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J. Biol. Chem., Vol. 278, Issue 27, 24509-24520, July 4, 2003

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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^*

Christine M. Szymanski, Frank St. Michael, Harold C. Jarrell, Jianjun Li, Michel Gilbert, Suzon Larocque, Evgeny Vinogradov and Jean-Robert Brisson 

From the Institute for Biological Sciences, National Research Council of Canada, Ottawa, Ontario K1A 0R6, Canada

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-1,4-GalNAc-1,4-[Glc-1,3-]GalNAc-1,4-GalNAc-1,4-GalNAc-1,3-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.

Received for publication, February 5, 2003 , and in revised form, April 25, 2003.

^* This work was funded by the National Research Council Genomics and Health Initiative. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

To whom correspondence should be addressed: Institute for Biological Sciences, National Research Council of Canada, 100 Sussex Dr., Ottawa, Ontario K1A 0R6, Canada. Tel.: 613-990-3244; Fax: 613-941-1327; E-mail: jean-robert.brisson{at}nrc-cnrc.gc.ca.

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