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Detection of conserved N-linked glycans and phase-variable lipooligosaccharides and capsules from campylobacter cells by mass spectrometry and high resolution magic angle spinning NMR spectroscopy
Author(s):
Affiliation:
NRC Institute for Biological Sciences; National Research Council Canada; NRC Institute for Biological Sciences
Language:
English
Type:
Article
Journal:
Journal Of Biological Chemistry
Date:
2003
Pages :
24509-24520
Identifier #:
SZYMANSKI2003A
NPArC #:
9363421
Abstract:
Glycomics, the study of microbial polysaccharides and genes responsible for
their formation, requires the continuous development of rapid and sensitive methods
for the identification of glycan structures. In this study, methods for the direct
analysis of sugars from 108 to 1010 cells are outlined using the human
gastrointestinal pathogen, Campylobacter jejuni. Using capillary-electrophoresis
coupled with sensitive electrospray mass spectrometry, we demonstrate variability in
the lipid A component of C. jejuni lipooligosaccharides (LOSs). In addition, these
sensitive methods have permitted the detection of phase-variable LOS core structures
that were not observed previously. High resolution magic angle spinning (HR-MAS) NMR
was used to examine capsular polysaccharides directly from campylobacter cells and
showed profiles similar to those observed for purified polysaccharides analyzed by
solution NMR. This method also exhibited the feasibility of campylobacter
serotyping, mutant verification, and preliminary sugar analysis. HR-MAS NMR
examination of growth from individual colonies of C. jejuni NCTC11168 indicated that
the capsular glycan modifications are also phase-variable. These variants show
different staining patterns on deoxycholate-PAGE and reactivity with immune sera.
One of the identified modifications was a novel -OP=O(NH2)OMe phosphoramide, not
observed previously in nature. In addition, HR-MAS NMR detected the N-linked glycan,
GalNAc-alpha1,4-GalNAc-alpha1,4-[Glc-beta1,3-]GalNAc-alpha1,4-GalNAc-alpha
1,4-GalNAc-alpha1,3-Bac, where Bac is
2,4-diacetamido-2,4,6-trideoxy-d-glucopyranose, in C. jejuni and Campylobacter coli.
The presence of this common heptasaccharide in multiple campylobacter isolates
demonstrates the conservation of the N-linked protein glycosylation pathway in this
organism and describes the first report of HR-MAS NMR detection of N-linked glycans
on glycoproteins from intact bacterial cells
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