Skip to main content Accessibility help

Trypanosoma cruzi heparin-binding proteins present a flagellar membrane localization and serine proteinase activity

  • F. O. R. OLIVEIRA-JR (a1), C. R. ALVES (a2), F. S. SILVA (a2), L. M. C. CÔRTES (a2), L. TOMA (a3), R. I. BOUÇAS (a3), T. AGUILAR (a3), H. B. NADER (a3) and M. C. S. PEREIRA (a1)...


Heparin-binding proteins (HBPs) play a key role in Trypanosoma cruzi-host cell interactions. HBPs recognize heparan sulfate (HS) at the host cell surface and are able to induce the cytoadherence and invasion of this parasite. Herein, we analysed the biochemical properties of the HBPs and also evaluated the expression and subcellular localization of HBPs in T. cruzi trypomastigotes. A flow cytometry analysis revealed that HBPs are highly expressed at the surface of trypomastigotes, and their peculiar localization mainly at the flagellar membrane, which is known as an important signalling domain, may enhance their binding to HS and elicit the parasite invasion. The plasmon surface resonance results demonstrated the stability of HBPs and their affinity to HS and heparin. Additionally, gelatinolytic activities of 70 kDa, 65·8 kDa and 59 kDa HBPs over a broad pH range (5·5–8·0) were revealed using a zymography assay. These proteolytic activities were sensitive to serine proteinase inhibitors, such as aprotinin and phenylmethylsulfonyl fluoride, suggesting that HBPs have the properties of trypsin-like proteinases.


Corresponding author

*Corresponding author: Laboratório de Ultra-estrutura Celular, Instituto Oswaldo Cruz, FIOCRUZ, Av. Brasil 4365, Manguinhos, 21045-900 Rio de Janeiro, RJ, Brazil. Tel: +5521 2598 4330. Fax: +5521 2598 4330. E-mail:


