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Biochem J. Jul 1, 1997; 325(Pt 1): 129–137.
PMCID: PMC1218537

A Trypanosoma cruzi-secreted 80 kDa proteinase with specificity for human collagen types I and IV.

Abstract

Specific interactions between parasites and extracellular matrix components are an important mechanism in the dissemination of Chagas' disease. Binding of the extracellular matrix proteins to Trypanosoma cruzi receptors has been described as a significant step in this phenomenon. In this study, a specific proteinase activity was identified in cell-free extracts of amastigote, trypomastigote and epimastigote forms of T. cruzi using the collagenase fluorogenic substrate N-Suc-Gly-Pro-Leu-Gly-Pro-7-amido-4-methylcoumarin. Isolation of this activity was achieved by a four-step FPLC procedure. Optimal enzyme activity was found to occur at pH 8.0 and was associated with a single T. cruzi 80 kDa protein (Tc 80 proteinase) on SDS/PAGE under reducing conditions. An internal peptide sequence of Tc 80 proteinase was obtained (AGDNYTPPE), and no similarity was found to previously described proteinases of T. cruzi. This enzyme activity is strongly inhibited by HgCl2, tosyl-lysylchloromethane ('TLCK') p-chloromercuribenzoate and benzyloxycarbonyl-Phe-Ala-diazomethane. The purified enzyme was able to hydrolyse purified human [14C]collagen types I and IV at neutral pH, but not 14C-labelled BSA, rat laminin, rabbit IgG or small proteins such as insulin or cytochrome c. In addition, Tc 80 proteinase activity was found to be secreted by T. cruzi forms infective to mammalian cells. Furthermore we demonstrated that purified Tc 80 proteinase mediates native collagen type I hydrolysis in rat mesentery. This feature is compared with that of Clostridium histolyticum collagenase. These findings suggest that Tc 80 proteinase may facilitate T. cruzi host-cell infection by degrading the collagens of the extracellular matrix and could represent a good target for Chagas' disease chemotherapy.

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Selected References

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  • Schenkman S, Robbins ES, Nussenzweig V. Attachment of Trypanosoma cruzi to mammalian cells requires parasite energy, and invasion can be independent of the target cell cytoskeleton. Infect Immun. 1991 Feb;59(2):645–654. [PMC free article] [PubMed]
  • Tardieux I, Webster P, Ravesloot J, Boron W, Lunn JA, Heuser JE, Andrews NW. Lysosome recruitment and fusion are early events required for trypanosome invasion of mammalian cells. Cell. 1992 Dec 24;71(7):1117–1130. [PubMed]
  • Andrews NW, Abrams CK, Slatin SL, Griffiths G. A T. cruzi-secreted protein immunologically related to the complement component C9: evidence for membrane pore-forming activity at low pH. Cell. 1990 Jun 29;61(7):1277–1287. [PubMed]
  • Hall BF, Webster P, Ma AK, Joiner KA, Andrews NW. Desialylation of lysosomal membrane glycoproteins by Trypanosoma cruzi: a role for the surface neuraminidase in facilitating parasite entry into the host cell cytoplasm. J Exp Med. 1992 Aug 1;176(2):313–325. [PMC free article] [PubMed]
  • Liotta LA, Rao CN, Barsky SH. Tumor invasion and the extracellular matrix. Lab Invest. 1983 Dec;49(6):636–649. [PubMed]
