Expression and partial characterization of a cathepsin B-like enzyme (Sm31) and a proposed 'haemoglobinase' (Sm32) from Schistosoma mansoni.
Schistosoma mansoni protein Sm31 is a cysteine proteinase similar to mammalian lysosomal cathepsin B, proposed to be a key enzyme in schistosome metabolism. Protein Sm32 has been identified as a putative cysteine proteinase termed a 'haemoglobinase'. Since neither Sm31 nor Sm32 have been completely purified, some controversy of the nature of the 'true' digestive enzyme still exists. By incubating a radiolabelled cysteine-proteinase active-site-directed synthetic inhibitor with total S. mansoni proteins, the target of inhibition was Sm31 and not Sm32. The selectivity and irreversibility of inactivation make affinity labelling an invaluable tool for exploring key differences among closely related enzymes and also for studying proteinase activity in a cellular environment. In order to confirm these results, we expressed the complete cDNA sequences of Sm31 and Sm32 in insect cells and analysed the recombinant gene products for proteolytic activities. Cell extracts containing S. mansoni cathepsin B, but not those expressing 'haemoglobinase', were demonstrated to cleave a synthetic substrate benzyloxycarbonyl-arginylarginylaminomethylcoumarin in fluorescence assays. Our findings confirm previous assertions that a cysteine proteinase resembling cathepsin B is the haemoglobinase involved in digestion of host proteins. Thus, the original proposal that Sm32 is a cysteine proteinase has not been verified, and its function remains unknown.
Götz
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Institute of Cell Biology, Consiglio Nazionale delle Ricerche, Rome, Italy.
Klinkert
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D000595:Amino Acid Sequence; D000818:Animals; D000947:Antigens, Helminth; D016367:Baculoviridae / Q000235:genetics; D001665:Binding Sites; D002401:Cathepsin B / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D002460:Cell Line; D003001:Cloning, Molecular; D003374:Coumarins / Q000378:metabolism; D003546:Cysteine Endopeptidases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D004151:Dipeptides / Q000378:metabolism; D015870:Gene Expression; D015801:Helminth Proteins / Q000235:genetics* / Q000378:metabolism; D006454:Hemoglobins / Q000378:metabolism; D006868:Hydrolysis; D015151:Immunoblotting; D008969:Molecular Sequence Data; D009036:Moths / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D012550:Schistosoma mansoni / Q000201:enzymology*; D013379:Substrate Specificity; D014162:Transfection
D000947:Antigens, Helminth; D003374:Coumarins; D004151:Dipeptides; D015801:Helminth Proteins; D006454:Hemoglobins; D011994:Recombinant Proteins; C069012:benzyloxycarbonylarginyl-arginine 4-methylcoumarin-7-ylamide; D003546:Cysteine Endopeptidases; C059747:Sm31 protein, Schistosoma mansoni; D002401:Cathepsin B