Experimental Allergic Uveitis. Isolation, Characterization, and Localization of a Soluble Uveitopathogenic Antigen from Bovine Retina

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Abstract
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.