Aspartate Residues Far From the Active Site Drive O-GlcNAc Transferase Substrate Selection

Abstract: 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. In this study, Invitrogen's Human ProtoArray protein microarray was used as a platform for high-throughput enzyme activity profiling. Multiple batches of microarrays were used over the course of the study, thus the platform represents a composite of the proteins on all microarray batches. 3 microarrays each were treated with wild-type OGT, the mutant dubbed D2A, or treated with reagents without enzyme. Enzyme microarrays were normalized 2 different ways; both modes of normalization are represented here.

摘要：O-连接N-乙酰葡糖胺（O-GlcNAc）是一类广泛存在于所有后生动物细胞核与细胞质蛋白中的丰度较高的翻译后修饰。该修饰调控诸多细胞生命过程，而O-GlcNAc水平升高已被证实与癌症进展密切相关。仅有一种必需酶——O-连接N-乙酰葡糖胺转移酶（O-GlcNAc transferase, OGT）——负责所有核质层面的O-GlcNAc糖基化修饰。阐明该酶如何选择底物，对于理解其功能乃至实现功能调控均具有关键意义。本研究借助蛋白质芯片技术与全蛋白质组糖基化谱分析，证实OGT的四肽重复序列（tetratricopeptide repeat, TPR）内腔中保守的天冬氨酸残基介导了底物选择过程。将这些残基突变为丙氨酸后，OGT的底物选择性发生改变，且意外提升了蛋白质糖基化反应速率。本研究结果支持如下模型：多数OGT底物的糖基化位点，由TPR结构域与糖基化位点C端5至15位残基侧链的相互作用所决定。该发现不仅可为靶向OGT TPR结构域的抑制剂设计提供指导，还将助力通过底物工程化改造探索其生物学功能的相关研究。本研究采用Invitrogen公司的人源ProtoArray蛋白质芯片作为高通量酶活分析平台。研究过程中使用了多批次芯片，因此该平台涵盖了所有批次芯片上的全部蛋白质组分。实验分别设置3张芯片经野生型OGT处理、3张芯片经命名为D2A的突变体OGT处理，以及3张芯片仅添加不含酶的反应试剂作为对照。酶反应芯片采用两种不同方式进行归一化处理，本研究均呈现了这两种归一化模式的结果。