The effect of dextran on subunit exchange of the molecular chaperone alphaA-crystallin.

Alpha-crystallin, a member of small heat shock protein (sHsp) family, is comprised of alphaA and alphaB subunits and acts as a molecular chaperone by interacting with unfolding proteins to prevent their aggregation. The alphaA-crystallin homopolymer consists of 30-40 subunits that are undergoing dynamic exchange. In vivo, alpha-crystallin elicits its chaperone action in a crowded cellular environment (e.g. in the lens). In vitro, inert molecular crowding agents (e.g. dextran) are often used to mimic crowded conditions. In this study, it was found that alpha-crystallin and alphaA-crystallin are poorer chaperones in the presence of dextran. Using fluorescence resonance energy transfer, it is shown that the alphaA-crystallin subunit exchange rate strongly increases with temperature. Binding of reduced ovotransferrin to alphaA-crystallin markedly decreases the rate of subunit exchange, as does the presence of dextran. In addition, in the presence of dextran the effect of reduced ovotransferrin on decreasing the rate of subunit exchange of alphaA-crystallin is greater than in the absence of dextran. Under the conditions of molecular crowding, the alphaA-crystallin subunit exchange rate is not temperature-dependent. In the absence of dextran, the exchange rate of alphaA-crystallin subunits correlates with its chaperone efficiency, i.e. the chaperone ability of alphaA-crystallin increases with temperature. However in the presence of dextran, the temperature dependence of the chaperone ability of alphaA-crystallin is eliminated.

Comparison of Lp82- and m-calpain-mediated proteolysis during cataractogenesis in Shumiya cataract rat (SCR).

PURPOSE: It is well known that m-calpain, a ubiquitous calpain, is involved in cataract formation in rodent lens. Involvement of Lp82, a lens-specific calpain, in the cataract formation is also suggested. However, the exact relationship between Lp82-mediated proteolysis and lens opacification has not yet been established. We therefore compared Lp82- and m-calpain-mediated proteolyses of alphaA-crystallin during cataractogenesis to clarify whether Lp82 is involved in cataract formation. METHODS: In order to analyze the Lp82- and m-calpain-mediated proteolyses, we developed antibodies exclusively specific to the proteolytic products of alphaA-crystallin produced by Lp82 and m-calpain actions, respectively. The proteolytic profiles of alphaA-crystallin by Lp82 and m-calpain during cataractogenesis in SCR lenses were analyzed by Western blotting and immunohistochemical staining. RESULTS: While m-calpain-mediated proteolysis was detected predominantly in cataractous lenses, Lp82-mediated proteolysis was detected not only in cataractous but in normal lenses. The m-calpain-mediated proteolysis was observed in restricted areas developing and destined to develop opacification, i.e., the nuclear and perinuclear regions of lens. On the other hand, Lp82-mediated proteolysis was observed not only in the same regions but also in the cortical region where opacity does not develop. Unlike m-calpain-mediated proteolysis, Lp82-mediated proteolysis was not inhibited by the oral administration of aminoguanidine (AG), which acts to prevent lens opacification. CONCLUSIONS: From these results, it is shown that there is no direct contribution of Lp82-mediated proteolysis to cataract formation in SCR. Rather, Lp82 may function in fiber cell development and/or fiber cell remodeling during lens maturation under physiological conditions, since Lp82-mediated proteolysis occurs in the cortical region of normal lens.