Biochemical Properties And Physiological Function Of OfHex2from The Insect Ostrinia Furnacalis

β-N-acetyl-D-hexosaminidases (EC3.2.1.52) are widely distributed in bacteria, fungi, plants and animals, cleaving the β-linked N-acetyl-D-glucosamine (GlcNAc) or N-acetyl-D-galactosamine (GalNAc) from the non-reducing ends of substrates including oligosaccharides, glycoproteins, glycolipids and glycosaminoglycans. Insect β-N-acetyl-D-hexosaminidases function in several critical physiological processes such as chitin degradation, Golgi N-glycan modification, lysosomal glycoconjugates degradation and gamete recognition. Lysosomal β-N-acetyl-D-hexosaminidases are also believed to exist in insect due to the existence of various physiological glycoconjugate substrates. The β-N-acetyl-D-hexosaminidase that involved in insect glycoconjugate degradation will be a potential target for developing green pesticides. Moreover, insect lysosomal β-N-acetyl-D-hexosaminidase is of research interests for its similar physiological role as mammal analog. The investigation of insect lysosomal β-N-acetyl-D-hexosaminidase is belived to contribute to the knowledge of lysosomal β-N-acetyl-D-hexosaminidase and the lysosomal storage disorders caused by β-N-acetyl-D-hexosaminidase defeciency.This dissertation studied the lysosomal β-N-acetyl-D-hexosaminidase from the destructive pest, Ostrinia furnacalis (Guenee). The β-N-acetyl-D-hexosaminidase gene (OfHEX2) was obtained and recombinantly expressed. The characterization of the comprehensive properties of OfHex2, the investigation of developmental expression pattern of OfHEX2and the RNA interference were performed. Here are the results and conclusions:1. Sequence phylogenetic analysis of OfHEX2(1) The cDNA gene encoding β-N-acetyl-D-hexosaminidase termed OfHEX2was cloned from the fifth instar larvae of O. furnacalis using specific primers. The1734-bp nucleotide sequence of the OfHEX2contains a1674-bp ORF encoding577amino acids (Genbank assession number EF469203).(2) The residues of OfHex2involved in catalysis arc highly conserved. The segment I (Cys300-Thr315) and segment Ⅱ (Trp435-Trp446) cxibit unique sequence features when compared to the humanβ-N-acetyl-D-hexosaminidases. Phylogenetic analysis indicate that OfHEX2is phylogenetically distant from insect chitinolytic β-N-acetyl-D-hexosaminidase and the N-processing β-N-acetyl-D-hexosaminidase.2. Recombinant expression and purification of OfHex2(1)The plasmids pPIC9-OfHex1and pPIC9-OfHex2were constructed and transformated into the methylotrophic yeast Pichia pastoris GS115. After fermentation for120h, approximately12.47mg of OfHex1with362.16U of enzymatic activity was obtained from1L of P. pastoris culture supernatant. After fermentation for144h, approximately1.85mg of OfHex2with16.06U of enzymatic activity was obtained from1L of P. pastoris culture supernatant.(2) By Ni2+IMAC,7.67mg of OfHex1was purified by11.56folds with a final yield of61.5%. By ammonium sulfate precipitation at65%saturation and Ni2+IMAC,1.2mg of OfHex2was purified by28.72folds with a final yield of65%.By ammonium sulfate precipitation at65%saturation and Ni2+IMAC,1.2mg of OfHex2was purified by28.72folds with a final yield of65%.3. Biochemical properties of OfHex2(1) The molecular mass of OfHex2was determined to be65kDa by SDS-PAGE and134.6kDa by analytical gel filtration chromatography, indicating OfHex2is a homodimer. pl of OfHex2was determined to be4.88.(2) The optimal temperature and pH of OfHex2are30℃and5.5, respectively. The acidic optimum suggests that OfHex2can exhibit high catalytic activity in lysosomal acidic condition.(3) OfHex2shows low selectivity toward-1sugar ring of the substrates, and hydrolyzes MU-β-GlcNAc and MU-β-GalNAc at comparable speeds. OfHex2degrades negatively charged MU-β-GlcNAc-SO4-with rather low activity, and the ratio of degrading speed for MU-β-GlcNAc to that for MU-β-GlcNAc-6-SO4-is250/1.(4) OfHex2degrades N-glycan GnGn and GlcNAcβ1,2Man completely. OfHex2shows preference to the α3branch-GlcNAc, and the ratio of appeaent degrading speed for α3branch-GlcNAc to that for α6branch-GlcNAc is4.97/1.(5) OfHex2degrades artifical single-acetylglucosamine substrates vary efficiently and exhibits the highest enzymatic affinity to (GlcNAc)2, and the Km value was0.169±0.008mM. Among the chitinooligosaccharides with different DP grade, OfHex2prefers to degrade (GlcNAc)3first, which causes an accumulation of (GlcNAc)3during (GlcNAc)3degradation.(6) OfHex2has the same substrate spectrum as human HsHexB dose. OfHex2cleaves different types of β-glycosidic bonds and shows lower selectivity to the-1and+1sugar of the substrate, suggesting that OfHex2is capable of degrading physiological neutrol glycoconjugates in O. furnacalis. 4. The transcription of OfHEX2and RNA interference(1) The expression profiles of OfHEX2during larval-pupation-adult metamorphosis were determined by Real-time PCR. High levels of OfHEX2transcript were detected both at feeding stage of the5th instar larva and pupa. The transcription levels were dramatically downregulated during molting. This exprssion pattern indicates the physiological role of OfHex2may be related to insect metabolism except chitin degradation.(2) The RNAi against OfHEX2was induced by the injection of dsOfHEX2at the5th larvae day1. The gene silencing of OfHEX2in the5th instar larva was non-lethal but severely affected the development of larval abdomen, pupal wing and adult appendages. The phenotypes indicate the defecency of OfHex2might interrupt the glycocojugate degradaion on appendage dies, causing abnormal appendage mutants.5. The sequence characteristics, the in vitro biochemical properties of recombinant OfHex2and the in vivo expression pattern as well as gene silence phenotype suggest OfHex2may be a lysosomal enzyme that is involved in glycoconjugate degradation in the insect appendage discs.