  • Velge P, Ouaissi MA, Cornette J, Afchain D, Capron A. Identification and isolation of Trypanosoma cruzi trypomastigote collagen-binding proteins: possible role in cell-parasite interaction. Parasitology. 1988 Oct;97(Pt 2):255–268. [PubMed]
  • Ouaissi MA, Afchain D, Capron A, Grimaud JA. Fibronectin receptors on Trypanosoma cruzi trypomastigotes and their biological function. Nature. 1984 Mar 22;308(5957):380–382. [PubMed]
  • Ouaissi MA, Cornette J, Afchain D, Capron A, Gras-Masse H, Tartar A. Trypanosoma cruzi infection inhibited by peptides modeled from a fibronectin cell attachment domain. Science. 1986 Oct 31;234(4776):603–607. [PubMed]
  • Giordano R, Chammas R, Veiga SS, Colli W, Alves MJ. An acidic component of the heterogeneous Tc-85 protein family from the surface of Trypanosoma cruzi is a laminin binding glycoprotein. Mol Biochem Parasitol. 1994 May;65(1):85–94. [PubMed]
  • Ortega-Barria E, Pereira ME. A novel T. cruzi heparin-binding protein promotes fibroblast adhesion and penetration of engineered bacteria and trypanosomes into mammalian cells. Cell. 1991 Oct 18;67(2):411–421. [PubMed]
  • Avila JL, Bretaña A, Casanova MA, Avila A, Rodríguez F. Trypanosoma cruzi: defined medium for continuous cultivation of virulent parasites. Exp Parasitol. 1979 Aug;48(1):27–35. [PubMed]
  • Bonaldo MC, d'Escoffier LN, Salles JM, Goldenberg S. Characterization and expression of proteases during Trypanosoma cruzi metacyclogenesis. Exp Parasitol. 1991 Jul;73(1):44–51. [PubMed]
  • Piras MM, Henriquez D, Piras R. The effect of proteolytic enzymes and protease inhibitors on the interaction Trypanosoma cruzi-fibroblasts. Mol Biochem Parasitol. 1985 Feb;14(2):151–163. [PubMed]
  • Santana JM, Grellier P, Rodier MH, Schrevel J, Teixeira A. Purification and characterization of a new 120 kDa alkaline proteinase of Trypanosoma cruzi. Biochem Biophys Res Commun. 1992 Sep 30;187(3):1466–1473. [PubMed]
  • Burleigh BA, Andrews NW. A 120-kDa alkaline peptidase from Trypanosoma cruzi is involved in the generation of a novel Ca(2+)-signaling factor for mammalian cells. J Biol Chem. 1995 Mar 10;270(10):5172–5180. [PubMed]
  • Teixeira AR, Córdoba JC, Souto Maior I, Solórzano E. Chagas' disease: lymphoma growth in rabbits treated with Benznidazole. Am J Trop Med Hyg. 1990 Aug;43(2):146–158. [PubMed]
  • Cicarelli RM, Lopes JD. Characterization of a protein from Trypanosoma cruzi trypomastigotes that cleaves non-immune IgG bound through its Fab fragment. J Immunol. 1989 Mar 1;142(5):1685–1690. [PubMed]
  • Bontempi E, Cazzulo JJ. Digestion of human immunoglobulin G by the major cysteine proteinase (cruzipain) from Trypanosoma cruzi. FEMS Microbiol Lett. 1990 Aug;58(3):337–341. [PubMed]
  • Eakin AE, Mills AA, Harth G, McKerrow JH, Craik CS. 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]
  • Meirelles MN, Juliano L, Carmona E, Silva SG, Costa EM, Murta AC, Scharfstein J. Inhibitors of the major cysteinyl proteinase (GP57/51) impair host cell invasion and arrest the intracellular development of Trypanosoma cruzi in vitro. Mol Biochem Parasitol. 1992 Jun;52(2):175–184. [PubMed]
  • Harth G, Andrews N, Mills AA, Engel JC, Smith R, McKerrow JH. Peptide-fluoromethyl ketones arrest intracellular replication and intercellular transmission of Trypanosoma cruzi. Mol Biochem Parasitol. 1993 Mar;58(1):17–24. [PubMed]
