Kunihiko Yamaki

The sequence of human retinal S-antigen reveals similarities with α-transducin

The complete amino acid sequence of human retinal S‐antigen (48 kDa protein), a retinal protein involved in the visual process has been determined by cDNA sequencing. The largest cDNA was 1590 base pairs (bp) and it contained an entire coding sequence. The similarity of nucleotide sequence between the human and bovine is approximately 80%. The predicted amino acid sequence indicates that human S‐antigen has 405 residues and its molecular mass is 45050 Da. The amino acid sequence homology between human and bovine is 81%. There is no overall sequence similarity between S‐antigen and other proteins listed in the National Biomedical Research Foundation (NBRF) protein data base. However, local regions of sequence homology with α‐transducin (Tα) are apparent including the putative rhodopsin binding and phosphoryl binding sites. In addition, human S‐antigen has sequences identical to bovine uveitopathogenic sites, indicating that some types of human uveitis may in part be related to the animal model of experimental autoimmune uveitis (EAU).

Molecular mimicry between uveitopathogenic site of retinal S-antigen and Escherichia coli protein: induction of experimental autoimmune uveitis and lymphocyte cross-reaction.

Experimental autoimmune uveitis (EAU) is caused by the immunization of microgram amounts of a soluble retinal protein, known as S-antigen, in susceptible animal strains including primates. The disease serves as an animal model of ocular inflammation. We induced EAU and pinealitis in Lewis rats with small synthetic peptides, corresponding to the amino acid sequence in Escherichia coli protein, which contains six consecutive amino acids identical to a uveitopathogenic site in human S-antigen (peptide M). EAU and pinealitis induced in rats by synthetic peptide derived from E. coli was indistinguishable from those induced by native S-antigen or other uveitopathogenic synthetic peptides corresponding to the amino acid sequence of S-antigen. Furthermore, lymph node cells from animals immunized with either peptide M or peptide derived from E. coli protein showed significant proliferation in the presence of either peptide when tested in vitro for lymphocyte mitogenesis using [3H]thymidine. Thus, molecular mimicry, a process by which an immune response directed against a nonself protein cross-reacts with a normal host protein, may play a role in autoimmunity.

Suppression of experimental autoimmune uveitis in rats by the oral administration of the uveitopathogenic S-antigen fragment or a cross-reactive homologous peptide

The oral administration of S-antigen fragment (a synthetic peptide designated as peptide M and known to be uveitopathogenic for rat, guinea pig, and monkey) to Lewis rats prior to challenge with an emulsion of peptide M and CFA resulted in either a total or partial suppression of experimental autoimmune uveitis (EAU), a T cell-mediated autoimmune disease studied as a model for human uveitis and experimental autoimmune pinealitis (EPA). Both the clinical and histopathologic manifestations of the disease were suppressed in a dose-dependent manner. Pinealitis associated with EAU was also suppressed by the oral administration of peptide M. Additionally, ingestion of a fragment of bakers yeast (Saccharomyces cerevisiae) histone H3, which has five consecutive amino acids identical to peptide M and which has been found to be uveitopathogenic in Lewis rats, induced tolerance to either peptide M or synthetic histone H3 peptide. In addition, the proliferative response to peptide M was inhibited in peptide M-fed rats. The suppression of EAU and in vitro lymphocyte proliferative responses to peptide M were observed to be antigen specific, since oral feeding of a control protein (BSA) exerted no suppressive effect. Furthermore, the T cells isolated from the spleen and lymph nodes of animals rendered tolerant by oral administration of peptide M can transfer protection against EAU adoptively. These results demonstrate that the oral administration of an autoantigen or its homologous peptide initiates an antigen-specific cellular mechanism which may ameliorate EAU.

S-antigen: From gene to autoimmune uveitis

Retinal S-antigen (S-Ag) is capable of inducing experimental autoimmune uveitis (EAU) in laboratory animals. EAU may serve as an animal model for studying human uveitis. As a first step we have determined the nucleotide sequence of an S-Ag gene and its cDNAs. The amino acid sequences were deduced from the cDNAs of various animals and human. Four uveitopathogenic sites in bovine S-Ag were characterized. One of the sites (peptide M) has sequence homology with non-self proteins from bakers yeast, potato, E. coli, hepatitis B virus, moloney murine leukemia virus, Moloney murine sarcoma virus, AKR murine leukemia virus and baboon endogenous virus. Mononuclear cells from animals immunized with peptide M showed significant proliferation when incubated with synthetic peptides corresponding to the amino acid sequences of the above-mentioned foreign proteins. In addition, all the peptides induced EAU in Lewis rats with a dose of 10-2000 micrograms. Moreover, native histone H3 from bakers yeast histone H3 induced EAU in Lewis rats. Thus, we found several examples of antigenic mimicry between self and non-self proteins. These findings establish a base to study further the mechanism of autoimmune inflammation.

