αB-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 αA-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 αB-crystallin are 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 αB as Thr 170. We have further shown that this modification is not restricted to lens αB-crystallin but occurs on αB isolated from rat heart tissue and human astroglioma cells. Two-dimensional electrophoresis of rat heart αB-crystallin revealed two O-GlcNAcylated forms with mobilities corresponding to the unphosphorylated form (αB2) and an unidentified, slightly more acidic form. Phosphorylated αB-crystallin (αB1) was not detected in the rat heart preparation. The major O-GlcNAcylation site on αB-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 αB-crystallin is not sialic 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.
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