Mutational analysis of the catalytic domain of O-linked N-acetylglucosaminyl transferase
Mutational analysis of the catalytic domain of O-linked N-acetylglucosaminyl transferase
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DOI：
10.1074/jbc.m504948200
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发表时间：
2005-10-21
影响因子：
4.8
通讯作者：
Hanover, JA
中科院分区：
文献类型：
--
作者：
Lazarus, BD;Roos, MD;Hanover, JA
O-连接的N-乙酰葡糖胺转移酶（OGT）催化O-连接的GlcNAc转移到多种靶蛋白的丝氨酸/苏氨酸残基，其中许多靶蛋白与糖尿病和神经变性等疾病有关。O-GlcNAc向蛋白质的添加响应于UDP-GlcNAc的细胞浓度的波动而发生，这是由营养物进入己糖胺生物合成途径引起的。然而，涉及糖核苷酸识别和转移到蛋白质的分子机制知之甚少。我们采用定点突变，目标潜在的重要氨基酸残基内的两个保守的催化结构域的OGT（CDI和CDII），然后通过体外糖基化测定，以评估细菌表达后的N-乙酰氨基葡萄糖转移酶活性。虽然许多氨基酸取代引起的酶的失活，我们确定了三个氨基酸残基（两个在CD I和一个在CD II），突变时产生活性酶。基于结构的同源性建模显示，这些允许的突变体可能是在或附近的糖核苷酸结合位点。我们的研究结果表明，催化结构域的两个保守区域，CDI和CDII，有助于形成一个UDP-GlcNAc结合口袋，催化O-GlcNAc转移到底物蛋白的模型。鉴定可行的OGT突变体可能有助于研究其在营养传感和信号转导级联中的作用。
O-Linked N-acetylglucosaminyltransferase (OGT) catalyzes the transfer of O-linked GlcNAc to serine/threonine residues of a variety of target proteins, many of which have been implicated in such diseases as diabetes and neurodegeneration. The addition of O-GlcNAc to proteins occurs in response to fluctuations in cellular concentrations of UDP-GlcNAc, which result from nutrients entering the hexosamine biosynthetic pathway. However, the molecular mechanisms involved in sugar nucleotide recognition and transfer to protein are poorly understood. We employed site-directed mutagenesis to target potentially important amino acid residues within the two conserved catalytic domains of OGT(CDI and CDII), followed by an in vitro glycosylation assay to evaluate N-acetylglucosaminyltransferase activity after bacterial expression. Although many of the amino acid substitutions caused inactivation of the enzyme, we identified three amino acid residues ( two in CD I and one in CD II) that produced viable enzymes when mutated. Structure-based homology modeling revealed that these permissive mutants may be either in or near the sugar nucleotide-binding site. Our findings suggest a model in which the two conserved regions of the catalytic domain, CDI and CDII, contribute to the formation of a UDP-GlcNAc-binding pocket that catalyzes the transfer of O-GlcNAc to substrate proteins. Identification of viable OGT mutants may facilitate examination of its role in nutrient sensing and signal transduction cascades.