  • Ley V, Andrews NW, Robbins ES, Nussenzweig V. Amastigotes of Trypanosoma cruzi sustain an infective cycle in mammalian cells. J Exp Med. 1988 Aug 1;168(2):649–659. [PMC free article] [PubMed]
  • CAMARGO EP. GROWTH AND DIFFERENTIATION IN TRYPANOSOMA CRUZI. I. ORIGIN OF METACYCLIC TRYPANOSOMES IN LIQUID MEDIA. Rev Inst Med Trop Sao Paulo. 1964 May-Jun;6:93–100. [PubMed]
  • Bradford MM. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem. 1976 May 7;72:248–254. [PubMed]
  • Kojima K, Kinoshita H, Kato T, Nagatsu T, Takada K, Sakakibara S. A new and highly sensitive fluorescence assay for collagenase-like peptidase activity. Anal Biochem. 1979 Nov 15;100(1):43–50. [PubMed]
  • Laemmli UK. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. [PubMed]
  • Giulian GG, Moss RL, Greaser M. Improved methodology for analysis and quantitation of proteins on one-dimensional silver-stained slab gels. Anal Biochem. 1983 Mar;129(2):277–287. [PubMed]
  • Bonner WM. Fluorography for the detection of radioactivity in gels. Methods Enzymol. 1984;104:460–465. [PubMed]
  • Dottavio-Martin D, Ravel JM. Radiolabeling of proteins by reductive alkylation with [14C]formaldehyde and sodium cyanoborohydride. Anal Biochem. 1978 Jul 1;87(2):562–565. [PubMed]
  • Tiollier J, Dumas H, Tardy M, Tayot JL. Fibroblast behavior on gels of type I, III, and IV human placental collagens. Exp Cell Res. 1990 Nov;191(1):95–104. [PubMed]
  • Schägger H, von Jagow G. Tricine-sodium dodecyl sulfate-polyacrylamide gel electrophoresis for the separation of proteins in the range from 1 to 100 kDa. Anal Biochem. 1987 Nov 1;166(2):368–379. [PubMed]
  • WILKINSON GN. Statistical estimations in enzyme kinetics. Biochem J. 1961 Aug;80:324–332. [PMC free article] [PubMed]
  • Inverso JA, Medina-Acosta E, O'Connor J, Russell DG, Cross GA. Crithidia fasciculata contains a transcribed leishmanial surface proteinase (gp63) gene homologue. Mol Biochem Parasitol. 1993 Jan;57(1):47–54. [PubMed]
  • Cazzulo JJ, Cazzulo Franke MC, Martínez J, Franke de Cazzulo BM. Some kinetic properties of a cysteine proteinase (cruzipain) from Trypanosoma cruzi. Biochim Biophys Acta. 1990 Feb 9;1037(2):186–191. [PubMed]
  • Schrével J, Deguercy A, Mayer R, Monsigny M. Proteases in malaria-infected red blood cells. Blood Cells. 1990;16(2-3):563–590. [PubMed]
  • Ashall F. Characterisation of an alkaline peptidase of Trypanosoma cruzi and other trypanosomatids. Mol Biochem Parasitol. 1990 Jan 1;38(1):77–87. [PubMed]
  • Healy N, Greig S, Enahoro H, Roberts H, Drake L, Shaw E, Ashall F. Detection of peptidases in Trypanosoma cruzi epimastigotes using chromogenic and fluorogenic substrates. Parasitology. 1992 Apr;104(Pt 2):315–322. [PubMed]
  • Green GD, Shaw E. Peptidyl diazomethyl ketones are specific inactivators of thiol proteinases. J Biol Chem. 1981 Feb 25;256(4):1923–1928. [PubMed]
  • Kirschke H, Wikstrom P, Shaw E. Active center differences between cathepsins L and B: the S1 binding region. FEBS Lett. 1988 Feb 8;228(1):128–130. [PubMed]
  • Crawford C, Mason RW, Wikstrom P, Shaw E. The design of peptidyldiazomethane inhibitors to distinguish between the cysteine proteinases calpain II, cathepsin L and cathepsin B. Biochem J. 1988 Aug 1;253(3):751–758. [PMC free article] [PubMed]
