Modification of proteins play a pivotal role in changing a normal lens into a cataractous lens. Proteolytic cleavage of peptide bonds in crystallins by endogenous enzymes is probably one of the most frequent and important modifications. So far, information about these cleavages in crystallins is limited in terms of their enzymatic nature, regulation at the enzyme and substrate levels, effect on crystallin functions and secondary modifications in crystallin fragments. Based on preliminary results, the P.I. has hypothesized that the degradation of crystallins is regulated at both enzyme and substrate levels and the cleaved fragments undergo secondary modification such as glycation via the Maillard reaction and therefore directly participate in the cross-linking of the lens proteins which is central to the development of lens opacity. To test this hypothesis, the following three specific aims will be pursued: (1) specific Aim #1: Determine the mechanism of autolytic activation of a membrane proteinase from a putative zymogen type activation of the BA3/A1-crystallin proteinase by a detergent such as sodium deoxycholate. (2) specific Aim #2: Determine the role of the membrane proteinase and BA3/A1-crystallin proteinase in the in vivo proteolysis of selected crystallins. (3) Specific Aim #3: Determine crosslinking of degraded polypeptides in general and specifically of a 9 kDa yD-crystallin fragment to form homologous (crystallin fragments alone) and heterologous (crystallin fragments plus native crystallins) multimers possibly via the glycation mechanism of the Maillard reaction. The above studies will address the questions regarding the in vivo regulation of crystallin degradation and the role of endogenous proteinases in this process. This study will also answer the questions whether the degraded polypeptides, on secondary modifications, acquire cross-linking properties and participate directly in protein cross-linking during the development of lens opacity. Since all of the proposed studies will be carried out in human lenses, the results will be directly relevant to the human senile cataract problem.
