The fucomic potential of mosquitoes: Fucosylated N-glycan epitopes and their cognate fucosyltransferases |
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| Institution: | 1. Department für Chemie, Universität für Bodenkultur, 1190 Wien, Austria;2. W. Harry Feinstone Department of Molecular Microbiology & Immunology, Johns Hopkins Bloomberg School of Public Health & The Malaria Research Institute, Baltimore, MD 21205, USA;1. Department of Radiation Oncology, Mount Sinai Hospital, New York, New York;2. Department of Cardiology, Mount Sinai Hospital, New York, New York;1. Structural Chemistry Program, Eskitis Institute, Griffith University, Brisbane, Queensland, Australia;2. Centre for Biodiscovery and Molecular Development of Therapeutics, Australian Institute of Tropical Health and Medicine, James Cook University, Cairns, Queensland, Australia;3. Université Lyon 1, Villeurbanne cedex, and Laboratoire Chimie et Biologie des Membranes et des Nanoobjets, Université Bordeaux, CBMN, UMR 5248, 33600 Pessac, France;4. Institute for Structural Biology, School of Biological Sciences, The University of Edinburgh, Scotland, UK;5. School of Veterinary Science, The University of Queensland, Gatton, Queensland, Australia;6. Queensland Institute of Medical Research, Herston, Queensland, Australia;7. Biology Division, California Institute of Technology, Pasadena, CA, USA;8. Faculty of Veterinary Science, The University of Melbourne, Parkville, Victoria, Australia;1. School of Marine Science, Ningbo University, Ningbo 315211, China;2. Institute of Preventive Veterinary Medicine, Zhejiang Provincial Key Laboratory of Preventive Veterinary Medicine, Zhejiang University, Hangzhou 310058, China |
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| 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. |
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| Keywords: | Fucosyltransferases Anti-HRP N-glycans Immunofluorescence Mass spectrometry |
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