  • Lonsdale-Eccles JD, Grab DJ. Lysosomal and non-lysosomal peptidyl hydrolases of the bloodstream forms of Trypanosoma brucei brucei. Eur J Biochem. 1987 Dec 15;169(3):467–475. [PubMed]
  • Bornstein P, Sage H. Structurally distinct collagen types. Annu Rev Biochem. 1980;49:957–1003. [PubMed]
  • French MF, Mookhtiar KA, Van Wart HE. Limited proteolysis of type I collagen at hyperreactive sites by class I and II Clostridium histolyticum collagenases: complementary digestion patterns. Biochemistry. 1987 Feb 10;26(3):681–687. [PubMed]
  • Bond MD, Van Wart HE. Characterization of the individual collagenases from Clostridium histolyticum. Biochemistry. 1984 Jun 19;23(13):3085–3091. [PubMed]
  • Lopes JD, dos Reis M, Brentani RR. Presence of laminin receptors in Staphylococcus aureus. Science. 1985 Jul 19;229(4710):275–277. [PubMed]
  • Wyler DJ, Sypek JP, McDonald JA. In vitro parasite-monocyte interactions in human leishmaniasis: possible role of fibronectin in parasite attachment. Infect Immun. 1985 Aug;49(2):305–311. [PMC free article] [PubMed]
  • Talamás-Rohana P, Meza I. Interaction between pathogenic amebas and fibronectin: substrate degradation and changes in cytoskeleton organization. J Cell Biol. 1988 May;106(5):1787–1794. [PMC free article] [PubMed]
  • Bouchara JP, Tronchin G, Annaix V, Robert R, Senet JM. Laminin receptors on Candida albicans germ tubes. Infect Immun. 1990 Jan;58(1):48–54. [PMC free article] [PubMed]
  • Vicentini AP, Gesztesi JL, Franco MF, de Souza W, de Moraes JZ, Travassos LR, Lopes JD. Binding of Paracoccidioides brasiliensis to laminin through surface glycoprotein gp43 leads to enhancement of fungal pathogenesis. Infect Immun. 1994 Apr;62(4):1465–1469. [PMC free article] [PubMed]
  • Liotta LA, Steeg PS, Stetler-Stevenson WG. Cancer metastasis and angiogenesis: an imbalance of positive and negative regulation. Cell. 1991 Jan 25;64(2):327–336. [PubMed]
  • Muńoz ML, Calderón J, Rojkind M. The collagenase of Entamoeba histolytica. J Exp Med. 1982 Jan 1;155(1):42–51. [PMC free article] [PubMed]
  • McKerrow JH, Brindley P, Brown M, Gam AA, Staunton C, Neva FA. Strongyloides stercoralis: identification of a protease that facilitates penetration of skin by the infective larvae. Exp Parasitol. 1990 Feb;70(2):134–143. [PubMed]
  • Hotez P, Haggerty J, Hawdon J, Milstone L, Gamble HR, Schad G, Richards F. Metalloproteases of infective Ancylostoma hookworm larvae and their possible functions in tissue invasion and ecdysis. Infect Immun. 1990 Dec;58(12):3883–3892. [PMC free article] [PubMed]
  • Kumar S, Pritchard DI. Secretion of metalloproteases by living infective larvae of Necator americanus. J Parasitol. 1992 Oct;78(5):917–919. [PubMed]
  • Martin GR, Timpl R. Laminin and other basement membrane components. Annu Rev Cell Biol. 1987;3:57–85. [PubMed]
  • Hudson L, Snary D, Morgan SJ. Trypanosoma cruzi: continuous cultivation with murine cell lines. Parasitology. 1984 Apr;88(Pt 2):283–294. [PubMed]
  • Andrews NW, Hong KS, Robbins ES, Nussenzweig V. Stage-specific surface antigens expressed during the morphogenesis of vertebrate forms of Trypanosoma cruzi. Exp Parasitol. 1987 Dec;64(3):474–484. [PubMed]

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