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Abstract
Various studies in the past have revealed that molluscs can produce a wide range of rather complex N-glycan structures, which vary from those occurring in other invertebrate animals; particularly methylated glycans have been found in gastropods, and there are some reports of anionic glycans in bivalves. Due to the high variability in terms of previously described structures and methodologies, it is a major challenge to establish glycomic workflows that yield the maximum amount of detailed structural information from relatively low quantities of sample. In this study, we apply differential release with peptide:N-glycosidases F and A followed by solid-phase extraction on graphitized carbon and reversed-phase materials to examine the glycome of Volvarina rubella (C. B. Adams, 1845), a margin snail of the clade Neogastropoda. The resulting four pools of N-glycans were fractionated on a fused core RP-HPLC column and subject to MALDI-TOF MS and MS/MS in conjunction with chemical and enzymatic treatments. In addition, selected N-glycan fractions, as well as O-glycans released by β-elimination, were analyzed by porous graphitized carbon-LC-MS and MS(n). This comprehensive approach enabled us to determine a number of novel modifications of protein-linked glycans, including N-methyl-2-aminoethylphosphonate on mannose and N-acetylhexosamine residues, core β1,3-linked mannose, zwitterionic moieties on core Galβ1,4Fuc motifs, additional mannose residues on oligomannosidic glycans, and bisubstituted antennal fucose; furthermore, typical invertebrate N-glycans with sulfate and core fucose residues are present in this gastropod.
Highlights
From the ‡Department fur Chemie, Universitat fur Bodenkultur Wien, 1190 Wien, Austria; §Institutionen for Biomedicin, Goteborgs universitet, 405 30 Goteborg, Sweden; ¶Haus des Meeres—Aqua Terra Zoo, 1060 Wien, Austria
There is only scattered information regarding the biosynthesis of mollusc N-glycan epitopes, based on assay of some fucosyl, xylosyl, N-acetylglucosaminyltransferases, and N-acetylgalactosaminyltransferases (16 –18); probably only two mollusc glycosyltransferases have ever been characterized in recombinant form [19, 20]
We have sought to maximize the potential of off-line MALDI-TOF MS and MS/MS by prefractionating Nglycans first on the basis of whether they can be released by peptide:N-glycosidase A or F (the former being able to remove glycans containing core ␣1,3-fucose [21]) and using solid-phase extraction on nonporous graphitized carbon (for an initial separation of anionic from neutral glycans [22]) and on a reversed-phase resin
Summary
From the ‡Department fur Chemie, Universitat fur Bodenkultur Wien, 1190 Wien, Austria; §Institutionen for Biomedicin, Goteborgs universitet, 405 30 Goteborg, Sweden; ¶Haus des Meeres—Aqua Terra Zoo, 1060 Wien, Austria. Molluscs represent one of the largest groups of animals on the planet; there is an estimated 200,000 species, which vary in morphology from gastropods (snails) through to cephalopods (octopus) and live in a range of marine, aquatic, and terrestrial environments [1]. The most studies on molluscs have been structural characterizations of the N-glycans on hemocyanins of a range of gastropods, such as from keyhole limpet (Megathura crenulata; KLH is an often-used carrier protein for immunization), Lymnaea stagnalis, Helix pomatia, and Rapana venosa [7,8,9,10]. The residual glycopeptides (posttreatment with peptide:N-glycosidases) were subject to -elimination to release the O-glycans followed by LC-MS
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