Eakin, A. E., Mills, A. A., Harth, G., McKerrow, J. H., Craik, C. S. (1992) The sequence, organization, and expression of the major cysteine protease (cruzain) from Trypanosoma cruzi. Journal of Biological Chemistry, 267 (11). pp. 7411-7420. ISSN 00219258 (ISSN)
Abstract
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-Padron, 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(α)-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.
| Item Type: | Paper |
|---|---|
| Uncontrolled Keywords: | 1,10 phenanthroline benzyloxycarbonylphenylalanylalanyl fluoromethyl ketone benzylsulfonyl fluoride cathepsin l cysteine proteinase hybrid protein leupeptin messenger rna n [n (3 carboxyoxirane 2 carbonyl)leucyl]agmatine papain pepstatin restriction endonuclease tosyllysyl chloromethyl ketone amastigote amino acid sequence article enzyme activity enzyme induction enzyme kinetics enzyme specificity enzyme structure gene expression microbial genetics nonhuman priority journal sequence homology trypanosoma cruzi Animal Bacterial Proteins Base Sequence Blotting, Northern Blotting, Southern Cysteine Endopeptidases DNA, Protozoan Electrophoresis, Polyacrylamide Gel Hydrolysis Kinetics Membrane Proteins Molecular Sequence Data Plasmids Protozoan Proteins Recombinant Fusion Proteins RNA, Protozoan Sequence Alignment Support, Non-U.S. Gov't Support, U.S. Gov't, Non-P.H.S. Support, U.S. Gov't, P.H.S. Mammalia Mouriri domingensis Murinae Trypanosoma Trypanosoma brucei |
| Subjects: | bioinformatics > genomics and proteomics > genetics & nucleic acid processing > DNA, RNA structure, function, modification diseases & disorders bioinformatics > genomics and proteomics > genetics & nucleic acid processing bioinformatics > genomics and proteomics diseases & disorders > parasitic diseases bioinformatics > genomics and proteomics > genetics & nucleic acid processing > DNA, RNA structure, function, modification > genes, structure and function bioinformatics > genomics and proteomics > genetics & nucleic acid processing > genomes |
| CSHL Authors: | |
| Communities: | CSHL labs > Mills lab |
| Depositing User: | Matt Covey |
| Date: | 1992 |
| Date Deposited: | 11 Mar 2013 20:37 |
| Last Modified: | 11 Mar 2013 20:37 |
| Related URLs: | |
| URI: | https://repository.cshl.edu/id/eprint/27764 |
Actions (login required)
 |
Administrator's edit/view item |