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论文类型：期刊论文

发表时间：2012-07-01

发表刊物：INTERNATIONAL JOURNAL OF BIOLOGICAL SCIENCES

收录刊物：PubMed、SCIE

卷号：8

期号：8

页面范围：1085-1096

ISSN号：1449-2288

关键字：beta-N-acetyl-D-hexosaminidase; insect; glycoside hydrolase; N-glycan; Ostrinia furnacalis

摘要：Insect beta-N-acetyl-D-hexosaminidases with broad substrate-spectrum (IBS-Hex) are the homologues of human beta-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 beta-N-acetyl-D-hexosaminidase subunits alpha/beta 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-beta-D-glucosaminide, ganglioside GM2 and peptidoglycan. OfHex2 showed a broad substrate-spectrum by hydrolyzing beta 1-2 linked N-acetyl-D-glucosamines from both alpha 3 and alpha 6 branches of biantennary N-glycan and beta 1-4 linked GlcNAc from chitooligosaccharides as well as beta 1-3 linked or beta 1-4 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.