Targeted release and fractionation reveal glucuronylated and sulphated N- and O-glycans in larvae of dipteran insects

Abstract

Mosquitoes are important vectors of parasitic and viral diseases with Anopheles gambiae transmitting malaria and Aedes aegypti spreading yellow and Dengue fevers. Using two different approaches (solid-phase extraction and reversed-phase or hydrophilic interaction HPLC fractionation followed by MALDI-TOF MS or permethylation followed by NSI-MS), we examined the N-glycans of both A. gambiae and A. aegypti larvae and demonstrate the presence of a range of paucimannosidic glycans as well as bi- and tri-antennary glycans, some of which are modified with fucose or with sulphate or glucuronic acid residues; the latter anionic modifications were also found on N-glycans of larvae from another dipteran species (Drosophila melanogaster). The sulphate groups are attached primarily to core α-mannose residues (especially the α1,6-linked mannose), whereas the glucuronic acid residues are linked to non-reducing β1,3-galactose. Also, O-glycans were found to possess glucuronic acid and sulphate as well as phosphoethanolamine modifications. The presence of sulphated and glucuronylated N-glycans is a novel feature in dipteran glycomes; these structures have the potential to act as additional anionic glycan ligands involved in parasite interactions with the vector host. Biological significance statement Glycans of various types are relevant in many vector-parasite systems and it was previously shown that proteoglycans play roles in malaria transmission, whereas some fungal lectins are toxic towards mosquito larvae. The presence of sulphated and glucuronylated N- and O-glycans in mosquito larvae show that there are anionic glycans other than glycosaminoglycans; these could be ligands in interactions between pathogens (protozoan parasites and viruses) and the tissues of adult mosquitoes.

The fucomic potential of mosquitoes: Fucosylated N-glycan epitopes and their cognate fucosyltransferases

“…In our recent study (Kurz et al, 2015), we examined the N-glycome of An. gambiae by using PNGase F and subsequently PNGase A (post PNGase F) to release N-glycans.…”

“…gambiae larvae (Keele) N-glycans was performed as described recently (Kurz et al, 2015). Briefly, frozen larvae were boiled in deionised water before grinding and proteolysis using porcine pepsin (Sigma-Aldrich).…”

“…As required, neutral N-glycans were further purified by a second solid-phase extraction using a Lichroprep RP18 cartridge column (25–40 μm; Merck) and pyridylaminated. The fluorescently-labelled N-glycans were fractionated by HPLC using either an AscentisR Express RP-Amide column (150 × 4.6 mm, 2.7 μm; Supelco) with a gradient of 0.3% methanol per minute at a flow rate of 0.8 mL/min for neutral glycans or an IonPac AS11 column (HIAX, Dionex) for anionic glycans (Kurz et al, 2015). Collected HPLC fractions were lyophilized, reconstituted in water and analysed by MALDI-TOF MS in positive and negative ion modes (Bruker Autoflex Speed or Bruker UltrafleXtreme) with 6-aza-2-thiothymine (ATT) as matrix.…”

“…Selected glycan fractions obtained from HPLC analysis of the recent study (Kurz et al, 2015) were subjected to chemical and enzymatic treatments. Thereby, hydrofluoric acid was used as it specifically releases α1,3-linked fucose residues (Haslam et al, 2000).…”

“…gambiae larvae and revealed expected and novel epitopes including a large set of anionic glycans modified with sulphate, glucuronic acid or both which might represent additional glycan ligands, other than proteoglycans, involved in parasite-vector interactions (Kurz et al, 2015). Although, the detection of fucosylated N-glycans was somehow expected, considering that An.…”