Molecular and Biochemical Characterization of a Novel β-N-Acetyl-D-Hexosaminidase with Broad Substrate-Spectrum from the Aisan Corn Borer, Ostrinia Furnacalis

Abstract

Insect β-N-acetyl-D-hexosaminidases with broad substrate-spectrum (IBS-Hex) are the homologues of human β-N-acetyl-D-hexosaminidase A/B (HsHex A/ B). These enzymes are distributed in most insect species and vary in physiological roles. In this study, the gene encoding an IBS-Hex, OfHEX2, was cloned from the Asian corn borer, Ostrinia furnacalis. Recombinant OfHex2 was expressed in Pichia pastoris and purified to homogeneity. By structure-based sequence alignment, three sequence segments with high diversity among IBS-Hexs were firstly concluded. Furthermore, the residue pair N423-R424/ D452-L453 important for the specificity of human β-N-acetyl-D-hexosaminidase subunits α/β toward charged/ non-charged substrates was not conserved in OfHex2 and other IBS-Hexs. Unlike HsHex A, OfHex2 could not degrade charged substrates such as 4-methylumbelliferyl-6-sulfo-N-acetyl-β-D-glucosaminide, ganglioside GM2 and peptidoglycan. OfHex2 showed a broad substrate-spectrum by hydrolyzing β1-2 linked N-acetyl-D-glucosamines from both α3 and α6 branches of biantennary N-glycan and β1-4 linked GlcNAc from chitooligosaccharides as well as β1-3 linked or β1-4 linked N-acetyl-D-galactosamine from oligosaccharides of glycolipids. Real-time PCR analysis demonstrated that the expression of OfHEX2 was up-regulated in the intermolt stages (both larva and pupa), and mainly occurred in the carcass rather than in the midgut during the feeding stage of fifth (final) instar larva. This study reported a novel IBS-Hex with specific biochemical properties, suggesting biodiversity of this class of enzymes.

The β‐N‐acetylhexosaminidase gene family in the brown planthopper, Nilaparvata lugens

“…HEXs are also widely distributed in insects. Indeed, Hex genes have been identified and purification of the heterologously expressed proteins has been performed in lepidopterans, including Manduca sexta (Zen et al ., ), Spodoptera frugiperda (Tomiya et al ., ), Choristoneum fumiferana (Zheng et al ., ) and Ostrinia furnacalis (Yang et al ., ; Liu et al ., ; Liu et al ., ); dipterans, including Drosophila melanogaster (Cattaneo et al ., ) and Aedes aegypti (Filho et al ., ); coleopterans, including Tribolium castaneum (Hogenkamp et al ., ); and orthopterans, including Locusta migratoria (Rong et al ., ). The purification of an insect HEX protein has also been performed in Bombyx mori (Nagamatsu et al ., ).…”

A Sperm–Plasma β-N-Acetyl-D-Hexosaminidase Interacting with a Chitinolytic β-N-Acetyl-D-Hexosaminidase in Insect Molting Fluid

“…Group II comprised Hexes with a broad substrate spectrum, e.g. OfHex2 (GenBank ID: ABO65045) [25]. Group IV consisted of N-glycan modifying Hexes, e.g.…”

“…The reaction mixtures were incubated at 25°C for an appropriate time, and then the reaction was immediately stopped by incubation on ice. The hydrolysis products were analyzed by HPLC using a TSKgel Amide-80 column [25]. The hydrolytic products were quantified by converting the peak area to concentration values according to a standard curve of GlcNAc made with known concentrations.…”

“…The reaction mixtures were incubated at 25°C for 24 h and the hydrolysis products were analyzed by HPLC as previously described [25].…”

“…Proteins were extracted from the samples and separated by 10% SDS-PAGE. Western blotting was performed as described previously [25]. The antibodies used for western blot were 1∶400 dilution for OfHex3 and 1∶2000 dilution for tubulin.…”