title: Molecular analysis of an alternative N-glycosylation machinery by functional transfer from Actinobacillus pleuropneumoniae to Escherichia coli.
abstract: N-Linked protein glycosylation is a frequent post-translational modification that can be found in all three domains of life. In a canonical, highly conserved pathway, an oligosaccharide is transferred by a membrane-bound oligosaccharyltransferase from a lipid donor to asparagines in the sequon NX(S/T) of secreted polypeptides. The δ-proteobacterium Actinobacillus pleuropneumoniae encodes an unusual pathway for N-linked protein glycosylation. This pathway takes place in the cytoplasm and is mediated by a soluble N-glycosyltransferase (NGT) that uses nucleotide-activated monosaccharides to glycosylate asparagine residues. To characterize the process of cytoplasmic N-glycosylation in more detail, we studied the glycosylation in A. pleuropneumoniae and functionally transferred the glycosylation system to Escherichia coli. N-Linked glucose specific human sera were used for the analysis of the glycosylation process. We identified autotransporter adhesins as the preferred protein substrate of NGT in vivo, and in depth analysis of the modified sites in E. coli revealed a surprisingly relaxed peptide substrate specificity. Although NX(S/T) is the preferred acceptor sequon, we detected glycosylation of alternative sequons, including modification of glutamine and serine residues. We also demonstrate the use of NGT to glycosylate heterologous proteins. Therefore, our study could provide the basis for a novel route for the engineering of N-glycoproteins in bacteria.
authors.affiliation: From the Institute of Microbiology, Swiss Federal Institute of Technology, ETH Zurich, CH-8093 Zurich, Switzerland and.
authors.forename: Andreas
authors.initials: A
authors.lastname: Naegeli
authors.forename: Christine
authors.initials: C
authors.lastname: Neupert
authors.forename: Yao-Yun
authors.initials: YY
authors.lastname: Fan
authors.forename: Chia-Wei
authors.initials: CW
authors.lastname: Lin
authors.forename: Kristina
authors.initials: K
authors.lastname: Poljak
authors.forename: Anna Maria
authors.initials: AM
authors.lastname: Papini
authors.forename: Flavio
authors.initials: F
authors.lastname: Schwarz
authors.forename: Markus
authors.initials: M
authors.lastname: Aebi
mesh_terms: D016977:Actinobacillus pleuropneumoniae / Q000201:enzymology* / Q000235:genetics; D018829:Adhesins, Bacterial / Q000235:genetics / Q000378:metabolism; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D029968:Escherichia coli Proteins / Q000235:genetics / Q000378:metabolism*; D006031:Glycosylation; D006602:Hexosyltransferases / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism*; D015202:Protein Engineering; D013379:Substrate Specificity / Q000502:physiology
chemical_list: D018829:Adhesins, Bacterial; D029968:Escherichia coli Proteins; D008565:Membrane Proteins; D006602:Hexosyltransferases; C025620:dolichyl-diphosphooligosaccharide - protein glycotransferase
keywords: Actinobacillus pleuropneumoniae; Bacteria; Glucose; Glycosylation; Glycosyltransferases; Post-translational Modification