Mass spectral evidence for N-glycans with branching on fucose in a molluscan hemocyanin.
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(3)GlcNAc(2) for N-glycosylation, structures with a supplementary GlcNAc linked to either the Man(alpha1-3) or the Man(alpha1-6) 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.
Gielens
Constant
C
Laboratory of Biochemistry, Chemistry Department, Katholieke Universiteit Leuven, Celestijnenlaan 200 G, 3001 Leuven-Heverlee, Belgium. constant.gielens@chem.kuleuven.ac.be
Idakieva
Krassimira
K
Van den Bergh
Viviane
V
Siddiqui
Nurul I
NI
Parvanova
Katja
K
Compernolle
Frans
F
D000818:Animals; D002240:Carbohydrate Sequence; D005643:Fucose / Q000032:analysis / Q000737:chemistry*; D006020:Glycopeptides / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D006031:Glycosylation; D006433:Hemocyanins / Q000737:chemistry*; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D011134:Polysaccharides / Q000032:analysis* / Q000737:chemistry*; D012908:Snails / Q000737:chemistry*; D014357:Trypsin / Q000378:metabolism
D006020:Glycopeptides; D011134:Polysaccharides; D005643:Fucose; D006433:Hemocyanins; D014357:Trypsin