Elucidating the protein substrate recognition of O-GlcNAc transferase (OGT) toward O-GlcNAcase (OGA) using a GlcNAc electrophilic probe.
Elucidating the protein substrate recognition of O-GlcNAc transferase (OGT) toward O-GlcNAcase (OGA) using a GlcNAc electrophilic probe.
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使用GLCNAC亲电探针阐明O-GLCNAC转移酶（OGT）朝O-Glcnacase（OGA）阐明蛋白质底物的识别。
DOI：
10.1016/j.ijbiomac.2020.12.078
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发表时间：
2021-02-01
影响因子：
8.2
通讯作者：
Jiang J
中科院分区：
文献类型：
--
作者：
Kositzke A;Fan D;Wang A;Li H;Worth M;Jiang J
人必需的O-连接β-N-乙酰葡糖胺（O-GlcNAc）转移酶（OGT）是负责用单糖O-GlcNAc修饰数千种细胞内蛋白质的唯一酶。这种独特的修饰在人类健康和疾病中起着至关重要的作用，但OGT的底物识别仍然知之甚少。有趣的是，据报道唯一去除这种修饰的人类酶O-GlcNAc酶（OGA）是O-GlcNAc修饰的。在这里，我们利用GlcNAc亲电探针（GEP 1A），以快速筛选OGT突变体的荧光测定，可以区分改变OGT-糖和蛋白质底物结合，以帮助阐明OGT对OGA蛋白质底物的结合模式。由于OGT三肽重复（TPR）结构域在OGT-OGA结合中起关键作用，我们筛选了30个OGT TPR突变体，它们显示15个“梯形”天冬酰胺或天冬氨酸残基，跨越TPR 3-7和10-13.5，影响OGA O-GlcNAc化。通过应用截短的OGA构建体，我们发现OGA的N-末端区域或假组蛋白乙酰转移酶结构域不是其O-GlcNAc化所必需的，这表明OGT通过其催化和/或茎结构域与OGA功能性地相互作用。这项工作代表了第一次努力，系统地调查每个OGT TPR和我们的研究结果将促进新的策略的发展，调查底物特异性O-GlcNAc酰化的作用。
The essential human O-linked β-N-acetylglucosamine (O-GlcNAc) transferase (OGT) is the sole enzyme responsible for modifying thousands of intracellular proteins with the monosaccharide O-GlcNAc. This unique modification plays crucial roles in human health and disease, but the substrate recognition of OGT remains poorly understood. Intriguingly, the only human enzyme reported to remove this modification, O-GlcNAcase (OGA), is O-GlcNAc modified. Here, we exploited a GlcNAc electrophilic probe (GEP1A) to rapidly screen OGT mutants in a fluorescence assay that can discriminate between altered OGT-sugar and -protein substrate binding to help elucidate the binding mode of OGT toward OGA protein substrate. Since OGT tetratricopeptide repeat (TPR) domain plays a key role in OGT-OGA binding, we screened 30 OGT TPR mutants, which revealed 15 “ladder like” asparagine or aspartate residues spanning TPRs 3-7 and 10-13.5 that affect OGA O-GlcNAcylation. By applying a truncated OGA construct, we found that OGA’s N-terminal region or pseudo histone acetyltransferase domain is not required for its O-GlcNAcylation, suggesting OGT functionally interacts with OGA through its catalytic and/or stalk domains. This work represents the first effort to systemically investigate each OGT TPR and our findings will facilitate the development of new strategies to investigate the role of substrate-specific O-GlcNAcylation.
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影响因子：
15
作者：
Darabedian N;Gao J;Chuh KN;Woo CM;Pratt MR
通讯作者：
Pratt MR
影响因子：
5.6
作者：
Fontes, MRM;Teh, T;Kobe, B
通讯作者：
Kobe, B
影响因子：
15
作者：
Joiner, Cassandra M.;Levine, Zebulon G.;Walker, Suzanne
通讯作者：
Walker, Suzanne
DOI：
10.1002/chem.202000155
发表时间：
2020-09-21
期刊：
Chemistry (Weinheim an der Bergstrasse, Germany)
影响因子：
--
作者：
Estevez A;Zhu D;Blankenship C;Jiang J
通讯作者：
Jiang J
影响因子：
12.8
作者：
Hewagama A;Gorelik G;Patel D;Liyanarachchi P;McCune WJ;Somers E;Gonzalez-Rivera T;Michigan Lupus Cohort;Strickland F;Richardson B
通讯作者：
Richardson B