Abstract
Erythrocytic malaria parasites degrade hemoglobin as a principal source of amino acids for parasite protein synthesis. We have previously shown that a Plasmodium falciparum trophozoite cysteine proteinase, now termed falcipain, is required for hemoglobin degradation, and we have hypothesized that this proteinase is responsible for initial cleavages of hemoglobin. To further evaluate the biological role of falcipain, we expressed the enzyme in bacterial and viral expression systems. After expression in the baculovirus system, falcipain was enzymatically active and had biochemical properties very similar to those of the native proteinase. Recombinant falcipain rapidly hydrolyzed both denatured and native hemoglobin. Hemoglobin hydrolysis was blocked by cysteine proteinase inhibitors but not by inhibitors of other classes of proteinases. Our results support our hypothesis that falcipain is a critical malarial hemoglobinase that is responsible for both initial cleavages of hemoglobin and the subsequent hydrolysis of globin into small peptides.
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- Bailly E., Jambou R., Savel J., Jaureguiberry G. Plasmodium falciparum: differential sensitivity in vitro to E-64 (cysteine protease inhibitor) and Pepstatin A (aspartyl protease inhibitor). J Protozool. 1992 Sep-Oct;39(5):593–599. doi: 10.1111/j.1550-7408.1992.tb04856.x. [DOI] [PubMed] [Google Scholar]
- Barrett A. J., Kirschke H. Cathepsin B, Cathepsin H, and cathepsin L. Methods Enzymol. 1981;80(Pt 100):535–561. doi: 10.1016/s0076-6879(81)80043-2. [DOI] [PubMed] [Google Scholar]
- Dame J. B., Reddy G. R., Yowell C. A., Dunn B. M., Kay J., Berry C. Sequence, expression and modeled structure of an aspartic proteinase from the human malaria parasite Plasmodium falciparum. Mol Biochem Parasitol. 1994 Apr;64(2):177–190. doi: 10.1016/0166-6851(94)90024-8. [DOI] [PubMed] [Google Scholar]
- Dluzewski A. R., Rangachari K., Wilson R. J., Gratzer W. B. Plasmodium falciparum: protease inhibitors and inhibition of erythrocyte invasion. Exp Parasitol. 1986 Dec;62(3):416–422. doi: 10.1016/0014-4894(86)90050-0. [DOI] [PubMed] [Google Scholar]
- Eakin A. E., Mills A. A., Harth G., McKerrow J. H., Craik C. S. The sequence, organization, and expression of the major cysteine protease (cruzain) from Trypanosoma cruzi. J Biol Chem. 1992 Apr 15;267(11):7411–7420. [PubMed] [Google Scholar]
- Francis S. E., Gluzman I. Y., Oksman A., Knickerbocker A., Mueller R., Bryant M. L., Sherman D. R., Russell D. G., Goldberg D. E. Molecular characterization and inhibition of a Plasmodium falciparum aspartic hemoglobinase. EMBO J. 1994 Jan 15;13(2):306–317. doi: 10.1002/j.1460-2075.1994.tb06263.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
- GROMAN N. B. Dynamic aspects of the nitrogen metabolism of Plasmodium gallinaceum in vivo and in vitro. J Infect Dis. 1951 Mar-Apr;88(2):126–150. doi: 10.1093/infdis/88.2.126. [DOI] [PubMed] [Google Scholar]
- Gluzman I. Y., Francis S. E., Oksman A., Smith C. E., Duffin K. L., Goldberg D. E. Order and specificity of the Plasmodium falciparum hemoglobin degradation pathway. J Clin Invest. 1994 Apr;93(4):1602–1608. doi: 10.1172/JCI117140. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Goldberg D. E., Slater A. F., Beavis R., Chait B., Cerami A., Henderson G. B. Hemoglobin degradation in the human malaria pathogen Plasmodium falciparum: a catabolic pathway initiated by a specific aspartic protease. J Exp Med. 1991 Apr 1;173(4):961–969. doi: 10.1084/jem.173.4.961. [DOI] [PMC free article] [PubMed] [Google Scholar]
- McKerrow J. H., Sun E., Rosenthal P. J., Bouvier J. The proteases and pathogenicity of parasitic protozoa. Annu Rev Microbiol. 1993;47:821–853. doi: 10.1146/annurev.mi.47.100193.004133. [DOI] [PubMed] [Google Scholar]
- Meister A., Anderson M. E. Glutathione. Annu Rev Biochem. 1983;52:711–760. doi: 10.1146/annurev.bi.52.070183.003431. [DOI] [PubMed] [Google Scholar]
- Raventos-Suarez C., Pollack S., Nagel R. L. Plasmodium falciparum: inhibition of in vitro growth by desferrioxamine. Am J Trop Med Hyg. 1982 Sep;31(5):919–922. doi: 10.4269/ajtmh.1982.31.919. [DOI] [PubMed] [Google Scholar]
- Rawlings N. D., Pearl L. H., Buttle D. J. The baculovirus Autographa californica nuclear polyhedrosis virus genome includes a papain-like sequence. Biol Chem Hoppe Seyler. 1992 Dec;373(12):1211–1215. doi: 10.1515/bchm3.1992.373.2.1211. [DOI] [PubMed] [Google Scholar]
