Complex anionic and zwitterionic N-glycans in Drosophila melanogaster

Drosophila melanogaster is arguably the most widely-employed invertebrate model organism; here we have focussed on its anionic N-glycome applying our off-line HPLC-MALDI-TOF-MS approach. Using MS/MS in combination with chemical and enzymatic treatments, we reveal not only sulphated and glucuronylated N-glycans further to those previously proposed, but demonstrate that this dipteran insect modifies some structures with phosphoethanolamine, a zwitterion. Although not as complex as some other insect N-glycomes, N-glycans with up to three antennae can feature also LacdiNAc or β1,3-galactosylation as the basis for anionic modifications, whereby some glycans also carry up to five anionic/zwitterionic moieties; in total approximately 180 N-linked oligosaccharide structures, including isomers and isobars, can be proposed. A prototype microarray based on fractionated N-glycans was printed and showed different fucose-specific lectin binding to N-glycans from a wild-type and a fucose-deficient strain, whereas serum amyloid P bound anionic glycans. In terms of enzymatic analyses, recombinant forms of both N-acetylglucosaminyltransferases II and IV were expressed and their activities correlate with the occurrence of up to three antennae on Drosophila N-glycans. Overall, in keeping with the accepted phylogeny, the fruit fly is indeed closest in glycomic terms to mosquitoes, but also shares the feature of phosphoethanolamine modifications with the honeybee, rather than the phosphorylcholine found in lepidopteran species. Nevertheless, there are many unanswered questions regarding N-glycosylation in Drosophila; a mix of genomic, biochemical and glycomic approaches will be required to solve the biosynthesis and functions of this key set of post-translational modifications in this and other insects.