Hide All
Alves, C. R., Marzochi, M. C. and Giovanni-de-Simone, S. (1993). Heterogeneity of cysteine proteinases in Leishmania braziliensis and Leishmania major. Brazilian Journal of Medical and Biological Research 26, 167171.
Antalis, T. M., Bugge, T. H. and Wu, Q. (2011). Membrane-anchored serine proteases in health and disease. Progress in Molecular Biology and Translational Science 99, 150.
Ashall, F. (1990). Characterisation of an alkaline peptidase of Trypanosoma cruzi and other trypanosomatids. Molecular and Biochemical Parasitology 38, 7787.
Bambino-Medeiros, R., Oliveira, F. O., Calvet, C. M., Vicente, D., Toma, L., Krieger, M. A., Meirelles, M. N. and Pereira, M. C. (2011). Involvement of host cell heparan sulfate proteoglycan in Trypanosoma cruzi amastigote attachment and invasion. Parasitology 138, 593601.
Bastos, I. M., Grellier, P., Martins, N. F., Cadavid-Restrepo, G., de Souza-Ault, M. R., Augustyns, K., Teixeira, A. R., Schrével, J., Maigret, B., da Silveira, J. F. and Santana, J. M. (2005). Molecular, functional and structural properties of the prolyl oligopeptidase of Trypanosoma cruzi (POP Tc80), which is required for parasite entry into mammalian cells. The Biochemical Journal 388, 2938
Bastos, I. M., Motta, F. N., Charneau, S., Santana, J. M., Dubost, L., Augustyns, K. and Grellier, P. (2010). Prolyl oligopeptidase of Trypanosoma brucei hydrolyzes native collagen, peptide hormones and is active in the plasma of infected mice. Microbes and Infection 12, 457466.
Bates, P. A. (2008). Leishmania sand fly interaction: progress and challenges. Current Opinion in Microbiology 11, 340344.
Bates, P. A. and Rogers, M. E. (2004). New insights into the developmental biology and transmission mechanisms of Leishmania. Current Molecular Medicine 4, 601609.
Bongertz, V. and Hungerer, K. D. (1978). Trypanosoma cruzi: isolation and characterization of a protease. Experimental Parasitology 45, 818.
Bosetto, M. C. and Giorgio, S. (2007). Leishmania amazonensis: multiple receptor-ligand interactions are involved in amastigote infection of human dendritic cells. Experimental Parasitology 116, 306310
Bouças, R. I., Trindade, E. S., Tersariol, I. L., Dietrich, C. P. and Nader, H. B. (2008). Development of an enzyme-linked immunosorbent assay (ELISA)-like fluorescence assay to investigate the interactions of glycosaminoglycans to cells. Analytica Chimica Acta 618, 218226.
Burleigh, B. A. and Andrews, N. W. (1998). Signaling and host cell invasion by Trypanosoma cruzi. Current Opinion in Microbiology 1, 461465.
Burleigh, B. A., Caler, E. V., Webster, P. and Andrews, N. W. (1997). A cytosolic serine endopeptidase from Trypanosoma cruzi is required for the generation of Ca2 + −signaling in mammalian cells. Journal of Cell Biology 136, 609620.
Buschiazzo, A., Muiá, R., Larrieux, N., Pitcovsky, T., Mucci, J. and Campetella, O. (2012). Trypanosoma cruzi trans-sialidase in complex with a neutralizing antibody: structure/function studies towards the rational design of inhibitors. PLoS Pathogens 8, e1002474.
Caler, E. V., Vaena de Avalos, S., Haynes, P. A., Andrews, N. W. and Burleigh, B. A. (1998). Oligopeptidase B-dependent signaling mediates host cell invasion by Trypanosoma cruzi. The EMBO Journal 17, 49754986.
Calvet, C. M., Toma, L., De Souza, F. R., Meirelles, Mde N. and Pereira, M. C. (2003). Heparan sulfate proteoglycans mediate the invasion of cardiomyocytes by Trypanosoma cruzi. Journal of Eukaryotic Microbiology 50, 97103.
Caradonna, K. L. and Burleigh, B. A. (2011). Mechanisms of host cell invasion by Trypanosoma cruzi. Advances in Parasitology 76, 3361.
Cazzulo, J. J. (2002). Proteinases of Trypanosoma cruzi: patential targets for the chemotherapy of Changas desease. Current Topics in Medicinal Chemistry 2, 12611271.
Cordero, E. M., Gentil, L. G., Crisante, G., Ramírez, J. L., Yoshida, N., Añez, N. and Franco da Silveira, J. (2008). Expression of GP82 and GP90 surface glycoprotein genes of Trypanosoma cruzi during in vivo metacyclogenesis in the insect vector Rhodnius prolixus. Acta Tropica 105, 8791.
