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

Abstract

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.