e martë, shkurt 22, 2005

Identification and characterization of the unique N-linked glycan common to

The flagellum of Methanococcus voltae is composed of 4 structural flagellin proteins FlaA, FlaB1, FlaB2, and FlaB3. These proteins possess a total of 15 potential N-linked sequons (N-X-S/T) and show a mass shift on SDS-PAGE 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. NanoLC-MS/MS 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 which is composed of three sugar residues with masses of 318 Da, 258 Da 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 beta-ManpNAcA6Thr-(1-4)-beta-GlcpNAc3NAcA-(1-3)-beta-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.

posted by Selfmaderadio @ 12:01 e pasdites