Experimental allergic uveitis. Isolation, characterization, and localization of a soluble uveitopathogenic antigen from bovine retina.
Experimental allergic uveitis. Isolation, characterization, and localization of a soluble uveitopathogenic antigen from bovine retina.
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实验性过敏性葡萄膜炎。
DOI：
10.4049/jimmunol.119.6.1949
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发表时间：
1977
影响因子：
4.4
通讯作者：
D. Organisciak
中科院分区：
文献类型：
--
作者：
W. Wacker;L. Donoso;C. Kalsow;J. A. Yankeelov;D. Organisciak
用硫酸铵沉淀、分子筛层析和离子交换层析从牛视网膜提取物中分离出与EAU发病有关的可溶性抗原。纯化的组分通过免疫扩散和免疫电泳形成双沉淀带，通过分析盘电泳形成双沉淀带。通过免疫扩散，其中一种成分与豚鼠S抗原形成同一性反应，另一种成分与豚鼠S抗原形成部分同一性反应。因为这两种组分具有相同的分子量。和相似的电迁移率，它们被认为是同一分子的微小结构变体。
纯化的抗原具有分子量通过分析超离心法测定约102，000，通过SDS电泳测定约50，500，通过凝胶过滤色谱测定约56，000。随着抗原浓度的增加，沉降速度研究表明自缔合。对于单体获得3.6的S20值，对应于近似的m.w. 50，000。
抗原被鉴定为具有非常少量脂质的蛋白质。数据上的吸收光谱，消光系数，和氨基酸组成。该分子缺乏半胱氨酸和色氨酸，但含有相对高比例的非极性氨基酸，对应于亲脂性蛋白质。通过免疫荧光将其定位于整个感光细胞周围的区域，其模式表明与质膜相关。该抗原不是视紫红质，视紫红质被认为是EAU发病机制中的可能抗原，但具有一些类似于最近描述的感光细胞视黄醇结合蛋白的性质，该蛋白暂时涉及维生素A从循环到视网膜的运输。
在豚鼠中用于产生EAU的纯化级分的测定表明其在微克范围内是高致病性的。与免疫荧光结果一致，在葡萄膜细胞浸润后，感光细胞被特异性破坏。
The soluble antigen involved in the pathogenesis of EAU was isolated from bovine retinal extract by precipitation with ammonium sulfate followed by molecular sieve and ion exchange chromatography. The purified fraction formed a double precipitin band by immunodiffusion and immunoelectrophoresis, and a doublet by analytical disc electrophoresis. By immunodiffusion one of these components formed a reaction of identity with guinea pig S antigen and the other a reaction of partial identity. Because the two components had the same m.w. and similar electric mobility, they are presumed to be minor structural variants of the same molecule.
The purified antigen had a m.w. of approximately 102,000 by analytical ultracentrifugation, 50,500 by SDS electrophoresis, and 56,000 by gel filtration chromatography. Sedimentation velocity studies with increasing concentrations of antigen indicated self-association. An S20 value of 3.6 was obtained for the monomer, corresponding to an approximate m.w. of 50,000.
The antigen was identified as a protein with a very small amount of lipid. Data are presented on the absorption spectrum, extinction coefficient, and amino acid composition. The molecule was lacking in cysteine and tryptophan, but contained a relatively high proportion of nonpolar amino acids corresponding with a lipophilic protein. It was localized by immunofluorescence to the area surrounding the entire photoreceptor cell in a pattern indicating association with the plasma membrane. The antigen was not rhodopsin, which has been implicated as a probable antigen in the pathogenesis of EAU, but has some properties resembling a recently described photoreceptor cell retinol-binding protein tentatively implicated in the transport of vitamin A from the circulation to the retina.
Assay of the purified fraction for production of EAU in the guinea pig indicated it was highly pathogenic in the microgram range. In agreement with immunofluorescent findings, the photoreceptors were specifically destroyed after cellular infiltration of the uveal tract.