Côrtes, L. M. C., Pereira, M. C. S., Oliveira-Jr, F. O., Corte-Real, S., da Silva, F. S., Pereira, B. A., Madeira, M. F., de Moraes, M. T., Brazil, R. P. and Alves, C. R. (2012). Leishmania (Viannia) braziliensis: insights on subcellular distribution and biochemical properties of heparin-binding proteins. Parasitology 139, 200207.
Cuevas, I. C., Cazzulo, J. J. and Sánchez, D. O. (2003). gp63 homologues in Trypanosoma cruzi: surface antigens with metalloprotease activity and a possible role in host cell infection. Infection and Immunity 71, 57395749.
De Souza, W., de Carvalho, T. M. and Barrias, E. S. (2010). Review on Trypanosoma cruzi: Host Cell Interaction. International Journal of Cell Biology 118.
Eugenia Giorgi, M. and de Lederkremer, R. M. (2011). Trans-sialidase and mucins of Trypanosoma cruzi: an important interplay for the parasite. Carbohydrate Research 346, 13891393.
Fampa, P., Santos, A. L. and Ramirez, M. I. (2010). Trypanosoma cruzi: ubiquity expression of surface cruzipain molecules in TCI and TCII field isolates. Parasitology Research 107, 443447.
Ferreira, D., Cortez, M., Atayde, V. D. and Yoshida, N. (2006). Actin cytoskeleton-dependent and -independent host cell invasion by Trypanosoma cruzi is mediated by distinct parasite surface molecules. Infection and Immunity 74, 55225528.
Ghosh, A. K. and Jacobs-Lorena, M. (2011). Surface-expressed enolases of Plasmodium and other pathogens. Memórias do Instituto Oswaldo Cruz 106, 8590.
Gonçalves, A. M., Nehme, N. S. and Morel, C. M. (1990). An improved silver staining procedure for schizodeme analysis in polyacrylamide gradient gels. Memórias do Instituto Oswaldo Cruz 85, 101106.
Grellier, P., Vendeville, S., Joyeau, R., Bastos, I. M., Drobecq, H., Frappier, F., Teixeira, A. R., Schrével, J., Davioud-Charvet, E., Sergheraert, C. and Santana, J. M. (2001). Trypanosoma cruzi prolyl oligopeptidase Tc80 is involved in nonphagocytic mammalian cell invasion by trypomastigotes. The Journal of Biological Chemistry 276, 47 07847 086.
Guedes, H. L., Rezende, J. M., Fonseca, M. A., Salles, C. M., Rossi-Bergmann, B. and De-Simone, S. G. (2007). Identification of serine proteases from Leishmania braziliensis. Zeitschrift für Naturforschung C 62, 373381.
Hedstrom, L. (2002). Serine protease mechanism and specificity. Chemical Reviews 102, 45014524.
Hemerly, J. P., Oliveira, V., Del Nery, E., Morty, R. E., Andrews, N. W., Juliano, M. A. and Juliano, L. (2003). Subsite specificity (S3, S2, S1′, S2′ and S3′) of oligopeptidase B from Trypanosoma cruzi and Trypanosoma brucei using fluorescent quenched peptides: comparative study and identification of specific carboxypeptidase activity. The Biochemical Journal 373, 933939.
Herrera, E. M., Ming, M., Ortega-Barria, E. and Pereira, M. E. (1994). Mediation of Trypanosoma cruzi invasion by heparan sulfate receptors on host cells and penetrin counter-receptors on the trypanosomes. Molecular and Biochemical Parasitology 65, 7383.
Heussen, C. and Dowdle, E. B. (1980). Electrophoretic analysis of plasminogen activators in polyacrylamide gels containing sodium dodecyl sulfate and copolymerized substrates. Analytical Biochemistry 102, 196202.
Kulkarni, M. M., Olson, C. H., Engman, D. M. and McGwire, B. S. (2009). Trypanosoma cruzi GP63 proteins undergo stage-specific differential posttranslational modification and are important for host cell invasion. Infection and Immunity 77, 21932200.
Kribs-Zaleta, C. M. (2010). Alternative transmission modes for Trypanosoma cruzi. Mathematical Biosciences and Engineering 7, 657673.
Laemmli, U. K. (1970). Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature, London 227, 680685.
Lowndes, C. M., Bonaldo, M. C., Thomaz, N. and Goldenberg, S. (1996). Heterogeneity of metalloprotease expression in Trypanosoma cruzi. Parasitology 112, 393399.
Madala, P. K., Tyndall, J. D., Nall, T. and Fairlie, D. P. (2010). Update 1 of: Proteases universally recognise beta strands in their active sites. Chemical Reviews 110, 131.
