S-ANTIGEN: STRUCTURE/FUNCTION RELATIONSHIPS IN UVEITIS

S 抗原：葡萄膜炎中的结构/功能关系

• 批准号: 3259871
• 负责人: LARRY A DONOSO
• 金额: $ 11.38万
• 依托单位: WILLS EYE HEALTH SYSTEM
• 依托单位国家: 美国
• 财政年份: 1984
• 资助国家: 美国
• 起止时间: 1984-09-30 至 1992-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3259871
• 关键词: DNA; antigens; autoimmune disorder; biological polymorphism; chemical binding; chemical structure function; conformation; disease /disorder model; enzyme linked immunosorbent assay; genetic library; genome; high performance liquid chromatography; histochemistry /cytochemistry; hybridomas; immunochemistry; immunofluorescence technique; monoclonal antibody; oligonucleotides; pathologic process; peptide chemical synthesis; peptides; spectrometry; tissue /cell culture; uveitis

S-antigen is a well-characterized retinal protein, intimately involved in the visual process, and highly pathogenic for the induction of experimental autoimmune uveitis (EAU), and intimately involved in the phototransduction of vision. In order to more fully understand structure/function relationships of S- antigen in the pathogenesis of EAU and in the phototransduction of vision we have (1) produced monoclonal antibodies (MAbs) which define different epitopes of S-antigen, (2) determined the amino acid sequence of bovine S-antigen, (3) localized one MAb binding site and (4) localized two uveitopathogenic sites in bovine S-antigen. In our laboratory, we have generated two MAbs, MAbA9-C6 and MAbA1-G5, which define different epitopes of S-antigens. These MAbs have been useful in our studies concerning S-antigen in the developing retina, pineal gland and in tumors arising from these tissues. The amino acid sequence of bovine S-antigen has now been determined. Analysis of our sequence data has revealed that; S-antigen exists primarily in a beta sheet conformation; contains two potential phorphorylation sites; three potential carbohydrate attachments sites; an ADP-ribosylation site; and sequence homologies to tranducin, another protein in the visual pathway. Such information is essential in order to understand the role of S-antigen in the phototransduction of vision. A knowledge of the amino acid sequence has also led to the identification of the MAbA9-C6 binding site and two uveitopathogenic sites. Peptides, corresponding to the amino acid sequence of S-antigen, have been synthesized chemically and two peptides designated peptide K and peptide M have been found to be highly uveitopathogenic. Immunization of Lewis rats with microgram amounts of either peptides induced an EAU which was clinically and histopathologically indistinguishable from the EAU induced by native S-antigen. In this continuation proposal we plan to refine and expand our initial observations regarding the uveitopathogenic sites in bovine, human and rat S-antigen. In addition, we propose to produce MAbs against the uveitopathogenic determinants and to refine the MAbA9-C6 binding site to the resolution of a single amino acid. These studies and the reagents we are developing will not only aid in understanding the role of S-antigen in the pathogenesis of EAU, but in the phototransduction of vision as well.

S抗原是一种特征性很强的视网膜蛋白， 参与视觉过程，并且对 诱导实验性自身免疫性葡萄膜炎（EAU），和 与视觉的光传导密切相关。 为了 更全面地了解S-的结构/功能关系 抗原在EAU发病机制和光传导中的作用 我们已经（1）生产了单克隆抗体（MAbs） 确定了S抗原的不同表位，（2）确定了 牛S抗原的氨基酸序列，（3）定位一个单克隆抗体 结合位点和（4）定位两个葡萄膜致病位点在牛 S抗原 在我们的实验室中，我们已经产生了两种单克隆抗体，MAbA 9-C6和 MAbA 1-G5，其定义S抗原的不同表位。 这些 单克隆抗体已在我们的研究有关S-抗原在 发育中的视网膜、松果体和由这些引起的肿瘤 组织中 牛S抗原的氨基酸序列现在已经 确定了 我们的序列数据分析显示 S-抗原主要以β折叠构象存在; 含有两个潜在的磷酸化位点;三个潜在的磷酸化位点。 ADP-核糖基化位点;和 序列同源性transucin，另一种蛋白质在视觉 通路 这些信息对于了解 S抗原在视觉光传导中的作用。 知识 的氨基酸序列也导致了鉴定 MAbA 9-C6结合位点和两个葡萄膜致病位点。 肽，对应于S-抗原的氨基酸序列， 已被化学合成，并命名为两种肽 已经发现肽K和肽M是高度 葡萄膜致病的 微克免疫刘易斯大鼠 量的肽诱导EAU，其在临床上是 并且在组织病理学上与由以下物质诱导的EAU无法区分： 天然S抗原 在本延续提案中，我们计划完善和扩大我们的 关于牛中葡萄膜致病位点的初步观察， 人和大鼠S抗原。 此外，我们建议生产 抗葡萄膜致病决定因素的单克隆抗体，并完善 MAbA 9-C6结合位点的解析为单个氨基酸。 这些研究和我们正在开发的试剂不仅有助于 在理解S抗原在EAU发病机制中的作用时， 也在视觉的光传导中。