Crystallins of the eye lens are long-lived proteins because they hardly turn over. Such proteins are constantly subjected to posttranslational modifications such as nonenzymatic glycosylation (glycation). Diabetic patients are four to six times more likely to develop cataract at a younger age than the normal population and glycation of lens crystallins could play a significant role in diabetic cataractogenesis. Glycation proceeds primarily in two stages, formation of Schiff-base and Amadori products or early glycation and formation of advanced glycation end products that are fluorescent and having protein cross-linking properties. The long-range goal of the proposal is to study the role of glycation in diabetic cataractogenesis. The immediate goals are to identify the amino acid sites of different crystallins that are being glycated and to determine the rates or extents of glycation of such sites during the progression of diabetes and cataract in streptozotocin-diabetic rats, in aging and diabetic humans, and during in vitro glycation of rat and human crystallins with various sugars and sugar phosphates. Such information is essential to understand why glycation would induce protein conformational changes, increased reactivity of thiols and enhanced thiol oxidation. To achieve these goals modern techniques in protein chemistry will be applied which are: molecular sieve HPLC for separation of [3H]NaBH4 reacted native crystallins including HMW aggregates, reverse-phase HPLC for separation of crystallin subunit polypeptides and tryptic and chymotryptic peptides, Affigel 601 affinity chromatography for purification of glycated peptides, determination of amino acid composition by a HPLC/post-column derivatization method and amino acid sequencing by a manual microsequencing method. For identification of the crystallins in the in vitro and in vivo generated soluble or insoluble fractions, in addition to classical chemical characterization, immunologic identification will be carried out with monoclonal antibodies that will be produced against various rat and human crystallins. With the aid of inhibitors of early glycation and advanced glycation such as acetylsalicylic acid (aspirin) and aminoguanidine it will be shown whether early glycation and advanced glycation play any role in diabetic cataractogenesis. Moreover, with aspirin treatment it will be shown that glycation of which crystallins are inhibited in vitro (in rat and human crystallins) and in vivo (only in rat crystallins) and whether the mechanism involves acetylation of specific amino acid residues.
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