Inhibition of a Lens Endogenous Proteinase by –Crystallin

Abstract

Abstract: : Purpose: The purpose of the study was to determine inhibition of a human lens Arg–bond hydrolyzing proteinase by α–crystallin. Methods:To determine the presence of an Arg–bond hydrolyzing proteinase, the α–crystallin fractions, isolated from young (20–year–old) and old (60–year–old) lenses, were activated by several detergents and both enzyme activity and proteolysis of αA– and αB–crystallins were monitored. The proteinase inhibition by α–crystallin was determined in in vitro experiments. For this purpose, the endogenous proteinase was purified from sodium deoxycholate– treated α–crystallin fraction by four consecutive chromatographic steps. For the use α–crystallin as an inhibitor, it was first denatured with urea (to inactivate an α–crystallin–associated proteinase) and then renatured by the removal of urea. Results: Except Triton X–100, other detergents such as sodium deoxycholate, octyl ß–glucopyranoside, and CHAPS activated the proteinase at almost the same levels in the α–crystallin fractions (isolated from young or older lenses), and also resulted in proteolysis of both αA and αB–crystallins. No enzyme activation and proteolysis of crystallins were observed in the absence of the detergents. The purified proteinase showed a doublet of abour 25 kDa on SDS–PAGE, which were identified as N–termianlly truncated ßA3/A1–crystallin by MALDI–TOF analysis. The recombinant αA and αB–crystallins were also proteolyzed by the purified proteinase during in vitro incubation. On sodium deoxycholate treatment and size–exclusion chromatography, the inhibitor activity of α–crystallin towards the proteinase was reduced by about 75%. Further, the purified α–crystallin showed 60% inhibition of the purified proteinase at a ratio of 1:6 (proteinase:α–crystallin). The inhibition was observed in both concentration– and time–dependent manner. Conclusions:A detergent–dissociable complex of α–crystallin and an Arg–bond hydrolyzing proteinase exists in vivo, and detergents such as sodium deoxycholate cause proteinase activation resulting in proteolysis of both αA– and αB–crystallins. The α–crystallin acts as an inhibitor of the lens endogenous proteinase.

Keywords: crystallins • proteolysis • cataract