Magdesian, M. H., Tonelli, R. R., Fessel, M. R., Silveira, M. S., Schumacher, R. I., Linden, R., Colli, W. and Alves, M. J. (2007). A conserved domain of the gp85/trans-sialidase family activates host cell extracellular signal-regulated kinase and facilitates Trypanosoma cruzi infection. Experimental Cell Research 313, 210218.
McKerrow, J. H., Caffrey, C., Kelly, B., Loke, P. and Sajid, M. (2006). Proteases in parasitic diseases. Annual Review of Pathology 1, 497536.
Meyer-Hoffert, U. and Schröder, J. M. (2011). Epidermal proteinases in the pathogenesis of rosacea. Journal of Investigative Dermatology Symposium Proceedings 15, 1623.
Morgado-Díaz, J. A., Silva-Lopez, R. E., Alves, C. R., Soares, M. J., Corte-Real, S. and De Simone, S. G. (2005). Subcellular localization of an intracellular serine protease of 68 kDa in Leishmania (Leishmania) amazonensis promastigotes. Memórias do Instituto Oswaldo Cruz 100, 377383.
Mortara, R. A., Minelli, L. M., Vandekerckhove, F., Nussenzweig, V. and Ramalho-Pinto, F. J. (2001). Phosphatidylinositol-speciWc phospholipase C (PI-PLC) cleavage of GPI-anchored surface molecules of Trypanosoma cruzi triggers in vitro morphological reorganization of trypomastigotes. Journal of Eukaryotic Microbiology 48, 2737.
Motta, F. N., Bastos, I. M., Faudry, E., Ebel, C., Lima, M. M., Neves, D., Ragno, M., Barbosa, J. A., de Freitas, S. M. and Santana, J. M. (2012). The Trypanosoma cruzi virulence factor oligopeptidase B (OPBTc) assembles into an active and stable dimer. PLoS One 7, e30431.
Nogueira de Melo, A. C., de Souza, E. P., Elias, C. G., dos Santos, A. L., Branquinha, M. H., d'Avila-Levy, C. M., dos Reis, F. C., Costa, T. F., Lima, A. P., Pereira, M. C. S., Meirelles, M. N. and Vermelho, A. B. (2010). Detection of matrix metallopeptidase-9-like proteins in Trypanosoma cruzi. Experimental Parasitology 125, 256263.
Oliveira-Jr, F. O., Alves, C. R., Calvet, C. M., Toma, L., Bouças, R. I., Nader, H. B., Castro Côrtes, L. M., Krieger, M. A., Meirelles, Mde N. and Pereira, M. C. S. (2008). Trypanosoma cruzi heparin-binding proteins and the nature of the host cell heparan sulfate-binding domain. Microbial Pathogenesis 44, 329338.
Oliveira, F. O., Alves, C. R., Souza-Silva, F., Calvet, C. M., Côrtes, L. M., Gonzalez, M. S., Toma, L., Bouças, R. I., Nader, H. B. and Pereira, M. C. S. (2012). Trypanosoma cruzi heparin-binding proteins mediate the adherence of epimastigotes to the midgut epithelial cells of Rhodnius prolixus. Parasitology 139, 735743.
Ortega-Barria, E. and Pereira, M. E. (1991). A novel T. cruzi heparin-binding protein promotes fibroblast adhesion and penetration of engineered bacteria and trypanosomes into mammalian cells. Cell 67, 411421.
Ortega-Barria, E. and Pereira, M. E. (1992). Entry of Trypanosoma cruzi into eukaryotic cells. Infectious Agents and Diseases 1, 136145.
Peña, C. P., Lander, N., Rodríguez, E., Crisante, G., Añez, N., Ramírez, J. L. and Chiurillo, M. A. (2009). Molecular analysis of surface glycoprotein multigene family TrGP expressed on the plasma membrane of Trypanosoma rangeli epimastigotes forms. Acta Tropica 111, 255262.
Pinho, R. T., Beltramini, L. M., Alves, C. R. and De-Simone, S. G. (2009). Trypanosoma cruzi: isolation and characterization of aspartyl proteases. Experimental Parasitology 122, 128133.
Rangel, H. A., Araújo, P. M., Repka, D. and Costa, M. G. (1981). Trypanosoma cruzi: isolation and characterization of a proteinase. Experimetal Parasitology 52, 199209.
Rathore, D., McCutchan, T. F., Garboczi, D. N., Toida, T., Hernáiz, M. J., LeBrun, L. A., Lang, S. C. and Linhardt, R. J. (2001). Direct measurement of the interactions of glycosaminoglycans and a heparin decasaccharide with the malaria circumsporozoite protein. Biochemistry 40, 11 51811 524.
Rocha, G. M., Brandão, B. A., Mortara, R. A., Attias, M., de Souza, W. and Carvalho, T. M. (2006). The flagellar attachment zone of Trypanosoma cruzi epimastigote forms. Journal of Structural Biology 154, 8999.
Ruiz, R. C., Favoreto-Jr, S., Dorta, M. L., Oshiro, M. E., Ferreira, A. T., Manque, P. M. and Yoshida, N. (1998). Infectivity of Trypanosoma cruzi strains is associated with differential expression of surface glycoproteins with differential Ca2+ signalling activity. The Biochemical Journal 330, 505511.
