Enzymatic properties and subtle differences in the substrate specificity of phylogenetically distinct invertebrate N-glycan processing hexosaminidases.
Bottom Line: 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.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.
Affiliation: Department of Chemistry, University of Natural Resources and Life Sciences, Vienna VetCore Facility for Research, University of Veterinary Medicine, Vienna, Austria.Show MeSH
Related in: MedlinePlus
Mentions: The recombinant insect FDL enzymes generally have lower pH optima (pH 4–5) than the Caenorhabditis hexosaminidases (pH 5–6) when using either pNP-β-GalNAc or pNP-β-GlcNAc as substrates (Figure 2). Interestingly, as with the Drosophila FDL (Léonard et al. 2006), the Apis FDL also has higher pH optima toward N-glycan substrates than toward pNP-β-GlcNAc/GalNAc; this was not observed for any of the Caenorhabditis enzymes described in this study (data not shown). Differences between the recombinant enzymes were also detected when measuring the temperature optima using either pNP-β-GalNAc (Caenorhabditis HEX-4) or pNP-β-GlcNAc [all recombinant FDL(-like) enzymes] as substrates; the Drosophila FDL enzyme displays the lowest temperature optimum (25–30°C; Figure 3). In addition to testing the recombinant enzymes pH and temperature optima, we have analyzed the sensitivity of the enzymes to presence of free GlcNAc and GalNAc in the reaction mixture. We could show that none of the tested enzymes are inhibited by these monosaccharides, apart from the CeHEX-4 which is inhibited by the GalNAc monosaccharide (ki = 1.0 mM; Supplementary data, Figure S3).Fig. 2.
Affiliation: Department of Chemistry, University of Natural Resources and Life Sciences, Vienna VetCore Facility for Research, University of Veterinary Medicine, Vienna, Austria.