Tissue-specific glycosylation in the honeybee: Analysis of the N-glycomes of Apis mellifera larvae and venom.

BACKGROUND: Previous glycophylogenetic comparisons of dipteran and lepidopteran species revealed variations in the anionic and zwitterionic modifications of their N-glycans; therefore, we wished to explore whether species- and order-specific glycomic variations would extend to the hymenoptera, which include the honeybee Apis mellifera, an agriculturally- and allergologically-significant social species. METHODS: In this study, we employed an off-line liquid chromatography/mass spectrometry approach, in combination with enzymatic and chemical treatments, to analyse the N-glycans of male honeybee larvae and honeybee venom in order to facilitate definition of isomeric structures. RESULTS: The neutral larval N-glycome was dominated by oligomannosidic and paucimannosidic structures, while the neutral venom N-glycome displayed more processed hybrid and complex forms with antennal N-acetylgalactosamine, galactose and fucose residues including Lewis-like epitopes; the anionic pools from both larvae and venom contained a wide variety of glucuronylated, sulphated and phosphoethanolamine-modified N-glycans with up to three antennae. In comparison to honeybee royal jelly, there were more fucosylated and fewer Man4/5-based hybrid glycans in the larvae and venom samples as well as contrasting antennal lengths. CONCLUSIONS: Combining the current data on venom and larvae with that we previously published on royal jelly, a total honeybee N-glycomic repertoire of some 150 compositions can be proposed in addition to the 20 previously identified on specific venom glycoproteins. SIGNIFICANCE: Our data are indicative of tissue-specific modification of the core and antennal regions of N-glycans in Apis mellifera and reinforce the concept that insects are capable of extensive processing to result in rather complex anionic oligosaccharide structures.

The underestimated N-glycomes of lepidopteran species.

BACKGROUND: Insects are significant to the environment, agriculture, health and biotechnology. Many of these aspects display some relationship to glycosylation, e.g., in case of pathogen binding or production of humanised antibodies; for a long time, it has been considered that insect N-glycosylation potentials are rather similar and simple, but as more species are glycomically analysed in depth, it is becoming obvious that there is indeed a large structural diversity and interspecies variability. METHODS: Using an off-line LC-MALDI-TOF MS approach, we have analysed the N-glycomes of two lepidopteran species (the cabbage looper Trichoplusia ni and the gypsy moth Lymantria dispar) as well as of the commonly-used T. ni High Five cell line. RESULTS: We detected not only sulphated, glucuronylated, core difucosylated and Lewis-like antennal fucosylated structures, but also the zwitterion phosphorylcholine on antennal GlcNAc residues, a modification otherwise familiar from nematodes; in L. dispar, N-glycans with glycolipid-like antennae containing α-linked N-acetylgalactosamine were also revealed. CONCLUSION: The lepidopteran glycomes analysed not only display core α1,3-fucosylation, which is foreign to mammals, but also up to 5% anionic and/or zwitterionic glycans previously not found in these species. SIGNIFICANCE: The occurrence of anionic and zwitterionic glycans in the Lepidoptera data is not only of glycoanalytical and evolutionary interest, but is of biotechnological relevance as lepidopteran cell lines are potential factories for recombinant glycoprotein production.

Development of a multifunctional aminoxy-based fluorescent linker for glycan immobilization and analysis.

Glycan arrays have become a technique of choice to screen glycan-protein interactions in a high-throughput manner with high sensitivity and low sample consumption. Here, the synthesis of a new multifunctional fluorescent linker for glycan labeling via aminoxy ligation and immobilization is described; the linker features a fluorescent naphthalene group suitable for highly sensitive high-performance liquid chromatography-based purification and an azido- or amino-modified pentanoyl moiety for the immobilization onto solid supports. Several glycoconjugates displaying small sugar epitopes via chemical or chemoenzymatic synthesis were covalently attached onto a microarray support and tested with lectins of known carbohydrate binding specificity. The glycan library was extended using glycosyltransferases (e.g. galactosyl-, sialyl- and fucosyltransferases); the resulting neoglycoconjugates, which are easily detected by mass spectrometry, mimic antennal elements of N- and O-glycans, including ABH blood group epitopes and sialylated structures. Furthermore, an example natural plant N-glycan containing core α1,3-fucose and ß1,2-xylose was also successfully conjugated to the fluorescent linker, immobilized and probed with lectins as well as antihorseradish peroxidase. These experiments validate our linker as being a potentially valuable tool to study glycozyme and lectin specificities, sensitive enough to allow purification of natural glycans.

'Click chemistry' synthesis of 1-(α-D-mannopyranosyl)-1,2,3-triazoles for inhibition of α-mannosidases.

Three new triazole conjugates derived from d-mannose were synthesized and assayed in in vitro assays to investigate their ability to inhibit α-mannosidase enzymes from the glycoside hydrolase (GH) families 38 and 47. The triazole conjugates were more selective for a GH47 α-mannosidase (Aspergillus saitoi α1,2-mannosidase), showing inhibition at the micromolar level (IC50 values of 50-250 µM), and less potent towards GH38 mannosidases (IC50 values in the range of 0.5-6 mM towards jack bean α-mannosidase or Drosophila melanogaster lysosomal and Golgi α-mannosidases). The highest selectivity ratio [IC50(GH38)/IC50(GH47)] of 100 was exhibited by the phenyltriazole conjugate. To understand structure-activity properties of synthesized compounds, 3-D complexes of inhibitors with α-mannosidases were built using molecular docking calculations.