Biosynthesis of the N-linked glycan in Campylobacter jejuni and addition onto protein through block transfer.
In eukaryotes, N-linked protein glycosylation is a universal modification involving addition of preformed oligosaccharides to select Asn-Xaa-Ser/Thr motifs and influencing multiple biological events. We recently demonstrated that Campylobacter jejuni is the first member of the Bacteria to possess an N-linked glycan pathway. In this study, high-resolution magic angle spinning nuclear magnetic resonance (HR-MAS NMR) was applied to probe and quantitate C. jejuni N-glycan biosynthesis in vivo. To confirm HR-MAS NMR findings, glycosylation mutants were screened for chicken colonization potential, and glycoproteins were examined by mass spectrometry and lectin blotting. Consistent with the mechanism in eukaryotes, the combined data indicate that bacterial glycans are assembled en bloc, emphasizing the evolutionary conservation of protein N glycosylation. We also show that under the conditions examined, PglG plays no role in glycan biosynthesis, PglI is the glucosyltransferase and the putative ABC transporter, and WlaB (renamed PglK) is required for glycan assembly. These studies underpin the mechanism of N-linked protein glycosylation in Bacteria and provide a simple model system for investigating protein glycosylation and for exploitation in glycoengineering.
Institute for Biological Sciences, National Research Council, 100 Sussex Dr., Ottawa, Ontario K1A 0R6, Canada.
John
J
Kelly
Harold
H
Jarrell
Lorna
L
Millar
Luc
L
Tessier
Laura M
LM
Fiori
Peter C
PC
Lau
Brenda
B
Allan
Christine M
CM
Szymanski
D000818:Animals; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D002169:Campylobacter Infections / Q000382:microbiology / Q000662:veterinary; D016123:Campylobacter jejuni / Q000235:genetics / Q000378:metabolism*; D002645:Chickens; D006031:Glycosylation; D037102:Lectins; D009154:Mutation; D019906:Nuclear Magnetic Resonance, Biomolecular; D011134:Polysaccharides / Q000096:biosynthesis* / Q000737:chemistry*; D011201:Poultry Diseases / Q000382:microbiology
D001426:Bacterial Proteins; D037102:Lectins; D011134:Polysaccharides