye, B., Chedid, L. and Faure. J,-P. ( 19 79). Effects of mycobacterial hydrosoluble adjuvants on the induction and prevention of ex-

perimental autoimmune uveoretinitis in guinea pigs. Ann. Immunol. (Paris) 130. 29-32. De Kozak, Y.. Yuan, W. S., Bogossian, M. and Faure, J.-P. (1976). Humoral and cellular immunity to retinal antigens in guinea pigs. Mod. Probl. Ophthafmol. 16. 51-8. DeLisi. C. and Berzofsky, J. A. (1985). T-cell antigenic sites tend to be amphipathic structure. Proc. Nut/. Acad. Sci. U.S.A. 82, 7042-52. Donoso. L. A., Merryman, C. F.. Sery. T., Shinohara, T. Dietzschold. B., Smith, A. and Kalsow. C. M. (1987a). SAntigen : characterization of a pathogenic epitope which mediates experimental autoimmune uveitis and pinealitis in Lewis rats. Curr. Eye Res. 6, 1151-9. Donoso. L. A., Merryman. C. F., Shinohara, T.. Dietzschold. B., Wistow. G.. Craft, C., Morley, W. and Henry, R. (1986). S-antigen: identification of the MAbA9-C6 monoclonal antibody binding site and the uveitopathogenie sites. Curr. Eye Res. 5. 995-1004. Donoso. L. A.. Merryman. C. F., Shinohara. T., Sery. T. W.

ET AL.

and Smith. A. (I Y87b1. S-Antigen: experimental autoimmune uveitis following immunization with a small synthetic peptide. Arch. Ophth~&no~. 105, 8 38-40. Dorey , c‘. and Faure. J.-P. (1977). Isolement ct caractkrisation d’un antigtne retinien de la rCtine induisant I’uvCo-rktinite auto-immune expt;rimentalr. ,4r1n. ~rmnuno/. (Paris) 128C. 229-32. Faure. J-P. ( 1980). Autoimmunity and the retina. Curr. ‘fop. Eye RPS. 2. 21 S-302. Fujinami, R. S. and Oldstone, B. A. ( 1985). Amino acid homology between the encephalitogenic site of myelin basic protein and virus: mechanism for autoimmunity. Science 230. 1043-j.

Gammon,G.. Shastri. N.. Cogswell.J.. Wilbur, S.. SadeghNasseri. S.. Krzych. U., Miller. A. and Sercaze. E. ( 1987). The choice of T-cell epitopes utilized on a protein antigen depend on multiple factors distant from, as well as at the determinant site. Immunol. Rev. 98. 53-73. Gery, I., Mochizuki. M. and Nussenblatt. R. B. (1986). Retinal specific antigens and immunopathogenic processes they provoke. In Progress in Retinal Research. Vol. 5. (Eds Osborne, N. and Chader. 1.). Pp. 75-109. Pergamon Press: New York. Kalsow, C. M. and Wacker, W. B. (1 Y 77). Pineal reactivity of anti-retina sera. lnvrst. Ophthalmol. KS. Sci. 16, 181-4. Kalsow, C. M. and Wacker. W. B. (1978). Pineal gland involvement in retina-induced experimental allergic uveitis. lrwest. Ophthalmol. Vis. Sci. 17. 774-83. Kuhn, H.. S. W. and Wilden, Il. (1984). Light-induced binding of 48-kDa protein to photoreceptor membranes is highly enhanced by phosphorylation of rhodopsin FEBS Left. 176, 473-8. Kyte, J. and Doolittle, R. F. ( 1982). A simple method for displaying the hydropathic character of a protein. 1. Mol. Biol. 157. 105-32. Margalit. H.. Spouge, J. I,.. Cornette. J. I,.. Cease, K. B., DiLisi. C. and Berzofsky, J. A. (I 987). Prediction of Immunodominant helper T-cell antigenic sites from the primary sequence. j. immunol. 138, 2213-19. Mochizuki. M.. Kuwabara. T.. Chan. C.-C.. Nussenblatt. R. B.. Metcalfe, D. D. and Gery, 1. (1984). An association between susceptibility to experimental autoimmune uveitis and choroidal mast cell numbers. 1. Zmmunol. 133, 16YY-1701. Mochizuki. M., Kuwabara. T.. McAllister, C., Nussenblatt. R. B. and Gery. I. (198 5). Adoptive transfer of experimental autoimmune uveoretinitis in rats: immunopathogenic mechanisms and histological features. Invest. Ophthalmol. Vis. Sci. 26. 1-Y. Mueller. P. P. and Hinnebusch. A. C. (1986). Multiple upstream AUG codons mediate translational control of GCN4. Cd 45, 201-7. Muga. A.. Suretyships, W., Wondrousness, P. T. T.. Mantsch. H.. Singh. V. K. and Shinohara. T. (1989). Structural studies with the uveitopathogenic peptide M derived from retinal S-antigen. Biochemistry (in press). Ngo. J. T.. Klisak, I., Mohandas, T.. Shinohara, T., Sparkes, R. S. and Bateman. J. B. (1990). Assignment of the Santigen gene to chromosome (S-Ag) to the human chromosome 2Q3 3-34. Genomics (in press). Oldstone. M. B. A. (lY87). Molecular mimicry and autoimmune disease. Cell 50. 8 19-X). Oldstone, M. B. A. and Notkins. A. L. (1986). Molecular mimicry. In Concepts in Viral Pathogenesis. Vol. II (Eds Notkins. A. L. and Oldstone. M. B. A.). Pp. 1 Y 5-202. Springer-Verlag: New York. Psfister. C., Chabre. M.. Plouet. J.. Tuyen. V. V.. De Kozak. Y., Faure. J. P. and Kuhn, H. (1985). Retinal S-antigen identified as the 48K protein regulating light-dependent phosphodiesterase in rod cells. Science 228, 891-3.

