Characterization of human liver α-D-mannosidase purified by affinity chromatography

Abstract

Human liver acidic α-D-mannosidase was purified 1400-fold by a relatively short procedure incorporating chromatography on concanavalin A-Sepharose and affinity chromatography on Sepharose 4B-epsilon-aminohexanoylmannosylamine. In contrast with the acidic enzymic activity the neutral α-mannosidase did not bind to the concanavalin A-Sepharose so the two types of α-mannosidase could be separated at an early stage in the purification. The only significant glycosidase contaminant after affinity chromatography on the mannosylamine ligand was α-L-fucosidase, which was selectively removed by affinity chromatography on the corresponding fucosylamine ligand. The final preparation was free of other glycosidase activities. The pI of the purified enzyme was increased from 6.0 to 6.45 on treatment with neuraminidase. Although the pI and the mol.wt. (220 000) suggested that α-mannosidase A had been purified selectively, ion-exchange chromatography on DEAE-cellulose indicated that the preparation consisted predominantly of α-mannosidase B. This discrepancy is discussed in relation to the basis of the multiple forms of human α-mannosidase. The purified enzyme completely removed the α-linked non-reducing terminal mannose from a trisaccharide isolated from the urine of a patient with mannosidosis. A comparison of the activity of the pure enzyme towards the natural substrate and synthetic substrates suggests that the same enzymic activity is responsible for hydrolysing all the substrates. These results validate the use of synthetic substrates for determining the mannosidosis genotype. They are also further evidence that mannosidosis is a lysosomal storage disease resulting from a deficiency of acidic α-mannosidase.