CHARACTERIZATION OF RECOMBINANT HUMAN MAN(9)-MANNOSIDASE EXPRESSED INESCHERICHIA-COLI

Man(9)-mannosidase is an alpha 1,2-specific exo-enzyme involved in the N-linked oligosaccharide processing pathway, In this study, the expre ssion of the truncated gene encoding for the human Man(9)-mannosidase (amino acids 140 to 625) in Escherichia coli facilitated further chara cterization of the enzyme. PCR primers were designed to isolate a trun cated 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 t he purified enzyme was examined by HPLC and pyridylaminated (PA) oligo saccharides as substrates. Results showed that Man(9) GlcNAc(2) is rap idly converted to Man(6)GlcNAc(2). Substrate specificity was analyzed using different isomers of Man(6)GlcNAc(2), Man(7)GlcNAc(2) and Man(8) GlcNAc(2), The truncated Man(9)-mannosidase exhibited very little acti vity towards the alpha 1,2-linked terminal mannose residue on the alph a 3 branch of the Man(9)GlcNAc(2), a characteristic of the pig and cal f liver Man(9)-mannosidases. Based on the HPLC analysis of the reactio n products using size-fractionation and reversed-phase columns, the pa thway for Man(9)GlcNAc(2) trimming was deduced. The recombinant enzyme had optimum activity at 37-40 degrees C and pH 6.5-7.0. Enzyme activi ty was inhibited by 100 mu M deoxymannojirimycin (dMNJ), Expression of the human kidney Man(9)-mannosidase gene in E. coli Facilitated the d etailed characterization of the enzyme which until now had not been ex pressed in amounts necessary for biochemical and physical analyses.