Cloning, expression, purification, and characterization of the acid alpha-mannosidase from Trypanosoma cruzi.
The acid alpha-mannosidase of Trypanosoma cruzi is a broad-specificity hydrolase involved in the catabolism of glycoconjugates, presumably in the digestive vacuole. We have cloned the alpha-mannosidase gene from a T.cruzi epimastigote genomic library. The alpha-mannosidase gene was determined to be single copy by Southern analysis, and similar sequences were not detected in genomic digests of either Trypanosoma brucei or Leishmania donovani. The coding region was subcloned into the Pichia pastoris expression vector pPICZ, and alpha-mannosidase activity was detected in the medium of induced cultures. The recombinant alpha-mannosidase demonstrated a pH optimum, inhibition by swainsonine, Km, and substrate specificity consistent with the characteristics of the alpha-mannosidase previously purified from T.cruzi epimastigotes. The recombinant enzyme was purified 103-fold from the culture medium of Pichia pastoris and had a native molecular mass of 359 kDa by gel filtration. A combination of SDS-PAGE, deglycosylation with endo H, and NH2-terminal sequencing indicates that the enzyme is originally synthesized as a homodimeric polypeptide that is subsequently cleaved to form a heterotetramer composed of 57 and 46 kDa subunits. A polyclonal antibody raised to the recombinant enzyme was shown to immunoprecipitate the alpha-mannosidase from T.cruzi cell extracts and will be used in future immunolocalization studies.
Vandersall-Nairn
A S
AS
Complex Carbohydrate Research Center and the Department of Biochemistry and Molecular Biology, University of Georgia, Athens, GA 30602, USA.
Merkle
R K
RK
O'Brien
K
K
Oeltmann
T N
TN
Moremen
K W
KW
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002240:Carbohydrate Sequence; D003001:Cloning, Molecular; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008361:Mannosidases / Q000235:genetics* / Q000378:metabolism; D008969:Molecular Sequence Data; D009844:Oligosaccharides / Q000378:metabolism; D011499:Protein Processing, Post-Translational / Q000502:physiology; D012333:RNA, Messenger / Q000235:genetics; D011994:Recombinant Proteins / Q000235:genetics; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity; D017026:Swainsonine / Q000494:pharmacology; D014349:Trypanosoma cruzi / Q000201:enzymology*; D043323:alpha-Mannosidase
D004791:Enzyme Inhibitors; D009844:Oligosaccharides; D012333:RNA, Messenger; D011994:Recombinant Proteins; D008361:Mannosidases; D043323:alpha-Mannosidase; D017026:Swainsonine