title: Enzymatic properties and subtle differences in the substrate specificity of phylogenetically distinct invertebrate N-glycan processing hexosaminidases.
abstract: Fused lobes (FDL) hexosaminidases are the most recently genetically defined glycosidases involved in the biosynthesis of N-glycans in invertebrates, and their narrow specificity is essential for the generation of paucimannosidic N-glycans in insects. In this study, we explored the potential of FDL hexosaminidases in the utilization of different artificial and natural substrates, both as purified, native compounds or generated in vitro using various relevant glycosyltransferases. In addition to the already-known FDL enzyme from Drosophila melanogaster, we now have identified and characterized the Apis mellifera FDL homolog. The enzymatic properties of the soluble forms of the affinity-purified insect FDL enzymes, expressed in both yeast and insect cells, were compared with those of the phylogenetically distinct recombinant Caenorhabditis elegans FDL-like enzymes and the N-acetylgalactosamine (GalNAc)-specific Caenorhabditis hexosaminidase HEX-4. In tests with a range of substrates, including natural N-glycans, we show that the invertebrate FDL(-like) enzymes are highly specific for N-acetylglucosamine attached to the α1,3-mannose, but under extreme conditions also remove other terminal GalNAc and N-acetylglucosamine residues. Recombinant FDL also proved useful in the analysis of complex mixtures of N-glycans originating from wild-type and mutant Caenorhabditis strains, thereby aiding isomeric definition of paucimannosidic and hybrid N-glycans in this organism. Furthermore, differences in activity and specificity were shown for two site-directed mutants of Drosophila FDL, compatible with the high structural similarity of chitinolytic and N-glycan degrading exohexosaminidases in insects. Our studies are another indication for the variety of structural and function aspects in the GH20 hexosaminidase family important for both catabolism and biosynthesis of glycoconjugates in eukaryotes.
authors.affiliation: Department of Chemistry, University of Natural Resources and Life Sciences, Vienna VetCore Facility for Research, University of Veterinary Medicine, Vienna, Austria.
authors.forename: Martin
authors.initials: M
authors.lastname: Dragosits
authors.affiliation: Department of Chemistry, University of Natural Resources and Life Sciences, Vienna.
authors.forename: Shi
authors.initials: S
authors.lastname: Yan
authors.affiliation: VetCore Facility for Research, University of Veterinary Medicine, Vienna, Austria.
authors.forename: Ebrahim
authors.initials: E
authors.lastname: Razzazi-Fazeli
authors.affiliation: Department of Chemistry, University of Natural Resources and Life Sciences, Vienna.
authors.forename: Iain B H
authors.initials: IB
authors.lastname: Wilson
authors.affiliation: Department of Chemistry, University of Natural Resources and Life Sciences, Vienna dubravko.rendic@boku.ac.at.
authors.forename: Dubravko
authors.initials: D
authors.lastname: Rendic
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001516:Bees / Q000201:enzymology; D055162:Biocatalysis; D017173:Caenorhabditis elegans / Q000201:enzymology; D029742:Caenorhabditis elegans Proteins / Q000737:chemistry; D002236:Carbohydrate Conformation; D002240:Carbohydrate Sequence; D002460:Cell Line; D029721:Drosophila Proteins / Q000737:chemistry; D004331:Drosophila melanogaster / Q000201:enzymology; D006020:Glycopeptides / Q000737:chemistry; D006031:Glycosylation; D006596:Hexosaminidases / Q000737:chemistry*; D006863:Hydrogen-Ion Concentration; D008969:Molecular Sequence Data; D010802:Phylogeny; D011134:Polysaccharides / Q000737:chemistry; D013379:Substrate Specificity
chemical_list: D029742:Caenorhabditis elegans Proteins; D029721:Drosophila Proteins; D006020:Glycopeptides; D011134:Polysaccharides; D006596:Hexosaminidases
keywords: N-glycans; fused lobes; hexosaminidase; insect; invertebrate