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Nature Communications Apr 2019Chemoenzymatic modification of cell-surface glycan structures has emerged as a complementary approach to metabolic oligosaccharide engineering. Here, we identify...
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 (sLe) 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 sLe, which may account for the enhanced host cell killing of that mutant.
Topics: Animals; Bacterial Proteins; Biological Assay; CHO Cells; Cricetulus; Dogs; Glycosyltransferases; Healthy Volunteers; Helicobacter mustelae; Hemagglutinins; Host-Pathogen Interactions; Humans; Influenza A Virus, H1N1 Subtype; Influenza A Virus, H3N2 Subtype; Influenza, Human; Intravital Microscopy; Luciferases, Bacterial; Lung; Madin Darby Canine Kidney Cells; Metabolic Engineering; Microscopy, Fluorescence; Molecular Imaging; Oligosaccharides; Pasteurella multocida; Recombinant Proteins; Sialyl Lewis X Antigen; Staining and Labeling
PubMed: 30996301
DOI: 10.1038/s41467-019-09608-w -
Carbohydrate Research Apr 2021Simple protocols for attaching and detaching carbobenzyloxy (Cbz) groups at the reducing end of sugars was developed. Briefly, lactose was converted into its...
Simple protocols for attaching and detaching carbobenzyloxy (Cbz) groups at the reducing end of sugars was developed. Briefly, lactose was converted into its glycosylamine, which was then acylated with carbobenzyloxy chloride in high overall yield. The obtained lactose Cbz derivative was used in sequential glycosylations using glycosyltransferases and nucleotide sugars in aqueous buffers. Isolation of the reaction products after each step was by simple C-18 solid-phase extraction. The Cbz group was removed by catalytic hydrogenolysis or catalytic transfer hydrogenation followed by in situ glycosylamine hydrolysis. In this way, a trisaccharide (GlcNAc-lactose), a human milk tetrasaccharide (LNnT), and a human milk pentasaccharide (LNFPIII) were prepared in a simple and efficient way.
Topics: Benzene Derivatives; Fucosyltransferases; Glucosamine; Helicobacter mustelae; Humans; Hydrolysis; Oligosaccharides; Sugars
PubMed: 33711724
DOI: 10.1016/j.carres.2021.108272