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 constitutively expressed at high levels in the vertebrate eye lens, where it serves a structural role, and at low levels in several non-lenticular tissues. Like other members of the small heat shock protein family, alpha-crystallin has a chaperone-like activity in suppressing nonspecific aggregation of denaturing proteins in vitro. Apart from the major alpha A- and alpha B-subunits, alpha-crystallin of rodents contains an additional minor subunit resulting from alternative splicing, alpha A(ins)-crystallin. 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 coli. The multimeric 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 thermostability and morphology, as revealed by gel permeation chromatography, tryptophan fluorescence measurements, and electron microscopy. However, as compared with alpha A, the alpha A(ins)-particles display a diminished chaperone-like activity in the protection of heat-induced aggregation of beta low-crystallin. Our experiments indicate that alpha A(ins)-multimers have a 3-4-fold reduced substrate binding capacity, which might be correlated to their increased particle size and to a shielding of binding sites by the insert peptides. The structure-function relationship of the natural mutant alpha A(ins)-crystallin may shed light on the mechanism of chaperone-like activity displayed by all small heat shock proteins.
Smulders
R H
RH
Department of Biochemistry, University of Nijmegen, The Netherlands.
van Geel
I G
IG
Gerards
W L
WL
Bloemendal
H
H
de Jong
W W
WW
D017398:Alternative Splicing; D000818:Animals; D001483:Base Sequence; D003001:Cloning, Molecular; D003459:Crystallins / Q000737:chemistry / Q000494:pharmacology*; D018832:Molecular Chaperones / Q000494:pharmacology*; D008969:Molecular Sequence Data; D051381:Rats; D013329:Structure-Activity Relationship
D003459:Crystallins; D018832:Molecular Chaperones