Purification and enzymatic properties of endo-alpha 1,2-mannosidase from pig liver involved in oligosaccharide processing

An endo-alpha 1,2-mannosidase, which is involved in N-linked oligosaccharide processing, has been purified to homogeneity from crude pig liver microsomes using conventional techniques. Two catalytically active polypeptides, of 48 kDa and 50 kDa, have been isolated which degrade [C-14]Glc(3-1)-Man(9)-GlcNAc(2) to [C-14]Glc(3-1)-Man and a specific Man(8)-GlcNAc(2) isomer. They are not, however, active on synthetic alpha-mannosides. [C-14]Glc(1)-Man(9)-GlcNAc(2) was found to be approximately sevenfold more rapidly hydrolyzed than the [C-14]Glc(2)- and [C-14]Glc(3)-homologues. The 48 kDa and 50 kDa proteins are not N-glycosylated and ran on Superdex((TM)) 75 as monomers. Kinetic studies showed that these proteins had similar catalytic properties: (i) the pH optima were found to be close to 6.5; (ii) neither activity was metal ion dependent; (iii) hydrolysis of [C-14]Glc(3)-Man(9)-GlcNAc(2) was inhibited strongly by Glc-alpha 1,3-Man (app. K-i approximate to 120 mu M), but not by 1-deoxymannojirimycin or swainsonine, Other evidence, including immunological data, strongly suggests that the 48 kDa and 50 kDa polypeptides are proteolytic degradation products of a single endo-alpha 1,2-mannosidase, rather than distinct subunits of an oligomeric complex, Possible functions of the endo-alpha 1,2-mannosidase in N-linked oligosaccharide processing are discussed.