The N-glycosylation of structural unit 1 of Rapana venosa hemocyanin was studied. Enzymatically liberated N-glycans were analyzed by matrix-assisted laser desorption ionization-time-of-flight-mass spectrometry (MALDI-TOF-MS) and capillary electrophoresis (CE)-MS following 8-aminopyrene-1,3,6-trisulfonate labeling and labeling with 3-aminopyrazole, a new dedicated sugar reagent. Structural information was obtained by exoglycosidase sequencing, on-line MS/MS, permethylation, and amidation. A mixture of high-mannose and complex glycans with so far unknown and unusual acidic terminal structures was revealed. As the hemocyanin protein sequence is currently unknown, de novo sequencing of the glycopeptides had to be carried out. The N-glycans were therefore enzymatically removed with simultaneous partial (50%) (18)O-labeling of glycosylated asparagine residues prior to proteolysis. Following nano-liquid chromatography-MALDI-TOF-MS, the originally glycosylated peptides could be revealed and their sequences determined by MS/MS. The site occupancies were subsequently elucidated by precursor ion scanning of the intact glycopeptides using a Q-Trap mass spectrometer.

Molluscan hemocyanins (Hcs) have recently received particular interest due to their significant immunostimulatory properties. This is mainly related to their high carbohydrate content and specific monosaccharide composition. We have now analyzed the oligosaccharides and the carbohydrate linkage sites of the Rapana venosa hemocyanin (RvH) using different approaches. We analyzed a number of glycopeptides by LC/ESI-MS/MS and identified the sugar chains and peptide sequences of 12 glycopeptides. Additionally, the potential carbohydrate linkage sites of 2 functional units, RvH-b and RvH-c, were determined by gene sequence analysis. Only RvH-c shows a potential N-glycosylation site. During this study, we discovered a highly conserved linker-intron, separating the coding exons of RVH-b and RvH-c. Following reports on antiviral properties from arthropod hemocyanin, we conducted a preliminary study of the antiviral activity of RvH and the functional units RvH-b and RvH-c. We show that the glycosylated FU RvH-c has antiviral properties against the respiratory syncytial virus (RSV), whereas native RvH and the nonglycosylated FU RvH-b have not. This is the first report of the fact that also molluscan hemocyanin functional units possess antiviral activity.