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Publikationstyp: Zeitschriftenaufsatz
Dokumentart: Originalarbeit

Publikationsjahr: 2015

AutorInnen: Dragosits, M; Yan, S; Razzazi-Fazeli, E; Wilson, IB; Rendic, D

Titel: Enzymatic properties and subtle differences in the substrate specificity of phylogenetically distinct invertebrate N-glycan processing hexosaminidases.

Quelle: Glycobiology. 2015; 25(4):448-464



Autor/innen der Vetmeduni Vienna:

Razzazi-Fazeli Ebrahim
Yan Shi

Beteiligte Vetmed-Organisationseinheiten
VetCore


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.

Keywords Pubmed: Amino Acid Sequence
Animals
Bees/enzymology
Biocatalysis
Caenorhabditis elegans/enzymology
Caenorhabditis elegans Proteins/chemistry
Carbohydrate Conformation
Carbohydrate Sequence
Cell Line
Drosophila Proteins/chemistry
Drosophila melanogaster/enzymology
Glycopeptides/chemistry
Glycosylation
Hexosaminidases/chemistry*
Hydrogen-Ion Concentration
Molecular Sequence Data
Phylogeny
Polysaccharides/chemistry
Substrate Specificity


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