- Riggs A. Preparation of blood hemoglobins of vertebrates. Methods Enzymol. 1981;76:5–29. doi: 10.1016/0076-6879(81)76111-1. [DOI] [PubMed] [Google Scholar]
- Rosenthal P. J., Lee G. K., Smith R. E. Inhibition of a Plasmodium vinckei cysteine proteinase cures murine malaria. J Clin Invest. 1993 Mar;91(3):1052–1056. doi: 10.1172/JCI116262. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Rosenthal P. J., McKerrow J. H., Aikawa M., Nagasawa H., Leech J. H. A malarial cysteine proteinase is necessary for hemoglobin degradation by Plasmodium falciparum. J Clin Invest. 1988 Nov;82(5):1560–1566. doi: 10.1172/JCI113766. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Rosenthal P. J., McKerrow J. H., Rasnick D., Leech J. H. Plasmodium falciparum: inhibitors of lysosomal cysteine proteinases inhibit a trophozoite proteinase and block parasite development. Mol Biochem Parasitol. 1989 Jun 15;35(2):177–183. doi: 10.1016/0166-6851(89)90120-5. [DOI] [PubMed] [Google Scholar]
- Rosenthal P. J., Nelson R. G. Isolation and characterization of a cysteine proteinase gene of Plasmodium falciparum. Mol Biochem Parasitol. 1992 Mar;51(1):143–152. doi: 10.1016/0166-6851(92)90209-3. [DOI] [PubMed] [Google Scholar]
- Rosenthal P. J. Plasmodium falciparum: effects of proteinase inhibitors on globin hydrolysis by cultured malaria parasites. Exp Parasitol. 1995 Mar;80(2):272–281. doi: 10.1006/expr.1995.1033. [DOI] [PubMed] [Google Scholar]
- Rosenthal P. J., Wollish W. S., Palmer J. T., Rasnick D. Antimalarial effects of peptide inhibitors of a Plasmodium falciparum cysteine proteinase. J Clin Invest. 1991 Nov;88(5):1467–1472. doi: 10.1172/JCI115456. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Roth E. F., Jr, Brotman D. S., Vanderberg J. P., Schulman S. Malarial pigment-dependent error in the estimation of hemoglobin content in Plasmodium falciparum-infected red cells: implications for metabolic and biochemical studies of the erythrocytic phases of malaria. Am J Trop Med Hyg. 1986 Sep;35(5):906–911. doi: 10.4269/ajtmh.1986.35.906. [DOI] [PubMed] [Google Scholar]
- Silen J. L., Frank D., Fujishige A., Bone R., Agard D. A. Analysis of prepro-alpha-lytic protease expression in Escherichia coli reveals that the pro region is required for activity. J Bacteriol. 1989 Mar;171(3):1320–1325. doi: 10.1128/jb.171.3.1320-1325.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Slater A. F., Cerami A. Inhibition by chloroquine of a novel haem polymerase enzyme activity in malaria trophozoites. Nature. 1992 Jan 9;355(6356):167–169. doi: 10.1038/355167a0. [DOI] [PubMed] [Google Scholar]
- Smith S. M., Gottesman M. M. Activity and deletion analysis of recombinant human cathepsin L expressed in Escherichia coli. J Biol Chem. 1989 Dec 5;264(34):20487–20495. [PubMed] [Google Scholar]
- Vander Jagt D. L., Baack B. R., Hunsaker L. A. Purification and characterization of an aminopeptidase from Plasmodium falciparum. Mol Biochem Parasitol. 1984 Jan;10(1):45–54. doi: 10.1016/0166-6851(84)90017-3. [DOI] [PubMed] [Google Scholar]
- Vander Jagt D. L., Caughey W. S., Campos N. M., Hunsaker L. A., Zanner M. A. Parasite proteases and antimalarial activities of protease inhibitors. Prog Clin Biol Res. 1989;313:105–118. [PubMed] [Google Scholar]
- Vernet T., Tessier D. C., Richardson C., Laliberté F., Khouri H. E., Bell A. W., Storer A. C., Thomas D. Y. Secretion of functional papain precursor from insect cells. Requirement for N-glycosylation of the pro-region. J Biol Chem. 1990 Sep 25;265(27):16661–16666. [PubMed] [Google Scholar]
- Walsh J. A. Disease problems in the Third World. Ann N Y Acad Sci. 1989;569:1–16. doi: 10.1111/j.1749-6632.1989.tb27354.x. [DOI] [PubMed] [Google Scholar]
- Zhu X. L., Ohta Y., Jordan F., Inouye M. Pro-sequence of subtilisin can guide the refolding of denatured subtilisin in an intermolecular process. Nature. 1989 Jun 8;339(6224):483–484. doi: 10.1038/339483a0. [DOI] [PubMed] [Google Scholar]
- vander Jagt D. L., Hunsaker L. A., Campos N. M., Scaletti J. V. Localization and characterization of hemoglobin-degrading aspartic proteinases from the malarial parasite Plasmodium falciparum. Biochim Biophys Acta. 1992 Aug 21;1122(3):256–264. doi: 10.1016/0167-4838(92)90401-x. [DOI] [PubMed] [Google Scholar]