Isolation and purification of a neutral alpha(1,2)-mannosidase from Trypanosoma cruzi.
Trypanosoma cruzi is an obligatory intracellular protozoan parasite that causes Chagas' disease in humans. Although a fair amount is known about the biochemistry of certain trypanosomes, very little is known about the enzymic complement of synthesis and processing of glycoproteins and/or functions of the subcellular organelles in this parasite. There have been very few reports on the presence of acid and neutral hydrolases in Trypanosoma cruzi. Here we report the first purification and characterization of a neutral mannosidase from the epimastigote stage of Trypanosoma cruzi. The neutral mannosidase was purified nearly 800-fold with an 8% recovery to apparent homogeneity from a CHAPS extract of epimastigotes by the following procedures: (1) metal affinity chromatography on Co+2-Sepharose, (2) anion exchange, and (3) hydroxylapatite. The purified enzyme has a native molecular weight of 150-160 kDa and is apparently composed of two subunits of 76 kDa. The purified enzyme exhibits a broad pH profile with a maximum at pH 5.9-6.3. It is inhibited by swainsonine (Ki, 0.1 microM), D-mannono-delta-lactam (Ki, 20 microM), kifunensine (Ki, 60 microM) but not significantly by deoxymannojirimycin. The enzyme is activated by Co2+and Ni2+and strongly inhibited by EDTA and Fe2+. The purified enzyme is active against p-nitrophenyl alpha-D-mannoside (km = 87 microM). High-mannose Man9GlcNAc substrate was hydrolyzed by the purified enzyme to Man7GlcNAc at pH 6.1. The purified enzyme does not show activity against alpha1,3- or alpha1,6-linked mannose residues. Antibodies against the recently purified lysosomal alpha-mannosidase from T.cruzi did not react with the neutral mannosidase reported here.
Bonay
P
P
Centro de Biologia Molecular "Severo Ochoa," Universidad Autonoma de Madrid, Cantoblanco, Madrid 28049, Spain.
Fresno
M
M
D000595:Amino Acid Sequence; D000818:Animals; D002240:Carbohydrate Sequence; D002845:Chromatography; D004789:Enzyme Activation; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008361:Mannosidases / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009844:Oligosaccharides / Q000737:chemistry; D011487:Protein Conformation; D013379:Substrate Specificity; D014349:Trypanosoma cruzi / Q000201:enzymology*
D004791:Enzyme Inhibitors; D009844:Oligosaccharides; D008361:Mannosidases; C046090:mannosyl-oligosaccharide 1,2-alpha-mannosidase