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.

Background:The eastern oyster, an important estuarine species, is parasitized by a protozoan in a galectin-dependent manner. Results: A variety of paucimannosidic, hybrid, and complex neutral and acidic N-linked oligosaccharides was found. Conclusion:The oyster possesses a complex repertoire of glycans with some features reminiscent of vertebrates. Significance: The N-glycome of the eastern oyster correlates well with the specificity of its own galectin CvGal1.

Galactosidases are widespread enzymes that are used for manifold applications, including production of prebiotics, biosynthesis of different transgalactosylated products, improving lactose tolerance and in various analytical approaches. The nature of these applications often require galactosidases to be present in a purified form with clearly defined properties, including precisely determined substrate specificities, low sensitivity to inhibitors, and high efficiency and stability under distinct conditions. In this study, we present the recombinant expression and purification of two previously uncharacterized β-galactosidases from Aspergillus nidulans as well as one β-galactosidase from Aspergillus niger. All enzymes were active toward p-nitrophenyl-β-d-galactopyranoside as substrate and displayed similar temperature and pH optima. The purified recombinant galactosidases digested various complex substrates containing terminal galactose β-1,4 linked to either N-acetylglucosamine or fucose, such as N-glycans derived from bovine fibrin and Caenorhabditis elegans. In our comparative study of the recombinant galactosidases with the commercially available galactosidase from Aspergillus oryzae, all enzymes also displayed various degrees of activity toward complex oligosaccharides containing β-1,3-linked terminal galactose residues. All recombinant enzymes were found to be robust in the presence of various organic solvents, temperature variations, and freeze/thaw cycles and were also tested for their ability to synthesize galactooligosaccharides. Furthermore, the use of fermentors considerably increased the yield of recombinant galactosidases. Taken together, we demonstrate that purified recombinant galactosidases from A. niger and from A. nidulans are suitable for various glycobiological and biotechnological applications.Electronic supplementary materialThe online version of this article (doi:10.1007/s00253-013-5192-3) contains supplementary material, which is available to authorized users.

HPLC has been employed for decades to enhance detection sensitivity and quantification of complex analytes within biological mixtures. Among these analytes, glycans released from glycoproteins and glycolipids have been characterized as underivatized or fluorescently tagged derivatives by HPLC coupled to various detection methods. These approaches have proven extremely useful for profiling the structural diversity of glycoprotein and glycolipid glycosylation but require the availability of glycan standards and secondary orthogonal degradation strategies to validate structural assignments. A robust method for HPLC separation of glycans as their permethylated derivatives, coupled with in-line multidimensional ion fragmentation (MS n ) to assign structural features independent of standards, would significantly enhance the depth of knowledge obtainable from biological samples. Here, we report an optimized workflow for LC-MS analysis of permethylated glycans that includes sample preparation, mobile phase optimization, and MS n method development to resolve structural isomers on-the-fly. We report baseline separation and MS n of isomeric N- and O-glycan structures, aided by supplementing mobile phases with Li + , which simplifies adduct heterogeneity and facilitates cross-ring fragmentation to obtain valuable monosaccharide linkage information. Our workflow has been adapted from standard proteomics-based workflows and, therefore, provides opportunities for laboratories with expertise in proteomics to acquire glycomic data with minimal deviation from existing buffer systems, chromatography media, and instrument configurations. Furthermore, our workflow does not require a mass spectrometer with high-resolution/accurate mass capabilities. The rapidly evolving appreciation of the biological significance of glycans for human health and disease requires the implementation of high-throughput methods to identify and quantify glycans harvested from sample sets of sufficient size to achieve appropriately powered statistical significance. The LC-MSn approach we report generates glycan isomeric separations and robust structural characterization and is amenable to autosampling with associated throughput enhancements.

Fucoconjugates are key mediators of protein-glycan interactions in prokaryotes and eukaryotes. As examples, N-glycans modified with the non-mammalian core α1,3-linked fucose have been detected in various organisms ranging from plants to insects and are immunogenic in mammals. The rabbit polyclonal antibody raised against plant horseradish peroxidase (anti-HRP) is able to recognize the α1,3-linked fucose epitope and is also known to specifically stain neural tissues in the fruit fly Drosophila melanogaster. In this study, we have detected and localized the anti-HRP cross-reactivity in another insect species, the malaria mosquito vector Anopheles gambiae. We were able to identify and structurally elucidate fucosylated N-glycans including core mono- and difucosylated structures (responsible for anti-HRP cross reactivity) as well as a Lewis-type antennal modification on mosquito anionic N-glycans by applying enzymatic and chemical treatments. The three mosquito fucosyltransferase open reading frames (FucT6, FucTA and FucTC) required for the in vivo biosynthesis of the fucosylated N-glycan epitopes were identified in the Anopheles gambiae genome, cloned and recombinantly expressed in Pichia pastoris. Using a robust MALDI-TOF MS approach, we characterised the activity of the three recombinant fucosyltransferases in vitro and demonstrate that they share similar enzymatic properties as compared to their homologues from D. melanogaster and Apis mellifera. Thus, not only do we confirm the neural reactivity of anti-HRP in a mosquito species, but also demonstrate enzymatic activity for all its α1,3- and α1,6-fucosyltransferase homologues, whose specificity matches the results of glycomic analyses.