Human OGA binds substrates in a conserved peptide recognition groove.
Human OGA binds substrates in a conserved peptide recognition groove.
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DOI：
10.1042/bj20101338
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发表时间：
2010-11-15
期刊：
影响因子：
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通讯作者：
van Aalten DM
中科院分区：
文献类型：
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作者：
Schimpl M;Schüttelkopf AW;Borodkin VS;van Aalten DM
O-GlcNAc（O-连接的 N-乙酰氨基葡萄糖）对细胞蛋白质的修饰会与蛋白质磷酸化竞争并调节大量的细胞过程。 O-GlcNAc 酰化是由两种相反的酶 O-GlcNAc 转移酶和 OGA（O-GlcNAcase 或 β-N-乙酰氨基葡萄糖苷酶）精心策划的，它们通过尚未确定的机制识别其靶蛋白。在本研究中，我们首次揭示了人类 OGA 底物识别机制。一种新型细菌 OGA 直系同源物的结构揭示了后生动物 OGA 中保守的假定底物结合沟。在该结构的指导下，人 OGA 中该凹槽内的保守氨基酸发生突变，并在体外去糖基化测定中测试了三种不同底物蛋白 [TAB1（转化生长因子-β-激活蛋白激酶 1 结合蛋白 1）、FoxO1（叉头盒 O1）和 CREB（cAMP 反应元件结合蛋白）] 的活性。这些结果提供了第一个证据，证明人类 OGA 可能具有底物识别机制，该机制涉及与 GlcNAc 结合位点之外的 O-GlcNAc 酰化蛋白的相互作用，这可能对不同蛋白上 O-GlcNAc 循环的差异调节产生影响。
Modification of cellular proteins with O-GlcNAc (O-linked N-acetylglucosamine) competes with protein phosphorylation and regulates a plethora of cellular processes. O-GlcNAcylation is orchestrated by two opposing enzymes, O-GlcNAc transferase and OGA (O-GlcNAcase or β-N-acetylglucosaminidase), which recognize their target proteins via as yet unidentified mechanisms. In the present study, we uncovered the first insights into the mechanism of substrate recognition by human OGA. The structure of a novel bacterial OGA orthologue reveals a putative substrate-binding groove, conserved in metazoan OGAs. Guided by this structure, conserved amino acids lining this groove in human OGA were mutated and the activity on three different substrate proteins [TAB1 (transforming growth factor-β-activated protein kinase 1-binding protein 1), FoxO1 (forkhead box O1) and CREB (cAMP-response-element-binding protein)] was tested in an in vitro deglycosylation assay. The results provide the first evidence that human OGA may possess a substrate-recognition mechanism that involves interactions with O-GlcNAcylated proteins beyond the GlcNAc-binding site, with possible implications for differential regulation of cycling of O-GlcNAc on different proteins.