Iterative Bump-and-Hole Engineering Creates a Bioorthogonal Reporter for <i>N</i> -Acetylglucosaminyltransferase I

High Resolution Image Download MS PowerPoint Slide Asparagine-linked protein glycosylation is among the most frequent modifications of proteins trafficking through the secretory pathway. These glycans are manufactured in an assembly line process, yielding a common precursor that is then subjected to individual modifications with different levels of complexity. An important biosynthetic modulator is the incorporation of N -acetylglucosamine (GlcNAc) at distinct positions in N-linked glycan biosynthesis, commencing with the activity of the glycosyltransferase MGAT1. While mapping of N-glycans to their corresponding protein attachment sites is generally possible, not much is known about the glycoprotein substrate choice for MGAT1 and related transferases. Analogs of GlcNAc with small bioorthogonal tags can be incorporated into N-glycans. However, due to the promiscuity of some GlcNAc transferases, incorporation is of little specificity toward individual positions. Here, we report an iterative bump-and-hole approach for the design of a bioorthogonal precision tool to study the activity of MGAT1 in mammalian cells. Structure-informed protein engineering abrogated the activity of MGAT1 toward the nucleotide-sugar UDP-GlcNAc while retaining activity toward bumped, azide-modified analogs. Kinetic and computational analyses using a neural network approach informed the synthesis of a tailored UDP-GlcNAc analog with preferential acceptance by the engineered enzyme. Following substrate biosynthesis, the strategy allowed selective incorporation of a chemical tag on MGAT1 substrate proteins in living mammalian cells with little background incorporation by other GlcNAc transferases. Our work expands the toolbox for glycan-based reporter compounds.

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Journal
Journal of the American Chemical Society
Published
2026-06-30
DOI
https://doi.org/10.1021/jacs.6c01114
Primary Topic
Carbohydrate Chemistry and Synthesis
Type
article
Field-Weighted Citation Impact
0.00

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article

Iterative Bump-and-Hole Engineering Creates a Bioorthogonal Reporter for N -Acetylglucosaminyltransferase I

Ganka Bineva‐Todd, Weston B. Struwe, André Ohara, Karen M. Polizzi et al.
Journal of the American Chemical Society
Carbohydrate Chemistry and Synthesis
article

Iterative Bump-and-Hole Engineering Creates a Bioorthogonal Reporter for N -Acetylglucosaminyltransferase I

Ganka Bineva‐Todd, Weston B. Struwe, André Ohara, Karen M. Polizzi, Anna Cioce, Carme Rovira, Benjamin Schumann, Carolyn R. Bertozzi, Mert Sagiroglugil, Liu Yu, Saskia Pieters, Freya Hoddle, Sean A. Burnap, Sophie D. Schmidt, Kevin Bruemmer
article en

Abstract

High Resolution Image Download MS PowerPoint Slide Asparagine-linked protein glycosylation is among the most frequent modifications of proteins trafficking through the secretory pathway. These glycans are manufactured in an assembly line process, yielding a common precursor that is then subjected to individual modifications with different levels of complexity. An important biosynthetic modulator is the incorporation of N -acetylglucosamine (GlcNAc) at distinct positions in N-linked glycan biosynthesis, commencing with the activity of the glycosyltransferase MGAT1. While mapping of N-glycans to their corresponding protein attachment sites is generally possible, not much is known about the glycoprotein substrate choice for MGAT1 and related transferases. Analogs of GlcNAc with small bioorthogonal tags can be incorporated into N-glycans. However, due to the promiscuity of some GlcNAc transferases, incorporation is of little specificity toward individual positions. Here, we report an iterative bump-and-hole approach for the design of a bioorthogonal precision tool to study the activity of MGAT1 in mammalian cells. Structure-informed protein engineering abrogated the activity of MGAT1 toward the nucleotide-sugar UDP-GlcNAc while retaining activity toward bumped, azide-modified analogs. Kinetic and computational analyses using a neural network approach informed the synthesis of a tailored UDP-GlcNAc analog with preferential acceptance by the engineered enzyme. Following substrate biosynthesis, the strategy allowed selective incorporation of a chemical tag on MGAT1 substrate proteins in living mammalian cells with little background incorporation by other GlcNAc transferases. Our work expands the toolbox for glycan-based reporter compounds.

Journal of the American Chemical Society
Institució Catalana de Recerca i Estudis Avançats (ES), Howard Hughes Medical Institute (US), Transport Research Laboratory (United Kingdom) (GB), The Francis Crick Institute (GB), Center for NanoScience (DE), University of Oxford (GB), Stanford Health Care (US), Geophysical Laboratory (US), Instituto de Química Orgánica General (ES), Imperial College London (GB), Universitat de Barcelona (ES), Technische Universität Dresden (DE), Stanford University (US)
Howard Hughes Medical Institute, Children's Tumor Foundation, Wellcome Trust, Francis Crick Institute, UK Research and Innovation, Ministerio de Ciencia, Innovación y Universidades, Cancer Research UK, Imperial College London, University of Oxford, Institució Catalana de Recerca i Estudis Avançats, Universitat de Barcelona, Barcelona Supercomputing Center, Peking University, National Institutes of Health, Horizon 2020 Framework Programme, HORIZON EUROPE Framework Programme, Medical Research Council, Biotechnology and Biological Sciences Research Council, Agencia Estatal de Investigación, H2020 Marie Skłodowska-Curie Actions, HORIZON EUROPE Marie Sklodowska-Curie Actions
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Carbohydrate Chemistry and Synthesis
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