Structure of the N-linked glycan present on multiple glycoproteins in the Gram-negative bacterium, Campylobacter jejuni.
Mass spectrometry investigations of partially purified Campylobacter jejuni protein PEB3 showed it to be partially modified with an Asn-linked glycan with a mass of 1406 Da and composed of one hexose, five N-acetylhexosamines and a species of mass 228 Da, consistent with a trideoxydiacetamidohexose. By means of soybean lectin affinity chromatography, a mixture of glycoproteins was obtained from a glycine extract, and two-dimensional gel proteomics analysis led to the identification of at least 22 glycoproteins, predominantly annotated as periplasmic proteins. Glycopeptides were prepared from the glycoprotein mixture by Pronase digestion and gel filtration. The structure of the glycan was determined by using nano-NMR techniques to be GalNAc-alpha1,4-GalNAc-alpha1,4-[Glcbeta1,3-]GalNAc-alpha1,4-GalNAc-alpha1,4-GalNAc-alpha1,3-Bac-beta1,N-Asn-Xaa, where Bac is bacillosamine, 2,4-diacetamido-2,4,6-trideoxyglucopyranose. Protein glycosylation was abolished when the pglB gene was mutated, providing further evidence that the enzyme encoded by this gene is responsible for formation of the glycopeptide N-linkage. Comparison of the pgl locus with that of Neisseria meningitidis suggested that most of the homologous genes are probably involved in the biosynthesis of bacillosamine.
Young
N Martin
NM
Institute for Biological Sciences, National Research Council of Canada, 100 Sussex Dr., Ottawa, Ontario K1A 0R6, Canada. young@nrc.ca
Brisson
Jean-Robert
JR
Kelly
John
J
Watson
David C
DC
Tessier
Luc
L
Lanthier
Patricia H
PH
Jarrell
Harold C
HC
Cadotte
Nicolas
N
St  Michael
Frank
F
Aberg
Erika
E
Szymanski
Christine M
CM
D001426:Bacterial Proteins / Q000737:chemistry / Q000302:isolation & purification; D016123:Campylobacter jejuni / Q000737:chemistry*; D002240:Carbohydrate Sequence; D006020:Glycopeptides / Q000737:chemistry / Q000302:isolation & purification; D006023:Glycoproteins / Q000737:chemistry* / Q000302:isolation & purification; D009682:Magnetic Resonance Spectroscopy; D008969:Molecular Sequence Data; D009844:Oligosaccharides / Q000737:chemistry; D010446:Peptide Fragments / Q000737:chemistry; D011134:Polysaccharides / Q000737:chemistry*; D014357:Trypsin
D001426:Bacterial Proteins; D006020:Glycopeptides; D006023:Glycoproteins; D009844:Oligosaccharides; D010446:Peptide Fragments; D011134:Polysaccharides; D014357:Trypsin