S-ANTIGEN

Rothbard, J. B. and Taylor, W. R. (1988). A sequence pattern common to T-cell epitopes. EMBO I. 7, 93-100. Schwartz, R. I. P. (1985). T-Lymphocyte recognition of antigen in association with gene products of the major histocompatibility complex. Annu. Rev. Immunol. 3, 237-61. Sette. A., BUS, S.. Colon, S., Smith, J. A.. Miles, C. and Grey, H. M. (1987). Structural characteristics of an antigen required for its interaction with Ia and recognition by T cells. Nature 328, 395-9. Shinohara, T., Dietzschold, B., Craft, C. M., Wistow, G., Early, J., Donoso. L. A., Horwitz, J. and Tao, R. (1987). Primary and secondary structure of bovine retinal Santigen (48-kDa protein). Proc. N&l. Acad. Sri. U.S.A. 84, 6975-9. Shinohara. T., Donoso, L. A., Tsuda, M., Yamaki, K. and Singh, V. K. (1988). S-antigen: structure. function and experimental autoimmune uveitis @AU). In Progress in Retinal Research. Vol. 8 (Eds Osborne, N. and Chader, J.). Pp. 51-66. Pergamon Press: New York. Singh, V. K., Nussenblatt, R. B., Donoso, L. A., Yamaki, K., Chen. C. and Shinohara, T. (1988a). Identification of a uveitopathogenic and lymphocyte proliferation site in bovine S-antigen. Cell. Immunol. 115, 413-19. Singh, V. K., Yamaki, K., Donoso, L. A. and Shinohara, T. ( 1988b). S-Antigen : experimental autoimmune uveitis induced in guinea pigs with two synthetic peptides. Curr. Eye Res. 1. 87-92. Singh. V. K., Donoso. L. A.. Yamaki, Y. and Shinohara, T. (1989a). IJveitopathogenic sites in bovine S-antigen. Autoimmunity 3, 177-87. Singh, V. K., Yamaki, K., Abe, T. and Shinohara, T. (1989b). Molecular mimicry between uveitopathogenic sites of retinal S-antigen and Escherichia coli protein : induction of experimental autoimmune uveitis and lymphocyte cross-reaction. Cell. Immunol. 122, 262-73. Singh, V. K.. Yamaki. K.. Donoso. L. A. and Shinohara. T.

757 (1989~). Molecular mimicry : yeast histone H3 induced experimental autoimmune uveitis. 1. Immunol. 142, 1512-17. Singh, V. K.. Yamaki, K., Donoso, L. A. and Shinohara, T. ( 19 89d). Sequence homology between yeast histone H 3 and uveitopathogenic site of S-antigen : lymphocyte cross-reaction and adaptive transfer of the disease. Cell. Immunol. 119, 211-21. Singh. V. K., Kalra, H. K., Yamaki, K., Abe, T., Donoso, L. A. and Shinohara, T. (1990). Molecular mimicry between a uveitopathogenic site of S-antigen and viral peptides ; induction of experimental autoimmune uveitis in Lewis rats, J. Immunol. 144, 1282-7. Tsuda, M., Syed, M.. Bugra, J., Whelan, J. P., McGinnis, J. F. and Shinohara, T. (1988). Structural analysis of mouse S-antigen. Gene 73. 11-20. Wacker, W. B.. Donoso, L. A., Kalsow, C. M.. Yankeelov, Jr., J. A. and Organisciak. D. T. (1977). Experimental allergic uveitis. Isolation, characterization and localization of a soluble uveitopathogenic antigen from bovine retina. I. Immunol. 119, 1949-58. Watts, T. H., Brian, A., Kappler, J. W.. Marrock. P. and McConnell, H. M. ( 1984). Antigen presentation by supported planar membranes containing affinitypurified I-Ad. Proc. Natl. Acad. Sci. U.S.A. 81. 7564-8. Wistow. G. J., Katial, A., Craft, C. and Shinohara. T. (1986). Sequence analysis of bovine retinal S-antigen. Relationship with a-transducin and G-proteins. FEBS Lett. 196, 23-8. Yamaki. K., Takahashi, M., Sakuragi, S. and Matsubara, K. (1987). Molecular cloning of the S-antigen cDNA from bovine retina. Biochem. Biophys. Res. Commun. 142, 904-10. Yamaki, K., Tsuda. M. and Shinohara, T. (1988). The sequence of human retinal S-antigen reveals similarites with a-transducin. FEBS Lett. 234, 3943.