Substrate Specificity of Cytoplasmic N-Glycosyltransferase |
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Authors: | Andreas Naegeli Ga?lle Michaud Mario Schubert Chia-Wei Lin Christian Lizak Tamis Darbre Jean-Louis Reymond Markus Aebi |
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Affiliation: | From the ‡Department of Biology, Institute of Microbiology, ETH Zurich, CH-8093 Zurich.;the §Department of Chemistry and Biochemistry, University of Berne, 3012 Berne, and ;the ¶Department of Biology, Institute of Molecular Biology and Biophysics, ETH Zurich, CH-8093 Zurich, Switzerland |
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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. |
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Keywords: | Bacteria Glycosylation Glycosyltransferase Post-translational Modification (PTM) Substrate Specificity Actinobacillus Pleuropneumoniae |
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