Human S-antigen: Characterization of uveitopathogenic sites

Human S-antigen (HSA) is a 50,000 molecular weight photoreceptor cell protein capable of inducing an experimental autoimmune uveitis (EAU) in susceptible animal strains. In order to determine specific sites responsible for its uveitopathogenicity, we synthesized 39 overlapping peptides corresponding to its entire 404 amino acid sequence and tested each peptide for its ability to induce an EAU in Lewis rats. Two synthetic peptides designated peptide HSA 319 (amino acid positions 286 to 305) and peptide HSA 320 (amino acid positions 306 to 325) were uveitopathogenic when used at 50 and 100 micrograms immunizing doses. Smaller peptides corresponding to the amino, mid, and carboxy terminal portions of each peptide further refined each uveitopathogenic site to 12 amino acids. A computerized analysis of the amino acid sequence of S-antigen indicates that these uveitopathogenic sites are complex and may be related to a two-fold symmetry in the molecule. Our present and previous studies provide a basis for the uveitopathogenicity of human and bovine S-antigen in the pathogenesis of EAU as well as the pathogenesis of certain forms of uveitis in humans.

Rat pineal S-antigen: Sequence analysis reveals presence of α-transducin homologous sequence

S‐antigen (S‐Ag) is a soluble, highly antigenic protein, the administration of which induces autoimmune uveitis. This protein is found in the retina and pineal. Retinal S‐Ag from three species has been sequenced. In this study rat pineal S‐Ag was sequenced. Clones were isolated from a rat pineal λgt11 cDNA library by probing with a 300 bp fragment of mouse retinal S‐Ag cDNA containing the 5′‐coding region. The largest clone isolated (RPS‐118; 1364 bp) contained the entire coding sequence. Comparison of the rat pineal and mouse retinal S‐Ag nucleotide sequences indicated a high homology (95%). The deduced amino acid sequence was found to contain 403 residues (≌44 992 Da). Comparison of the rat pineal and mouse retinal S‐Ag amino acid sequences also revealed high homology (97%). The similarity of both the nucleotide and amino acid sequences of rat pineal and mouse retinal S‐Ag indicates that expression of the S‐Ag gene in both tissues is similar. Further analysis of the rat pineal S‐Ag sequence indicated that it contained essentially the same major uveitopathogenic region of S‐Ag present in bovine retina; minor uveitopathogenic sites were somewhat different. As is true of retinal S‐Ag, rat pineal S‐Ag contains the same consensus phosphoryl‐binding site present in many GTP/ GDP‐binding proteins and a homologous sequence found in the C‐terminus of α‐transducin. These sequences may play a role in the action of pineal S‐Ag in transmembrane signal transduction.

S-antigen: experimental autoimmune uveitis induced in guinea pigs with two synthetic peptides

Experimental autoimmune uveitis (EAU) was observed in Hartley guinea pigs following immunization with two small synthetic peptides, peptide M and peptide M15L, which correspond to the amino acid sequence of a well-characterized region of bovine S-antigen. Groups of guinea pigs were immunized with 100 micrograms of each peptide in complete Freunds adjuvant and examined at regular intervals for the development of disease. Approximately two weeks later, an EAU was present which was characterized clinically by iris and pericorneal hyperemia. Histopathologically, a severe inflammatory response involving the uveal tract and retina was observed. In these eyes the photoreceptor cell layer of the retina was destroyed. A subretinal exudate containing mononuclear cells and polymorphonuclear leukocytes was also present. In addition, animals with EAU showed an associated pinealitis characterized by a lymphocytic infiltration of the subcapsular and central area of the pineal gland. Furthermore, draining lymph node cells of guinea pigs immunized with peptide M showed strong in vitro proliferative responses towards peptide M as measured by 3H thymidine uptake. These results demonstrate the existence of at least one common pathogenic epitope in bovine S-antigen for the induction of EAU in Hartley guinea pigs and Lewis rats.

Assignment of the S-antigen gene (SAG) to human chromosome 2q24–q37

We report the mapping of the gene coding for the S-antigen (48-kDa protein) to human chromosome 2 using somatic cell hybrids. In situ hybridization further confirms this assignment and regionally maps the gene to 2q24-q37.

Sequence homology between yeast histone H3 and uveitopathogenic site of S-antigen: Lymphocyte cross-reaction and adoptive transfer of the disease

Experimental autoimmune uveitis (EAU) serves as an animal model of ocular inflammation. The disease is caused by the immunization of microgram amounts of a soluble retinal protein, designated S-antigen, in susceptible animal strains, including primates. We induced EAU and experimental autoimmune pinealitis (EAP) in Lewis rats with a small synthetic peptide corresponding to amino acid positions 106-121 in yeast histone H3. This peptide contains five consecutive amino acids identical to a uveitopathogenic site (peptide M) in human S-antigen. Lymph node or mononuclear cells from different species of animals immunized either with histone H3 or with peptide M showed significant cross-reaction as measured by in vitro lymphocyte mitogenesis assay using [3H]thymidine. Also, we adoptively transferred the EAU and EAP in naive rats by immune lymph node cells. These findings support the fact that selected bacterial, viral, or fungal proteins with amino acid sequence homologies to normal retinal proteins are uveitopathogenic and, as such, provide a basis for autoimmune inflammatory diseases.

Developmental expression of S-antigen in fetal human and rat eye

Development expression of S-antigen and its mRNA in human and rat fetal retina was studied by immunocytochemical and in situ hybridization techniques. Immunocytochemistry indicated that S-antigen was present after 4 months gestation in the fetal human retina. In the rat, S-antigen was detected in the retina only after birth. In situ hybridization studies indicated that the S-antigen mRNA was present at 13 weeks gestational age in the human and at 15 days in the rat embryo. S-antigen mRNA was expressed not only in the retina but also in ocular tissues of neural crest origin in the fetus.