Mass spectral evidence for N-glycans with branching on fucose in a molluscan hemocyanin

• Published in: Biochemical and biophysical research communications 2005-06, Vol.331 (2), p.562-570
• Main Authors: Gielens, Constant, Idakieva, Krassimira, Van den Bergh, Viviane, Siddiqui, Nurul I., Parvanova, Katja, Compernolle, Frans
• Format: Article
• Language: English
• Subjects: Animals; Carbohydrate Sequence; Deglycosylation; Electrospray ionization mass spectrometry; Fucose; Fucose - analysis; Fucose - chemistry; Gas chromatography; Gastropod; Glycopeptides - chemistry; Glycopeptides - isolation & purification; Glycopeptides - metabolism; Glycosylation; Hemocyanin; Hemocyanins - chemistry; Mass Spectrometry; Molecular Sequence Data; Mollusc; MS/MS; N-Glycosylation; Polysaccharides - analysis; Polysaccharides - chemistry; Rapana thomasiana; Snails - chemistry; Trypsin - metabolism
• ISSN: 0006-291X; 1090-2104
• DOI: 10.1016/j.bbrc.2005.03.217

Glycopeptides, isolated from a trypsinolysate of functional unit (FU) RtH2- e of Rapana thomasiana hemocyanin subunit 2, were analysed by electrospray ionization mass spectrometry and MS/MS. From the molecular mass observed after deglycosylation, it was inferred that all glycopeptides shared the same peptide stretch 92–143 of FU RtH2- e with a glycosylation site at Asn-127. Besides the core structure Man 3GlcNAc 2 for N-glycosylation, structures with a supplementary GlcNAc linked to either the Man(α1–3) or the Man(α1–6) arm and/or an additional tetrasaccharide unit connected to the other Man arm were observed, indicating the existence of microheterogeneity at the glycan level. The tetrasaccharide unit contains a central fucose moiety substituted with 3- O-methylgalactose and N-acetylgalactosamine, and linked to GlcNAc at the reducing end. This structure represents a novel N-glycan motif and is likely to be immunogenic. A second potential site for N-glycosylation in FU RtH2- e at Asn-17 was shown to be not glycosylated.