Characterization of Recombinant Human Man sub(9)-Mannosidase Expressed in Escherichia coli

Man sub(9)-mannosidase is an alpha 1,2-specific exo-enzyme involved in the N-linked oligosaccharide processing pathway. In this study, the expression of the truncated gene encoding for the human Man sub(9)-mannosidase (amino acids 140 to 625) in Escherichia coli facilitated further characterization of the enzyme. PCR primers were designed to isolate a truncated human kidney Man sub(9)-mannosidase gene. The gene was fused to a T7 protein tag and six histidine residues at the N and C-terminal ends, respectively. The truncated Man sub(9)-mannosidase enzyme was purified by affinity chromatography and ion exchange chromatography. Activity of the purified enzyme was examined by HPLC and pyridylaminated (PA) oligosaccharides as substrates. Results showed that Man sub(9) GlcNAc sub(2) is rapidly converted to Man sub(6)GlcNAc sub(2). Substrate specificity was analyzed using different isomers of Man sub(6)GlcNAc sub(2), Man sub(7)GlcNAc sub(2) and Man sub(8)GlcNAc sub(2). The truncated Man sub(9)-mannosidase exhibited very little activity towards the alpha 1,2-linked terminal mannose residue on the alpha 3 branch of the Man sub(9)GlcNAc sub(2), a characteristic of the pig and calf liver Man sub(9)-mannosidases. Based on the HPLC analysis of the reaction products using size-fractionation and reversed-phase columns, the pathway for Man sub(9)GlcNAc sub(2) trimming was deduced. The recombinant enzyme had optimum activity at 37-40 degree C and pH 6.5-7.0. Enzyme activity was inhibited by 100 mu M deoxymannojirimycin (dMNJ). Expression of the human kidney Man sub(9)-mannosidase gene in E. coli facilitated the detailed characterization of the enzyme which until now had not been expressed in amounts necessary for biochemical and physical analyses.