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
Mentions: In an effort to produce the recombinant FDL enzyme of high purity suitable for a thorough study of its properties, we have analyzed the expression of various FDL(-like) enzymes using two different expression systems. Prompted by the success in the previous study (Léonard et al. 2006), we have initially expressed the D. melanogaster FDL in Pichia pastoris. The activity of the recombinant protein carrying the C-terminal HIS-tag could not be detected, whereas the purification of the protein carrying the N-terminal HIS-tag yielded moderate amounts (88 mU mL−1) of the partly degraded recombinant protein (Figure 1, lanes 2 and 3). In an effort to increase the yield and quality of the recombinant product, common parameters (culture temperature, medium type) were varied, followed by the bioreactor pilot expression of the recombinant enzyme (Supplementary data, Figure S1). In addition, we used a codon-optimized version of the open reading frame (ORF), which finally lead to a minor increase of 25% in yield using optimal medium (YP) and optimal temperature (25°C) (Table I and Supplementary data, Figure S2). Therefore, we decided to also express the Drosophila FDL using the Baculovirus expression system in insect cells. The affinity-purified recombinant enzyme, when analyzed by sodium dodecyl sulphate –polyacrylamide gel electrophoresis (SDS–PAGE), migrated as single band of an expected size (Figure 1, lanes 4 and 5). Moreover, the insect expression system yielded higher amounts of the recombinant Drosophila FDL (Table I).Table I.
Affiliation: Department of Chemistry, University of Natural Resources and Life Sciences, Vienna VetCore Facility for Research, University of Veterinary Medicine, Vienna, Austria.