AGE-RELATED-CHANGES IN BOVINE ALPHA-CRYSTALLIN AND HIGH-MOLECULAR-WEIGHT PROTEIN

The high-molecular-weight (HMW) protein from the lens is composed most ly of alpha-crystallin in a highly aggregated state. Bovine HMW protei n was carefully separated from alpha-crystallin by size-exclusion chro matography. alpha-Crystallin has chaperone-like ability whereby it sta bilizes other proteins under conditions of stress (e.g. heat). Compari son of bovine HMW protein and alpha-crystallin shows that the HMW prot ein has a markedly reduced chaperone ability compared to alpha-crystal lin. However, in contrast to the results of other workers, we observe no alteration with age in the ability of alpha-crystallin to act as a chaperone. Using electrospray ionisation mass spectrometry, changes in the phosphorylation of the alpha-crystallin subunits with age have be en quantified. Phosphorylation of alpha-crystallin occurs early in lif e but does not alter in proportion after about three years of age. In addition, phosphorylation of the A subunit of alpha-crystallin has lit tle effect on its chaperone ability. As is found in the artificially p repared HMW complex of alpha- and gamma-crystallin, NMR spectroscopy s hows that in the naturally occurring HMW protein, the short C-terminal extension of the alpha(B) subunit has lost its flexibility whereas th e alpha(A) subunit extension is still flexible. Post-translational mod ifications therefore seem to have little effect on the chaperone actio n of alpha-crystallin, but alterations in the quaternary structure of alpha-crystallin via incorporation into the HMW aggregate, lead to maj or changes in the chaperone ability of the protein. The results are co nsistent with the notion that one of the contributing factors to catar act formation in the lens is the depletion of alpha-crystallin with ag e as it is converted into the HMW protein. (C) 1996 Academic Press Lim ited