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S Kang, JH Sohn, HJ Kaplan, NS Bora; Characterization of Melanin Associated Antigen(MAA) in Experimental Autoimmune Anterior Uveitis . Invest. Ophthalmol. Vis. Sci. 2002;43(13):1548.
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Purpose: Experimental antoimmune anteior uveitis (EAAU) can be induced in Lewis rats by a low molecular weight protein associated with bovine iris/ciliary body melanin. The present study was undertaken to characterize the antigen(s). Methods: MAA isolated from bovine iris/CB was purified to homogeneity using a combination of cation exchange and size exclusion chromatography followed by isoelectric focusing and preparative-PAGE . Isoelectric focusing was performed using Rotofor with ampholyte pH3-10 or pH5-8 in reduced conditions. Purified protein was treated with two different enzymes-Endo-O-Glycosidase and Sialidase A, separately – to specifically remove O-linked carbohydrates and sialic acid. The importance of O-linked carbohydrates and sialic acid to the uveitogenecity of MAA was investigated by comparing the development of EAAU after immunization of Lewis rats with carbohydrate-depleted and glycosylated MAA. Adjuvants CFA and PTX were included in the immunization protocol. The efficiency of deglycosylation was monitored by SDS-PAGE analysis. Results: All Lewis rats immunized with O-linked carbohydrate-depleted MAA (n=6) or sialic acid depleted MAA (n=6) did not develop EAAU. Control animals (n=6) immunized with glycosylated MAA developed severe bilateral EAAU. SDS-PAGE analysis revealed a decrease in the Mr of O-linked carbohydrate depleted and sialic acid depleted MAA. Conclusion: The isoelectric pH of MAA is 6.75. Sialic acid and O-linked carbohydrates on the uveitogenic protein(s) are necessary for the development of EAAU.
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