The fucomic potential of mosquitoes: Fucosylated N-glycan epitopes and their cognate fucosyltransferases. (January 2016) - Kurz, Simone
Abstract: 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. Graphical abstract: Highlights: We revealed the neural specificity of anti-HRP in Anopheles gambiae . Identification of the relevant genes involved in biosynthesis of fucosylated N-glycans in the mosquito. Native fucosylated N-glycans of the malaria vector were characterised. Robust MALDI-TOF MS based assays demonstrated in vitro enzymatic activity of three fucosyltransferases.