PURIFICATION AND PROPERTIES OF A GOLGI-DERIVED (ALPHA-1,2)-MANNOSIDASE-I FROM BACULOVIRUS-INFECTED LEPIDOPTERAN INSECT CELLS (IPLB-SF21AE) WITH PREFERENTIAL ACTIVITY TOWARD MANNOSE(6)-N-ACETYLGLUCOSAMINE(2)

Because the availability and subcellular distribution of processing mannosidases in cells play such powerful roles in determining ultimate structures of glycoconjugates, we desired to identify, characterize, and investigate possible regulation of mannosidases in infected and noninfected lepidopteran insect cells. Since our previous observations that a mannosidase activity that converted Man(6)GlcNAc(2) to Man(5)GlcNAc(2) was enhanced in virus-infected cells, thus providing the necessary intermediate for further processing to complex-type oligosaccharides, we attempted purification of this enzyme. A mannosidase was isolated and purified from membranes, operationally defined as Golgi, of recombinant baculovirus-infected Spodoptera frugiperda (IPLB-SF-21AE) cells. The molecular mass of this protein was approximately 63 kDa. Assays performed by measuring the conversion of (NaBH4)-H-3-reduced Man(6)GlcNAc(2)-ol to Man(5)GlcNAc-[H-3]GlcNAc(2)-ol demonstrated that the mannosidase activity was dependent on the presence of divalent cations, which was optimal for Ca2+ at pH 6.0. Inclusion of 1-deoxymannojirimycin resulted in 50% inhibition at a concentration of 20 mu M, whereas swainsonine did not show such inhibition. No activity was observed with p-nitrophenyl alpha-D-mannoside (4 mM) as a substrate. The preferred reduced oligosaccharide substrate was Man(6)GlcNAc(2)-ol, with lower activities obtained with Man(9)GlcNAc(2)-ol, Man(8)GlcNAc(2)-ol, and Man(7)GlcNAc(2)-ol. With Man(6)GlcNAc(2)-ol as substrate, products smaller than reduced Man(5)GlcNAc(2)-ol were not observed. Mannose was also liberated from the glycoprotein, ovalbumin. These properties are consistent with an enzyme classification as a type I (alpha 1,2)-Man(6)-mannosidase.