Abstract
Chemoenzymatic modification of cell-surface glycan structures has emerged as a complementary approach to metabolic oligosaccharide engineering. Here, we identify Pasteurella multocida α2-3-sialyltransferase M144D mutant, Photobacterium damsela α2-6-sialyltransferase, and Helicobacter mustelae α1-2-fucosyltransferase, as efficient tools for live-cell glycan modification. Combining these enzymes with Helicobacter pylori α1-3-fucosyltransferase, we develop a host-cell-based assay to probe glycan-mediated influenza A virus (IAV) infection including wild-type and mutant strains of H1N1 and H3N2 subtypes. At high NeuAcα2-6-Gal levels, the IAV-induced host-cell death is positively correlated with haemagglutinin (HA) binding affinity to NeuAcα2-6-Gal. Remarkably, an increment of host-cell-surface sialyl Lewis X (sLeX) exacerbates the killing by several wild-type IAV strains and a previously engineered mutant HK68-MTA. Structural alignment of HAs from HK68 and HK68-MTA suggests formation of a putative hydrogen bond between Trp222 of HA-HK68-MTA and the C-4 hydroxyl group of the α1-3-linked fucose of sLeX, which may account for the enhanced host cell killing of that mutant.
Publication types
-
Research Support, Non-U.S. Gov't
MeSH terms
-
Animals
-
Bacterial Proteins / genetics
-
Bacterial Proteins / metabolism*
-
Biological Assay / methods
-
CHO Cells
-
Cricetulus
-
Dogs
-
Glycosyltransferases / genetics
-
Glycosyltransferases / metabolism*
-
Healthy Volunteers
-
Helicobacter mustelae / genetics
-
Helicobacter mustelae / metabolism
-
Hemagglutinins / immunology*
-
Hemagglutinins / metabolism
-
Host-Pathogen Interactions / immunology*
-
Humans
-
Influenza A Virus, H1N1 Subtype / genetics
-
Influenza A Virus, H1N1 Subtype / immunology
-
Influenza A Virus, H3N2 Subtype / genetics
-
Influenza A Virus, H3N2 Subtype / immunology
-
Influenza, Human / immunology*
-
Influenza, Human / virology
-
Intravital Microscopy / methods
-
Luciferases, Bacterial / genetics
-
Luciferases, Bacterial / metabolism
-
Lung / pathology
-
Madin Darby Canine Kidney Cells
-
Metabolic Engineering / methods
-
Microscopy, Fluorescence / methods
-
Molecular Imaging / methods
-
Oligosaccharides / immunology
-
Oligosaccharides / metabolism*
-
Pasteurella multocida / genetics
-
Pasteurella multocida / metabolism
-
Recombinant Proteins / genetics
-
Recombinant Proteins / metabolism
-
Sialyl Lewis X Antigen
-
Staining and Labeling / methods
Substances
-
Bacterial Proteins
-
Hemagglutinins
-
Oligosaccharides
-
Recombinant Proteins
-
Sialyl Lewis X Antigen
-
Luciferases, Bacterial
-
Glycosyltransferases