The major in vivo modifications of the human water-insoluble lens crystallins are disulfide bonds, deamidation, methionine oxidation and backbone cleavage

This investigation of the water-insoluble crystallins from human lenses has used multiple chromatographic separations to obtain proteins of sufficient purity for mass spectrometric analysis. Each fraction was analysed to dete rmine the molecular masses of the constituent proteins as well as peptides in tryptic digests of these proteins. The major components of the water-ins oluble crystallins were identified as alpha A-and alpha B-crystallins. In a ddition, gamma S-, beta B1-, gamma D-, beta A3/A1- and beta B2-crystallins were found, in order of decreasing abundance. Although there was evidence o f some backbone cleavage, the predominant forms of alpha A-, alpha B, beta B2-, gamma S- and gamma D-crystallins were the intact polypeptide chains, T he major modifications distinguishing the water-soluble crystallins were in creased disulfide bonding, oxidation of Met, deamidation of Gin and Asn and backbone cleavage, Of the many reactions hypothesized to lead to crystalli n insolubility and cataract, these results most strongly support metal-cata lysed oxidation, deamidation and truncation as initiators of conformational changes that Favor aggregation. (C) 2000 Academic Press.