Dynamic O-GlcNAcylation of the small heat shock protein alpha B-crystallin.
alphaB-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 alphaA-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 alphaB-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 alphaB as Thr 170. We have further shown that this modification is not restricted to lens alphaB-crystallin but occurs on alphaB isolated from rat heart tissue and human astroglioma cells. Two-dimensional electrophoresis of rat heart alphaB-crystallin revealed two O-GlcNAcylated forms with mobilities corresponding to the unphosphorylated form (alphaB2) and an unidentified, slightly more acidic form. Phosphorylated alphaB-crystallin (alphaB1) was not detected in the rat heart preparation. The major O-GlcNAcylation site on alphaB-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 alphaB-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.
Roquemore
E P
EP
Department of Biochemistry and Molecular Genetics, University of Alabama at Birmingham 35294-0005, USA.
Chevrier
M R
MR
Cotter
R J
RJ
Hart
G W
GW
D000117:Acetylglucosamine / Q000378:metabolism*; D000595:Amino Acid Sequence; D000818:Animals; D001253:Astrocytes / Q000378:metabolism; D003459:Crystallins / Q000378:metabolism*; D015180:Electrophoresis, Gel, Two-Dimensional; D006360:Heat-Shock Proteins / Q000378:metabolism*; D006801:Humans; D007908:Lens, Crystalline / Q000378:metabolism; D008253:Macaca mulatta; D008969:Molecular Sequence Data; D009206:Myocardium / Q000378:metabolism; D010446:Peptide Fragments / Q000737:chemistry; D011499:Protein Processing, Post-Translational; D051381:Rats; D017207:Rats, Sprague-Dawley; D013912:Threonine / Q000737:chemistry; D014407:Tumor Cells, Cultured
D003459:Crystallins; D006360:Heat-Shock Proteins; D010446:Peptide Fragments; D013912:Threonine; D000117:Acetylglucosamine