Glycobiology Advance Access originally published online on June 12, 2006
Glycobiology 2006 16(9):874-890; doi:10.1093/glycob/cwl011
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Isomer and glycomer complexities of core GlcNAcs in Caenorhabditis elegans
4 Department of Chemistry, Center for Structural Biology, University of New Hampshire, Durham, NH 03824
1 To whom correspondence should be addressed; e-mail: vnr{at}unh.edu
2 Present address: Wyeth BioPharma, Andover, MA 01833
3 Present address: Faculty of Medicine of Ribeirão Preto, University of São Paulo, Av. Bandeirantes 3900, 14049-900, Ribeirão Preto, São Paulo, Brazil
Received on April 16, 2006; revised on May 31, 2006; accepted on June 2, 2006
Analysis of protein glycosylation within the nematode Caenorhabditis elegans has revealed an abundant and unreported set of core chitobiose modifications (CCMs) to N-linked glycans. With hydrazine release, an array of glycomers and isobars were detected with hexose extensions on the 3- and 3,6-positions of the penultimate and reducing terminus, respectively. A full complement of structures includes a range of glycomers possessing a Galß(1-4)Fuc disaccharide at the 3- and 6-positions of the protein-linked GlcNAc. Importantly, enzymatic (PNGase F/A) release failed to liberate many of these extended structures from reduced and alkylated peptides and, as a consequence, such profiles were markedly deficient in a representation of the worm glycome. Moreover, the 3-linked Galß(1-4)Fuc moiety was notably resistant to a range of commercial galactosidases. For identification, the fragments were spectrum-matched with synthetic products and library standards using sequential mass spectrometry (MSn). A disaccharide observed at the 3-position of penultimate GlcNAc, indicating a Hex-Fuc branch on some structures, was not further characterized because of low ion abundance in MSn. Additionally, a Hex-Hex-Fuc trisaccharide on the 6-position of proximal GlcNAc was also distinguished on select glycomers. Similar branch extensions on 6-linked core fucosyl residues have recently been reported among other invertebrates. Natural methylation and numerous isobars complement the glycome, which totals well over 100 individual structures. Complex glycans were detected at lower abundance, indicating glucosaminyltransferase-I (GnT-I) and GnT-II activity. A range of phosphorylcholine (PC)-substituted complex glycans were also confirmed following a signature two-stage loss of PC during MSn analysis, although the precursor ion was not observed in the mass profiles. In a similar manner, numerous other minor glycans may be present but unobserved in hydrazine-release profiles dominated by fucosylated structures. All CCM structures, including multiple isomers, were determined without chromatography by gas-phase disassembly (MSn) in Paul and linear ion trap (IT) instruments.
Key words: C. elegans / core chitobiose modifications / Galß(1-4)Fuc / MSn / paucimannose
Dedicated to Professor Charles Edward Warren, 1963–2005.
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