title: Substrate specificity of cytoplasmic N-glycosyltransferase.
abstract: N-Linked protein glycosylation is a very common post-translational modification that can be found in all kingdoms of life. The classical, highly conserved pathway entails the assembly of a lipid-linked oligosaccharide and its transfer to an asparagine residue in the sequon NX(S/T) of a secreted protein by the integral membrane protein oligosaccharyltransferase. A few species in the class of γ-proteobacteria encode a cytoplasmic N-glycosylation system mediated by a soluble N-glycosyltransferase (NGT). This enzyme uses nucleotide-activated sugars to modify asparagine residues with single monosaccharides. As these enzymes are not related to oligosaccharyltransferase, NGTs constitute a novel class of N-glycosylation catalyzing enzymes. To characterize the NGT-catalyzed reaction, we developed a sensitive and quantitative in vitro assay based on HPLC separation and quantification of fluorescently labeled substrate peptides. With this assay we were able to directly quantify glycopeptide formation by Actinobacillus pleuropneumoniae NGT and determine its substrate specificities: NGT turns over a number of different sugar donor substrates and allows for activation by both UDP and GDP. Quantitative analysis of peptide substrate turnover demonstrated a strikingly similar specificity as the classical, oligosaccharyltransferase-catalyzed N-glycosylation, with NX(S/T) sequons being the optimal NGT substrates.
authors: From the Department of Biology, Institute of Microbiology, ETH Zurich, CH-8093 Zurich.
authors: Andreas
authors: A
authors: Naegeli
authors: the Department of Chemistry and Biochemistry, University of Berne, 3012 Berne, and.
authors: Gaëlle
authors: G
authors: Michaud
authors: the Department of Biology, Institute of Molecular Biology and Biophysics, ETH Zurich, CH-8093 Zurich, Switzerland.
authors: Mario
authors: M
authors: Schubert
authors: From the Department of Biology, Institute of Microbiology, ETH Zurich, CH-8093 Zurich.
authors: Chia-Wei
authors: CW
authors: Lin
authors: the Department of Biology, Institute of Molecular Biology and Biophysics, ETH Zurich, CH-8093 Zurich, Switzerland.
authors: Christian
authors: C
authors: Lizak
authors: the Department of Chemistry and Biochemistry, University of Berne, 3012 Berne, and.
authors: Tamis
authors: T
authors: Darbre
authors: the Department of Chemistry and Biochemistry, University of Berne, 3012 Berne, and.
authors: Jean-Louis
authors: JL
authors: Reymond
authors: From the Department of Biology, Institute of Microbiology, ETH Zurich, CH-8093 Zurich, markus.aebi@micro.biol.ethz.ch.
authors: Markus
authors: M
authors: Aebi
mesh_terms: D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry / Q000378:metabolism*; D003593:Cytoplasm / Q000201:enzymology*; D005964:Glucosyltransferases / Q000737:chemistry / Q000378:metabolism*; D006031:Glycosylation; D006868:Hydrolysis; D007700:Kinetics; D008969:Molecular Sequence Data; D019906:Nuclear Magnetic Resonance, Biomolecular; D066244:Proton Magnetic Resonance Spectroscopy; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity
chemical_list: D001426:Bacterial Proteins; D005964:Glucosyltransferases; C410788:N-glycosyltransferase, Nocardia aerocolonigenes
keywords: Actinobacillus Pleuropneumoniae; Bacteria; Glycosylation; Glycosyltransferase; Post-translational Modification (PTM); Substrate Specificity