Iterative Bump-and-Hole Engineering Creates a Bioorthogonal Reporter for N -Acetylglucosaminyltransferase I
Year: 2026
Type: article
Abstract: 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 to a common precursor that is then subject 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 towards individual positions. Here, we report an iterative bump-and-hole approach in the design of a bioorthogonal precision tool for the activity of MGAT1 in mammalian cells. Structure-informed protein engineering abrogated the activity of MGAT1 towards the nucleotide-sugar UDP-GlcNAc while retaining activity towards 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.
DOI: 10.1021/jacs.6c01114
Language: English
Subfield: Molecular Biology
Domain: Life Sciences
Open Access status: hybrid
Estimated APC paid: $4,500
Funders: Howard Hughes Medical Institute, Children's Tumor Foundation, Alnylam Pharmaceuticals, Wellcome Trust, Francis Crick Institute, Government of the United Kingdom, 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
Awards: G1105844351, G1143298633, G1175115578, G141312279, G1767616181, A genetically encoded reporter platform to dissect the O-glycoproteome, G2189472788, G2223539600, G2256575814, G2262748287, G2594910703, G300979063, G3375431942, G3480869486, GLYCOprotein N-glycosylation from non-life to eukaryotes: a Doctoral Network to expand the knowledge on a ubiquitous posttranslational modification of proteins, Visualising Glycoprotein Interaction Dynamics, G4199003910, G4214726086, Evaluation of the impact of Seasonal Malaria Chemoprevention delivered by district health services in Southern Senegal., G425668148, G464105488, G4716061958, G491712853, G4950536974, G5002094762, G5080475149, G5259505699, G5362606921, G5451035857, G5635532810, Resource for Integrated Glycotechnology, G6032785003, G6066786398, G7084143925, G7169023097, G734693063, G7466406081, G7805626310, G7849468472, G7863371703, Exploring Filovirus-host interactions at single cell resolution, G8089423575, G8497050386, G8858395165, G9000286654
OpenAlex ID: W7166740659