Dynamic O-GlcNAcylation of the small heat shock protein alpha B-crystallin

alpha B-Crystallin, originally described as a structural lens protein, is now known to be a member of the small heat shock protein family and is expressed in a number of nonlens tissues. This highly conserved 20 kDa protein aggregates with homologous proteins, including alpha A-crystallin and the small heat shock protein HSP28, to form large heteromeric complexes. Recently, Roquemore et al. (1992) have established that both phosphorylated and unphosphorylated forms of lens alpha B-crystallin ate modified with O-linked N-acetylglucosamine, a dynamic posttranslational modification abundant on nuclear and cytoplasmic proteins. In this paper, we have identified the major site of O-GlcNAcylation on lens alpha B as Thr 170. We have further shown that this modification is not restricted to lens alpha B-crystallin but occurs on alpha B isolated from rat heart tissue and human astroglioma cells. Two-dimensional electrophoresis of rat heart alpha B-crystallin revealed two O-GlcNAcylated forms with mobilities corresponding to the unphosphorylated form (alpha B2) and an unidentified, slightly more acidic form. Phosphorylated alpha B-crystallin (alpha B1) was not detected in the rat heart preparation. The major O-GlcNAcylation site on alpha B-crystallins from rat heart also appears to be at Thr 170. Metabolic pulse-chase labeling studies of U373-MG astroglioma cells indicated that turnover of the carbohydrate on alpha B-crystallin is not static but proceeds many-fold more rapidly than turnover of the protein backbone itself, consistent with a regulatory role for O-GlcNAc on this small heat shock protein.