The sequence, organization, and expression of the major cysteine protease (cruzain) from Trypanosoma cruzi.
The complete sequence of the gene encoding the major cysteine protease from Trypanosoma cruzi is reported. The amino acid sequence predicted from the gene sequence aligns well with members of the papain family of cysteine proteases, suggesting the name cruzain. The sequence is most closely related to the cysteine protease of Trypanosoma brucei (59.3%) and the murine cathepsin L (42.2%). At least six copies of the gene are present in the genome and are organized in a tandem array of copies which are identical in all restriction endonuclease sites tested. The gene appears to be expressed in all developmental stages of T. cruzi with mRNA levels approximately 2-fold higher in the intracellular amastigote form. A copy of the T. cruzi gene was expressed in bacteria as an inactive, insoluble fusion polypeptide to approximately 5% of the total cell protein. The fusion protein was readily purified, solubilized in urea, and successfully refolded to produce a polyprotein which processed autocatalytically to yield approximately 1 mg of active protease per 3 g of wet cell paste. The processed form of the recombinant protease has an NH2-terminal sequence identical to that of the mature form of the protease purified from T. cruzi (Murta, A. C. M., Persechini, P. M., Souto-Padrón, T., de Souza, W., Guimaraes, J. A., and Scharfstein, J. (1990) Mol. Biochem. Parasitol. 43, 27-38; Cazzulo, J. J., Couso, R., Raimondi, A., Wernstedt, C., and Hellman, U. (1989) Mol. Biochem. Parasitol. 33, 33-42). This suggests that the recombinant protease possesses the requisite specificity and activity to correctly process the proform of the protease in vitro. Kinetic assays with peptide substrates demonstrate that the substrate specificity and kinetic parameters for the recombinant protease are consistent with those of the endogenous protease. The proteolytic activity of the recombinant protease is enhanced by dithiothreitol, inhibited by leupeptin, N alpha-p-tosyl-L-lysine chloromethyl ketone and trans-epoxysuccinyl-L-leucylamido(4-guanidino)butane (E-64) but is unaffected by phenylmethylsulfonyl fluoride, pepstatin, and 1,10-phenanthroline. More specifically, the recombinant enzyme was inhibited by benzyloxycarbonyl-Phe-Arg-fluoromethyl ketone, which inhibits replication and differentiation of T. cruzi within mammalian cells in culture.
Eakin
A E
AE
Department of Pharmaceutical Chemistry, University of California, San Francisco 94143.
Mills
A A
AA
Harth
G
G
McKerrow
J H
JH
Craik
C S
CS
D000595:Amino Acid Sequence; D000818:Animals; D001426:Bacterial Proteins; D001483:Base Sequence; D015152:Blotting, Northern; D015139:Blotting, Southern; D003546:Cysteine Endopeptidases / Q000235:genetics* / Q000378:metabolism; D016054:DNA, Protozoan / Q000235:genetics; D004591:Electrophoresis, Polyacrylamide Gel; D006868:Hydrolysis; D007700:Kinetics; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism; D000072236:Methyl-Accepting Chemotaxis Proteins; D008969:Molecular Sequence Data; D010957:Plasmids; D015800:Protozoan Proteins / Q000235:genetics* / Q000378:metabolism; D016053:RNA, Protozoan / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D016415:Sequence Alignment; D014349:Trypanosoma cruzi / Q000201:enzymology* / Q000235:genetics
D001426:Bacterial Proteins; D016054:DNA, Protozoan; D008565:Membrane Proteins; D000072236:Methyl-Accepting Chemotaxis Proteins; D015800:Protozoan Proteins; D016053:RNA, Protozoan; D011993:Recombinant Fusion Proteins; D003546:Cysteine Endopeptidases; C074032:cruzain, Trypanosoma cruzi