A Bioorthogonal Precision Tool for Human <i>N</i> -Acetylglucosaminyltransferase V
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 <i>N</i>-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.
Authors
- Ganka Bineva‐Todd (ORCID: https://orcid.org/0000-0001-8926-9417)
- G.J. Davies (ORCID: https://orcid.org/0000-0002-7343-776X)
- Weston B. Struwe (ORCID: https://orcid.org/0000-0003-0594-226X)
- André Ohara (ORCID: https://orcid.org/0000-0002-9792-284X)
- Karen M. Polizzi (ORCID: https://orcid.org/0000-0001-5435-2667)
- Chloë Roustan (ORCID: https://orcid.org/0000-0002-8439-8997)
- Richard W. Meek (ORCID: https://orcid.org/0000-0002-1370-0896)
- Sean A. Burnap (ORCID: https://orcid.org/0000-0002-3408-8608)
- Beatriz Piniello (ORCID: https://orcid.org/0000-0001-5557-3146)
- Carme Rovira (ORCID: https://orcid.org/0000-0003-1477-5010)
- Benjamin Schumann (ORCID: https://orcid.org/0000-0001-5504-0147)
- Olga V. Moroz (ORCID: https://orcid.org/0000-0002-0354-6119)
- Laura Mazo (ORCID: https://orcid.org/0009-0005-1896-3096)
- Liu Yu (ORCID: https://orcid.org/0009-0009-0722-182X)
- Svend Kjær (ORCID: https://orcid.org/0000-0001-9767-8683)
- Nadima Begum
- Sara Tomita
Institutions
- Institució Catalana de Recerca i Estudis Avançats (ES)
- The Francis Crick Institute (GB)
- University of Oxford (GB)
- University of Southampton (GB)
- Imperial College London (GB)
- University of York (GB)
- Universitat de Barcelona (ES)
Publication Details
- Journal
- Journal of the American Chemical Society
- Published
- 2024-09-17
- DOI
- https://doi.org/10.1021/jacs.4c05955
- Citations
- 7
- Primary Topic
- Glycosylation and Glycoproteins Research
- Type
- article
- Field-Weighted Citation Impact
- 1.64
Funders
- Wellcome Trust
- Francis Crick Institute
- UK Research and Innovation
- Ministerio de Ciencia, Innovación y Universidades
- Cancer Research UK
- Department of Health and Social Care
- Royal Society
- University of Southampton
- Barcelona Supercomputing Center
- National Institutes of Health
- Medical Research Council
- Engineering and Physical Sciences Research Council
- Biotechnology and Biological Sciences Research Council
- Agència de Gestió d'Ajuts Universitaris i de Recerca
- Agencia Estatal de Investigación
- H2020 European Research Council