Chemical Kinomics Research Center , Korea Institute of Science and Technology (KIST) , 5 Hwarangro 14-gil , Seongbuk-gu, Seoul 02792 , Republic of Korea.
Bio-Organic Chemistry Research Group, Department of Chemical Engineering and Chemistry , Eindhoven University of Technology , P.O. Box 513, 5600 MB Eindhoven , Netherlands.
J Am Chem Soc. 2019 Aug 28;141(34):13442-13453. doi: 10.1021/jacs.9b04695. Epub 2019 Aug 16.
O-Linked α--acetylgalactosamine (O-GalNAc) glycans constitute a major part of the human glycome. They are difficult to study because of the complex interplay of 20 distinct glycosyltransferase isoenzymes that initiate this form of glycosylation, the polypeptide -acetylgalactosaminyltransferases (GalNAc-Ts). Despite proven disease relevance, correlating the activity of individual GalNAc-Ts with biological function remains challenging due to a lack of tools to probe their substrate specificity in a complex biological environment. Here, we develop a "bump-hole" chemical reporter system for studying GalNAc-T activity in vitro. Individual GalNAc-Ts were rationally engineered to contain an enlarged active site (hole) and probed with a newly synthesized collection of 20 (bumped) uridine diphosphate -acetylgalactosamine (UDP-GalNAc) analogs to identify enzymesubstrate pairs that retain peptide specificities but are otherwise completely orthogonal to native enzymesubstrate pairs. The approach was applicable to multiple GalNAc-T isoenzymes, including GalNAc-T1 and -T2 that prefer nonglycosylated peptide substrates and GalNAcT-10 that prefers a preglycosylated peptide substrate. A detailed investigation of enzyme kinetics and specificities revealed the robustness of the approach to faithfully report on GalNAc-T activity and paves the way for studying substrate specificities in living systems.
O-连接的α-N-乙酰半乳糖胺(O-GalNAc)糖基化是人类糖组的主要组成部分。由于启动这种糖基化的 20 种不同糖基转移酶同工酶的复杂相互作用,它们很难研究,这些多肽-N-乙酰半乳糖胺基转移酶(GalNAc-Ts)。尽管已经证明与疾病有关,但由于缺乏在复杂的生物环境中探测其底物特异性的工具,仍然难以将单个 GalNAc-T 的活性与生物学功能相关联。在这里,我们开发了一种用于体外研究 GalNAc-T 活性的“凸起-空穴”化学报告系统。通过合理设计单个 GalNAc-T 使其包含扩大的活性位点(空穴),并用新合成的 20 个(凸起)尿苷二磷酸-N-乙酰半乳糖胺(UDP-GalNAc)类似物进行探测,以鉴定保留肽特异性但与天然酶-底物对完全正交的酶-底物对。该方法适用于多种 GalNAc-T 同工酶,包括优先非糖基化肽底物的 GalNAc-T1 和 -T2 以及优先预糖基化肽底物的 GalNAcT-10。对酶动力学和特异性的详细研究表明,该方法能够忠实地报告 GalNAc-T 活性,为在活系统中研究底物特异性铺平了道路。