Purification and properties of a Golgi-derived (alpha 1,2)-mannosidase-I from baculovirus-infected lepidopteran insect cells (IPLB-SF21AE) with preferential activity toward mannose6-N-acetylglucosamine2.
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 Man6GlcNAc2 to Man5GlcNAc2 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 NaB3H4-reduced Man6GlcNAc2-ol to Man5GlcNAc-[3H]GlcNAc2-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 microM, 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 Man6GlcNAc2-ol, with lower activities obtained with Man9GlcNAc2-ol, Man8GlcNAc2-ol, and Man7GlcNAc2-ol. With Man6GlcNAc2-ol as substrate, products smaller than reduced Man5GlcNAc2-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)-Man6-mannosidase.
Ren
J
J
Department of Chemistry and Biochemistry, University of Notre Dame, Indiana 46556.
Bretthauer
R K
RK
Castellino
F J
FJ
D000818:Animals; D016367:Baculoviridae / Q000201:enzymology / Q000235:genetics; D002240:Carbohydrate Sequence; D002460:Cell Line; D006056:Golgi Apparatus / Q000201:enzymology; D007700:Kinetics; D008361:Mannosidases / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D008969:Molecular Sequence Data; D009844:Oligosaccharides / Q000737:chemistry; D018411:Spodoptera / Q000201:enzymology / Q000235:genetics; D013379:Substrate Specificity
D009844:Oligosaccharides; D008361:Mannosidases; C046090:mannosyl-oligosaccharide 1,2-alpha-mannosidase