Aspartate Residues Far from the Active Site Drive O-GlcNAc Transferase Substrate Selection
Aspartate Residues Far from the Active Site Drive O-GlcNAc Transferase Substrate Selection
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DOI：
10.1021/jacs.9b06061
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发表时间：
2019-08-21
影响因子：
15
通讯作者：
Walker, Suzanne
中科院分区：
文献类型：
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作者：
Joiner, Cassandra M.;Levine, Zebulon G.;Walker, Suzanne
O-GlcNAc是一种丰富的翻译后修饰，存在于所有后生动物的核蛋白和细胞质蛋白上。这种修饰调节多种细胞过程，并且升高的O-GlcNAc水平与癌症进展有关。一种单一的必需酶，O-GlcNAc转移酶（OGT），负责所有核质O-GlcNAc化。了解这种酶如何选择其底物对于理解和潜在地操纵其功能至关重要。在这里，我们使用蛋白质微阵列技术和蛋白质组范围内的糖基化分析，以显示保守的天冬氨酸残基在tetratricopeptide重复（TPR）腔的OGT驱动底物选择。将这些残基改变为丙氨酸改变了底物选择性，并且出乎意料地增加了蛋白质糖基化的速率。我们的研究结果支持了一个模型，其中许多OGT底物的糖基化位点由TPR结构域与底物侧链的C-末端5至15个残基的接触决定。除了指导靶向OGT的TPR结构域的抑制剂的设计外，这些信息还将为工程底物以探索生物功能的努力提供信息。
O-GlcNAc is an abundant post-translational modification found on nuclear and cytoplasmic proteins in all metazoans. This modification regulates a wide variety of cellular processes, and elevated O-GlcNAc levels have been implicated in cancer progression. A single essential enzyme, O-GlcNAc transferase (OGT), is responsible for all nucleocytoplasmic O-GlcNAcylation. Understanding how this enzyme chooses its substrates is critical for understanding, and potentially manipulating, its functions. Here we use protein microarray technology and proteome-wide glycosylation profiling to show that conserved aspartate residues in the tetratricopeptide repeat (TPR) lumen of OGT drive substrate selection. Changing these residues to alanines alters substrate selectivity and unexpectedly increases rates of protein glycosylation. Our findings support a model where sites of glycosylation for many OGT substrates are determined by TPR domain contacts to substrate side chains five to fifteen residues C-terminal to the glycosite. In addition to guiding design of inhibitors that target OGT's TPR domain, this information will inform efforts to engineer substrates to explore biological functions.