Sahar, T., Reddy, K. S., Bharadwaj, M., Pandey, A. K., Singh, S., Chitnis, C. E. and Gaur, D. (2010). Plasmodium falciparum reticulocyte binding-like homologue protein 2 (PfRH2) is a key adhesive molecule involved in erythrocyte invasion. PLoS One 6, e17102.
Scharfstein, J. and Lima, A. P. (2008). Roles of naturally occurring protease inhibitors in the modulation of host cell signaling and cellular invasion by Trypanosoma cruzi. Subcellular Biochemistry 47, 140154.
Silva-Lopez, R. E., Coelho, M. G. and De Simone, S. G. (2005). Characterization of an extracellular serine protease of Leishmania (Leishmania) amazonensis. Memórias do Instituto Oswaldo Cruz 100, 377383.
Silva-López, R. E., dos Santos, T. R., Morgado-Díaz, J. A., Tanaka, M. N. and de Simone, S. G. (2010). Serine protease activities in Leishmania (Leishmania) chagasi promastigotes. Parasitology Research 107, 11511162.
Silva-Lopez, R. E., Morgado-Díaz, J. A., Alves, C. R., Côrte-Real, S. and Giovanni-De-Simone, S. (2004). Subcellular localization of an extracellular serine protease in Leishmania (Leishmania) amazonensis. Parasitology Research 93, 328331.
Silva-Lopez, R. E., Morgado-Díaz, J. A., dos Santos, P. T. and Giovanni-De-Simone, S. (2008). Purification and subcellular localization of a secreted 75 kDa Trypanosoma cruzi serine oligopeptidase. Acta Tropica 107, 159167.
Tanowitz, H. B., Machado, F. S., Jelicks, L. A., Shirani, J., de Carvalho, A. C., Spray, D. C., Factor, S. M., Kirchhoff, L. V. and Weiss, L. M. (2009). Perspectives on Trypanosoma cruzi-induced heart disease (Chagas disease). Progress in Cardiovascular Diseases 51, 524539.
Terao-Muto, Y., Yoneda, M., Seki, T., Watanabe, A., Tsukiyama-Kohara, K., Fujita, K. and Kai, C. (2008). Heparin-like glycosaminoglycans prevent the infection of measles virus in SLAM-negative cell lines. Antiviral Research 80, 370376.
Tonelli, R. R., Giordano, R. J., Barbu, E. M., Torrecilhas, A. C., Kobayashi, G. S., Langley, R. R., Arap, W., Pasqualini, R., Colli, W. and Alves, M. J. (2010). Role of the gp85/trans-sialidases in Trypanosoma cruzi tissue tropism: preferential binding of a conserved peptide motif to the vasculature in vivo. PLoS Neglected Tropical Diseases 4, e864.
Toso, M. A., Vial, U. F. and Galanti, N. (2011). Oral transmission of Chagas’ disease. Revista Médica do Chile 139, 258266.
Tossavainen, H., Pihlajamaa, T., Huttunen, T. K., Raulo, E., Rauvala, H., Permi, P. and Kilpeläinen, I. (2006). The layered fold of the TSR domain of P. falciparum TRAP contains a heparin binding site. Protein Science 15, 17601768.
Tyler, K. M., Fridberg, A., Toriello, K. M., Olson, C. L., Cieslak, J. A., Hazlett, T. L. and Engman, D. M. (2009). Flagellar membrane localization via association with lipid rafts. Journal of Cell Science 122, 859866.
Wu, C. and Wang, S. (2012). A pH-sensitive heparin-binding sequence from Baculovirus gp64 protein is important for binding to mammalian cells but not to Sf9 insect cells. Journal of Virology 86, 484491.
Yoshida, N. (2006). Molecular basis of mammalian cell invasion by Trypanosoma cruzi. Anais da Academia Brasileira de Ciências 78, 87111.
Yoshida, N. and Cortez, M. (2008). Trypanosoma cruzi: parasite and host cell signaling during the invasion process. Subcellular Biochemistry 47, 8291.
Yoshida, N., Tyler, K. M. and Llewellyn, M. S. (2011). Invasion mechanisms among emerging food-borne protozoan parasites. Trends in Parasitology 27, 459466.


Related content

Powered by UNSILO

Trypanosoma cruzi heparin-binding proteins present a flagellar membrane localization and serine proteinase activity

  • F. O. R. OLIVEIRA-JR (a1), C. R. ALVES (a2), F. S. SILVA (a2), L. M. C. CÔRTES (a2), L. TOMA (a3), R. I. BOUÇAS (a3), T. AGUILAR (a3), H. B. NADER (a3) and M. C. S. PEREIRA (a1)...


Full text views

Total number of HTML views: 0
Total number of PDF views: 0 *
Loading metrics...

Abstract views

Total abstract views: 0 *
Loading metrics...

* Views captured on Cambridge Core between <date>. This data will be updated every 24 hours.

Usage data cannot currently be displayed.