Cathepsin B of Schistosoma mansoni. Purification and activation of the recombinant proenzyme secreted by Saccharomyces cerevisiae.
Procathepsin B from the parasitic trematode Schistosoma mansoni was expressed as a glycosylation-minus mutant in yeast cells and purified by means of a histidine affinity tag which was added to the carboxyl terminus of the recombinant protein. The purified zymogen underwent autoprocessing but required an assisting protease for activation. Pepsin-activated schistosomal cathepsin B was further characterized with the cathepsin B-specific substrates N-benzyloxycarbonyl (Z)-Arg-Arg-p-nitroanilide, Z-Arg-Arg-7-amido-4-methyl-coumarin, and Z-Phe-Arg-7-amido-4-methylcoumarin. A proteolytic activity comparable to mammalian cathepsin B was observed. In addition, we analyzed the degradation of human hemoglobin by schistosomal cathepsin B, which has been suggested to be the physiological target of the protease.
Lipps
G
G
Biochemisches Institut am Klinikum, Justus-Liebig Universität Gissen, Federal Republic of Germany.
Füllkrug
R
R
Beck
E
E
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002401:Cathepsin B / Q000096:biosynthesis / Q000302:isolation & purification* / Q000378:metabolism*; D003001:Cloning, Molecular; D017931:DNA Primers; D004789:Enzyme Activation; D004792:Enzyme Precursors / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism*; D006454:Hemoglobins / Q000378:metabolism*; D006801:Humans; D007700:Kinetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010434:Pepsin A / Q000378:metabolism; D010957:Plasmids; D011480:Protease Inhibitors / Q000494:pharmacology; D011499:Protein Processing, Post-Translational; D051381:Rats; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism; D012441:Saccharomyces cerevisiae; D012550:Schistosoma mansoni / Q000201:enzymology*; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity
D017931:DNA Primers; D004792:Enzyme Precursors; D006454:Hemoglobins; D011480:Protease Inhibitors; D011994:Recombinant Proteins; C057832:procathepsin B; D002401:Cathepsin B; D010434:Pepsin A