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J. Biol. Chem., Vol. 280, Issue 17, 16586-16593, April 29, 2005

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Identification and Characterization of the Unique N-Linked Glycan Common to the Flagellins and S-layer Glycoprotein of Methanococcus voltae^*

Sebastien Voisin, R. Scott Houliston, John Kelly, Jean-Robert Brisson, David Watson, Sonia L. Bardy¶, Ken F. Jarrell, and Susan M. Logan||

From the Institute for Biological Sciences, National Research Council, Ottawa, Ontario K1A OR6 and the Department of Microbiology and Immunology, Queens University, Kingston, Ontario K7L 3N6, Canada

The flagellum of Methanococcus voltae is composed of four structural flagellin proteins FlaA, FlaB1, FlaB2, and FlaB3. These proteins possess a total of 15 potential N-linked sequons (NX(S/T)) and show a mass shift on an SDS-polyacrylamide gel indicating significant post-translational modification. We describe here the structural characterization of the flagellin glycan from M. voltae using mass spectrometry to examine the proteolytic digests of the flagellin proteins in combination with NMR analysis of the purified glycan using a sensitive, cryogenically cooled probe. Nano-liquid chromatography-tandem mass spectrometry analysis of the proteolytic digests of the flagellin proteins revealed that they are post-translationally modified with a novel N-linked trisaccharide of mass 779 Da that is composed of three sugar residues with masses of 318, 258, and 203 Da, respectively. In every instance the glycan is attached to the peptide through the asparagine residue of a typical N-linked sequon. The glycan modification has been observed on 14 of the 15 sequon sites present on the four flagellin structural proteins. The novel glycan structure elucidated by NMR analysis was shown to be a trisaccharide composed of -ManpNAcA6Thr-(1–4)--Glc-pNAc3NAcA-(1–3)--GlcpNAc linked to Asn. In addition, the same trisaccharide was identified on a tryptic peptide of the S-layer protein from this organism implicating a common N-linked glycosylation pathway.

Received for publication, January 10, 2005 , and in revised form, February 10, 2005.

^* This work was supported by genomic and health initiative funding (to S. M. L., S. V., and J. K.) and a discovery grant from the Natural Sciences and Engineering Council of Canada (NSERC) (to K. F. J.). 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.

The on-line version of this article (available at http://www.jbc.org) contains Supplemental Table 1.

^¶ Recipient of a Post-graduate Scholarship B from NSERC.

^|| To whom correspondence should be addressed: Inst. of Biological Sciences, National Research Council, 100 Sussex Dr., Ottawa, Ontario K1A OR6, Canada. Tel.: 613-990-0839; Fax: 613-952-9092; E-mail: susan.logan{at}nrc-cnrc.gc.ca.

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