title: Functional analysis of the Campylobacter jejuni N-linked protein glycosylation pathway.
abstract: We describe in this report the characterization of the recently discovered N-linked glycosylation locus of the human bacterial pathogen Campylobacter jejuni, the first such system found in a species from the domain Bacteria. We exploited the ability of this locus to function in Escherichia coli to demonstrate through mutational and structural analyses that variant glycan structures can be transferred onto protein indicating the relaxed specificity of the putative oligosaccharyltransferase PglB. Structural data derived from these variant glycans allowed us to infer the role of five individual glycosyltransferases in the biosynthesis of the N-linked heptasaccharide. Furthermore, we show that C. jejuni- and E. coli-derived pathways can interact in the biosynthesis of N-linked glycoproteins. In particular, the E. coli encoded WecA protein, a UDP-GlcNAc: undecaprenylphosphate GlcNAc-1-phosphate transferase involved in glycolipid biosynthesis, provides for an alternative N-linked heptasaccharide biosynthetic pathway bypassing the requirement for the C. jejuni-derived glycosyltransferase PglC. This is the first experimental evidence that biosynthesis of the N-linked glycan occurs on a lipid-linked precursor prior to transfer onto protein. These findings provide a framework for understanding the process of N-linked protein glycosylation in Bacteria and for devising strategies to exploit this system for glycoengineering.
authors.affiliation: Department of Infectious and Tropical Diseases, London School of Hygiene and Tropical Medicine, London WC1E 7HT, UK.
authors.forename: Dennis
authors.initials: D
authors.lastname: Linton
authors.forename: Nick
authors.initials: N
authors.lastname: Dorrell
authors.forename: Paul G
authors.initials: PG
authors.lastname: Hitchen
authors.forename: Saba
authors.initials: S
authors.lastname: Amber
authors.forename: Andrey V
authors.initials: AV
authors.lastname: Karlyshev
authors.forename: Howard R
authors.initials: HR
authors.lastname: Morris
authors.forename: Anne
authors.initials: A
authors.lastname: Dell
authors.forename: Miguel A
authors.initials: MA
authors.lastname: Valvano
authors.forename: Markus
authors.initials: M
authors.lastname: Aebi
authors.forename: Brendan W
authors.initials: BW
authors.lastname: Wren
mesh_terms: D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D016123:Campylobacter jejuni / Q000201:enzymology / Q000235:genetics / Q000378:metabolism*; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000235:genetics / Q000502:physiology; D005816:Genetic Complementation Test; D006023:Glycoproteins / Q000378:metabolism*; D006031:Glycosylation; D006602:Hexosyltransferases / Q000235:genetics / Q000378:metabolism; D013058:Mass Spectrometry; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism; D009154:Mutation; D011106:Polyisoprenyl Phosphates / Q000378:metabolism; D011134:Polysaccharides / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D011499:Protein Processing, Post-Translational; D013379:Substrate Specificity; D017855:Transferases (Other Substituted Phosphate Groups) / Q000235:genetics / Q000502:physiology; D014537:Uridine Diphosphate N-Acetylglucosamine / Q000378:metabolism
chemical_list: D001426:Bacterial Proteins; D029968:Escherichia coli Proteins; D006023:Glycoproteins; D008565:Membrane Proteins; D011106:Polyisoprenyl Phosphates; D011134:Polysaccharides; C009621:undecaprenyl phosphate; D014537:Uridine Diphosphate N-Acetylglucosamine; D006602:Hexosyltransferases; C025620:dolichyl-diphosphooligosaccharide - protein glycotransferase; D017855:Transferases (Other Substituted Phosphate Groups); C083617:wecA protein, E coli