The overall goals of this grant proposal is to determine role(s) of lens proteinases, their cognate inhibitors and proteolytic products of crystallins in the formation of high mo. wt. (HMW) proteins during the development of senile cataract. In this study, human lenses of different ages and cataractous lenses will be utilized. The following four specific aims will be pursued: (1) Regulation of proteolysis in lens: The regulation of proteolysis in the lens at the enzyme and protein substrate levels will be determined, i.e. by searching for zymogens and/or proteinase-inhibitor complexes of two lens proteinases, i.e.a 25 kDa and a membrane proteinase, and examining the effect of oxidation on endogenous proteinase inhibitors and the preferential proteolysis of native vs. oxidatively modified crystallins by endogenous proteinases. (2) Origin of degraded polypeptides from crystallins: The identity of """"""""parent crystallins"""""""" from which the three in vivo found degraded polypeptides of human lenses, i.e. 5.5 kDa, 9 kDa and 15 kDa, originated will be established by comparing their amino acid sequences. Lens crystallins will be proteolyzed in vitro with the two lens proteinases (25 kDa and a membrane proteinase) and proteolysis products compared with similar polypeptides found in vivo for their Mr's and partial N-terminal amino acid sequences. In addition, the origin of the degraded polypeptides from crystallins by a non-enzymatic reaction (Fenton reaction) will also be examined. (3) Cross-linking of degraded polypeptides to form HMW proteins: The mechanism of cross-linking to form HMW proteins in vitro by degraded polypeptides per se or between these polypeptides and individual crystallins will be determined. (4) Search for and quantitation of cross-linked products of degraded polypeptides in HMW and water insoluble proteins of normal aging and cataractous lenses. Antibodies raised against the specific cross-linked degraded polypeptides will be utilized to identify and quantitate the levels of aggregates of these polypeptides by Western blot analysis and a radioimmunoassay method respectively. The above studies will show: (i) how the lens proteolytic process is regulated in vivo; (ii) whether the degradation of crystallins in vivo is enzymatic or non-enzymatic, and (iii) if the degraded polypeptides cross-link per se and with individual crystallins to form the HMW proteins that exist in increasing levels in aging and cataractous lenses.
