Multistep Fractionation and Mass Spectrometry Reveal Zwitterionic and Anionic Modifications of the N- and O-glycans of a Marine Snail

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 MSⁿ. 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.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). Many molluscs are familiar due to their shells or being seafood. Less appreciated is perhaps their ecological role as filter feeders or scavengers and their being an indicator for water quality (2–4); also, some molluscs are intermediate hosts for pathogens such as viruses or schistosomes (5, 6).In glycobiological terms, 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 (7–10). Furthermore, glycans from cephalopod rhodopsins, proteins of bivalves involved in biomineralization, or whole snail viscera have also been analyzed. Including our recent study on the hemocytes and plasma of the eastern oyster (Crassostrea virginica), the variety of modifications of N-glycans in these organisms is immense and includes branched fucose residues, glucuronylation, sulfation, methylation, core xylose, and galactosylation of core fucose as well as LacdiNAc and blood-group-like motifs (11–15). On the other hand, 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); also, probably only two mollusc glycosyltransferases have ever been characterized in recombinant form (19, 20).The high variability and lack of predictability of mollusc glycomes mean that a suitable glycomic workflow has to be employed that takes account of the maxim “expect the unexpected.” Thereby, in comparison to mammalian glycomes with known major components, the analyses of those of lower eukaryotes can present major challenges. In the past, mollusc glycans from either a single glycoprotein or from tissue were very often analyzed in any single study by one or two methods (e.g. GC-MS and NMR or MALDI-TOF MS/MS of HPLC-fractionated N-glycans, LC-MS/MS of glycopeptides, or GC-MS and MSⁿ of permethylated N-glycans; see references above). In some cases, chemical and enzymatic treatments were employed. Here, we have sought to maximize the potential of off-line MALDI-TOF MS and MS/MS by prefractionating N-glycans 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 α1,3-fucose (21)) and then using solid-phase extraction on nonporous graphitized carbon (for an initial separation of anionic from neutral glycans (22)) and on a reversed-phase resin (which aids enrichment of glycans with substitutions of core α1,6-fucose). Subsequent use of a fused core reversed-phase (RP)-HPLC column (23) resulted in high-resolution separation into fractions containing either a single or very few glycan species that facilitated further MS-based analyses; as this RP column offers isomeric/isobaric separation, HPLC fractionation was a prerequisite for the definition of the individual N-glycan structures. Furthermore, the residual glycopeptides (posttreatment with peptide:N-glycosidases) were subject to β-elimination to release the O-glycans followed by LC-MS.On the basis of these considerations, we have examined the N- and O-glycomes of a margin snail (Volvarina rubella), a species of carnivorous and scavenging marine gastropod first described as Marginella rubella in 1845 (24). Using off-line LC-MALDI-TOF MS and on-line LC-ESI-MS, we reveal a particularly complex N-glycome encompassing a range of oligomannosidic, paucimannosidic, core-modified, and complex (up to triantennary) N-linked oligosaccharides with also a number of anionic and zwitterionic modifications, which are also present on O-glycans. Although some of these features are also found on N-glycans or lipid-linked glycans of other species, the majority of the ∼100 structures are described here for the first time.