REDUCED CHAPERONE-LIKE ACTIVITY OF ALPHA-A(INS)-CRYSTALLIN, AN ALTERNATIVE SPLICING PRODUCT CONTAINING A LARGE INSERT PEPTIDE

alpha-Crystallin is a multimeric protein complex which is constitutive ly expressed at high levels in the vertebrate eye lens, where it serve s a structural role, and at low levels in several non-lenticular tissu es. Like other members of the small heat shock protein family, alpha-c rystallin has a chaperone-like activity in suppressing nonspecific agg regation of denaturing proteins in vitro. Apart from the major alpha A - and alpha B-subunits, alpha-crystallin of rodents contains an additi onal minor subunit resulting from alternative splicing, alpha A(ins)-c rystallin. This polypeptide is identical to normal alpha A-crystallin except for an insert peptide of 23 residues. To explore the structural and functional consequences of this insertion, we have expressed rat alpha A- and alpha A(ins)-crystallin in Escherichia coil. The multimer ic particles formed by alpha A(ins) are larger and more disperse than those of alpha A, but they are native-like and display a similar therm ostability and morphology, as revealed by gel permeation chromatograph y, tryptophan fluorescence measurements, and electron microscopy, Howe ver, as compared with alpha A, the alpha A(ins)-particles display a di minished chaperone-like activity in the protection of heat-induced agg regation of beta(low)-crystallin. Our experiments indicate that alpha A(ins)-multimers have a 3-4-fold reduced substrate binding capacity, w hich might be correlated to their increased particle size and to a shi elding of binding sites by the insert peptides. The structure-function relationship of the natural mutant alpha A(ins)-crystallin may shed l ight on the mechanism of chaperone-like activity displayed by all smal l heat shock proteins.