Characterization of recombinant human Man 9-mannosidase expressed in Escherichia coli

Abstract

Man 9-mannosidase is an α1,2-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 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 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 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 9 GlcNAc 2 is rapidly converted to Man 6GlcNAc 2. Substrate specificity was analyzed using different isomers of Man 6GlcNAc 2, Man 7GlcNAc 2 and Man 8GlcNAc 2. The truncated Man 9-mannosidase exhibited very little activity towards the α1,2-linked terminal mannose residue on the α3 branch of the Man 9GlcNAc 2, a characteristic of the pig and calf liver Man 9-mannosidases. Based on the HPLC analysis of the reaction products using size-fractionation and reversed-phase columns, the pathway for Man 9GlcNAc 2 trimming was deduced. The recombinant enzyme had optimum activity at 37–40°C and pH 6.5–7.0. Enzyme activity was inhibited by 100 μM deoxymannojirimycin (dMNJ). Expression of the human kidney Man 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.