Mass Measurements of C-terminally Truncated α-Crystallins from Two-dimensional Gels Identify Lp82 as a Major Endopeptidase in Rat Lens

Molecular chaperone activity of lens α-crystallins is reduced by loss of the C terminus. The purpose of this experiment was to 1) determine the cleavage sites produced in vitro by ubiquitous m-calpain and lens-specific Lp82 on α-crystallins, 2) identify α-crystallin cleavage sites produced in vivo during maturation and cataract formation in rat lens, and 3) estimate the relative activities of Lp82 and m-calpain by appearance of protease-specific cleavage products in vivo . Total soluble protein from young rat lens was incubated with recombinant m-calpain or Lp82 and 2 m m Ca 2+ . Resulting fragmented α-crystallins were separated by two-dimensional gel electrophoresis. Eluted α-crystallin spots were analyzed by mass spectrometry. Cleavage sites on insoluble α-crystallins were determined similarly in mature rat lens nucleus and in cataractous rat lens nucleus induced by selenite. In vitro proteolysis of αA-crystallin by Lp82 and m-calpain produced unique cleavage sites by removing 5 and 11 residues, respectively, from the C terminus. In vivo , the protease-specific truncations removing 5 and 11 residues from αA were both found in maturing lens, whereas only the truncation removing 5 residues was found in cataractous lens. Other truncation sites, common to both calpain isoforms, resulted from the removal of 8, 10, 16, 17, and 22 residues from the C terminus of αA. Using uniquely truncated αA-crystallins as in vivo markers, Lp82 and m-calpain were both found to be active during normal maturation of rat lens, whereas Lp82 seemed especially active during selenite cataract formation. These C-terminal truncations decrease chaperone activity of α-crystallins, possibly leading to the observed increases in insoluble proteins during aging and cataract. The methodology that allowed accurate mass measurements of proteins eluted from 2D gels should be useful to examine rapidly other post-translational modifications.