A bioorthogonal precision tool for human N-acetylglucosaminyltransferase V
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Yu Liu Ganka Bineva-Todd Richard W Meek Laura Mazo Beatriz Piniello Olga Moroz Sean A Burnap Nadima Begum André Ohara Chloe Roustan Sara Tomita Svend Kjaer Karen Polizzi Weston B Struwe Carme Rovira Gideon J Davies Benjamin SchumannAbstract
Correct elaboration of N-linked glycans in the secretory pathway of human cells is essential in physiology. Early N-glycan biosynthesis follows an assembly line principle before undergoing crucial elaboration points that feature the sequential incorporation of the sugar N-acetylglucosamine (GlcNAc). The activity of GlcNAc transferase V (MGAT5) primes the biosynthesis of an N-glycan antenna that is heavily upregulated in cancer. Still, the functional relevance and substrate choice of MGAT5 are ill-defined. Here, we employ protein engineering to develop a bioorthogonal substrate analog for the activity of MGAT5. Chemoenzymatic synthesis is used to produce a collection of nucleotide-sugar analogs with bulky, bioorthogonal acylamide side chains. We find that WT-MGAT5 displays considerable activity toward such substrate analogues. Protein engineering yields an MGAT5 variant that loses activity against the native nucleotide sugar and increases activity toward a 4-azidobutyramide-containing substrate analogue. By such restriction of substrate specificity, we show that the orthogonal enzyme-substrate pair is suitable to bioorthogonally tag glycoproteins. Through X-ray crystallography and molecular dynamics simulations, we establish the structural basis of MGAT5 engineering, informing the design rules for bioorthogonal precision chemical tools.
Journal details
Volume
146
Issue number
39
Pages
26707-26718
Available online
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10.1021/jacs.4c05955
Europe PubMed Central
39287665
Pubmed
39287665
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The publication was previously a preprint.
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