Recombinant Expression And Biochemical Characterization Ofβ-N-Acetylhexosaminidase FDL From The Insect Ostrinia Furnacalis

Insect β-N-acetylhexosaminidases serve multifunctional roles, including chitin and glycoconjugates degradation, N-linked glycans modification and egg-sperm interactions. The β-N-acetylhexosaminidase FDL specifically removes the β-1,2-GlcNAc residue conjugated to the α-1,3-mannose residue of the core structure of insect N-glycans, playing significant physiological roles in post-translational modification in the Golgi apparatus. Little is known about its enzymatic properties.1) Here, we obtained the cDNA of OfFDL from the insect Ostrinia furnacalis by RT-PCR. The full length mRNA of FDL is2241bp carrying an opening reading frame of1923bp encoding640amino acids. The amino acids in the N-terminal from residues32-49are predicted to be the transmembrane domain. The transcriptional level of OfFDL is very low at all development stages according to Real-time PCR analysis.2) The expression vector of the truncated OfFDL (amino acids55-640) was constructed and introduced into the Pichia pastoris GS115competent cells by electroporation. After120h induction with methanol, the activity of the enzyme in the culture supernatant achieved the highest level. The recombinant protein OfFDL was obtained with high purity through ammonium sulfate precipitation followed by metal chelating affinity chromatography.3) Several kinetic parameters including kcat/Km values toward four artificial substrates were determined. The catalytic efficiency (as estimated by the kcat/Km values) for GlcNAc substrate was3.0to3.5-fold higher than toward GalNAc substrate. The recombinant OfFDL did not show any substrate inhibition when pNP-β-GlcNAc was used as the substrate. The recombinant OfFDL exclusively hydrolyzes the terminal β-1,2-GlcNAc residue from the α-1,3branch instead of the a-1,6branch of the substrate GnGn-PA. OfFDL did not digest oligosaccharide substrates ((GlcNAc)2-3) and (31-2linked disaccharide (GlcNAcβ1-2Man).4) Two new synthetic compounds named xl-10and xl-138-173were determined to be selective inhibitors against OfFDL but not its homologs:OfHex1and OfHex2.