Roquemore E P, Chevrier M R, Cotter R J, Hart G W
Department of Biochemistry and Molecular Genetics, University of Alabama at Birmingham 35294-0005, USA.
Biochemistry. 1996 Mar 19;35(11):3578-86. doi: 10.1021/bi951918j.
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.
αB-晶状体蛋白最初被描述为一种晶状体结构蛋白，现在已知它是小分子热休克蛋白家族的成员，并在许多非晶状体组织中表达。这种高度保守的20 kDa蛋白与同源蛋白聚集，包括αA-晶状体蛋白和小分子热休克蛋白HSP28，形成大型异聚体复合物。最近，Roquemore等人（1992年）证实，晶状体αB-晶状体蛋白的磷酸化和非磷酸化形式都被O-连接的N-乙酰葡糖胺修饰，这是一种在核蛋白和细胞质蛋白上大量存在的动态翻译后修饰。在本文中，我们确定了晶状体αB上O-连接的N-乙酰葡糖胺化的主要位点为苏氨酸170。我们进一步表明，这种修饰不仅限于晶状体αB-晶状体蛋白，在从大鼠心脏组织和人星形胶质瘤细胞中分离出的αB上也会发生。大鼠心脏αB-晶状体蛋白的二维电泳显示出两种O-连接的N-乙酰葡糖胺化形式，其迁移率分别对应于非磷酸化形式（αB2）和一种未知的、酸性稍强的形式。在大鼠心脏制剂中未检测到磷酸化的αB-晶状体蛋白（αB1）。大鼠心脏αB-晶状体蛋白上的主要O-连接的N-乙酰葡糖胺化位点似乎也在苏氨酸170。对U373-MG星形胶质瘤细胞的代谢脉冲追踪标记研究表明，αB-晶状体蛋白上碳水化合物的周转不是静态的，而是比蛋白质主链本身的周转快许多倍，这与O-连接的N-乙酰葡糖胺对这种小分子热休克蛋白的调节作用一致。
