Skip to main content Accessibility help
×
Home

Functional characterization of single-domain cystatin-like cysteine proteinase inhibitors expressed by the trematode Fasciola hepatica

  • MARTÍN CANCELA (a1), ILEANA CORVO (a2) (a3), EDILEUZA DA SILVA (a1), ALINE TEICHMANN (a1), LEDA ROCHE (a2), ALVARO DÍAZ (a4), JOSÉ FRANSISCO TORT (a2), HENRIQUE B. FERREIRA (a1) and ARNALDO ZAHA (a1)...

Summary

Cystatins are small, phylogenetically conserved proteins that are tight-binding inhibitors of cysteine proteinases. The liver fluke Fasciola hepatica uses a diverse set of cysteine proteinases of the papain superfamily for host invasion, immune evasion and nutrition, but little is known about the regulation of these enzymes. The aim of this work is to characterize the cystatin repertoire of F. hepatica. For this purpose, we first surveyed the available sequence databases, identifying three different F. hepatica single-domain cystatins. In agreement with the in silico predictions, at least three small proteins with cysteine proteinase binding activity were identified. Phylogenetic analyses showed that the three cystatins (named FhStf-1, -2 and -3) are members of the I25A subfamily (stefins). Whereas FhStf-1 grouped with classical stefins, FhStf-2 and 3 fell in a divergent stefin subgroup unusually featuring signal peptides. Recombinant rFhStf-1, -2 and -3 had potent inhibitory activity against F. hepatica cathepsin L cysteine proteinases but differed in their capacity to inhibit mammalian cathepsin B, L and C. FhStf-1 was localized in the F. hepatica reproductive organs (testes and ovary), and at the surface lamella of the adult gut, where it may regulate cysteine proteinases related with reproduction and digestion, respectively. FhStf-1 was also detected among F. hepatica excretion–secretion (E/S) products of adult flukes. This suggests that it is secreted by non-classical secretory pathway and that it may interact with host lysosomal cysteine proteinases.

Copyright

Corresponding author

*Corresponding author: Laboratório de Biologia Molecular de Cestódeos, Centro de Biotecnologia, UFRGS, Caixa Postal 15053, CEP 91501-970, Porto Alegre, RS, Brazil. E-mail: martin@cbiot.ufrgs.br

References

Hide All
Abrahamson, M. (1994). Cystatins. Methods in Enzymology 244, 685700.
Acosta, D., Goni, F. and Carmona, C. (1998). Characterization and partial purification of a leucine aminopeptidase from Fasciola hepatica . Journal of Parasitology 84, 17.
Andrews, S. J. (1999). The life cycle of Fasciola hepatica . In Fasciolosis (ed. Dalton, J. P.), pp. 129. CABI Publishing, Oxin, UK.
Barrett, A. J. (1986). The cystatins: a diverse superfamily of cysteine peptidase inhibitors. Biomedica Biochimica Acta 45, 13631374.
Beckham, S. A., Piedrafita, D., Phillips, C. I., Samarawickrema, N., Law, R. H., Smooker, P. M., Quinsey, N. S., Irving, J. A., Greenwood, D., Verhelst, S. H., Bogyo, M., Turk, B., Coetzer, T. H., Wijeyewickrema, L. C., Spithill, T. W. and Pike, R. N. (2009). A major cathepsin B protease from the liver fluke Fasciola hepatica has atypical active site features and a potential role in the digestive tract of newly excysted juvenile parasites. International Journal of Biochemistry and Cell Biology 41, 16011612.
Cancela, M., Carmona, C., Rossi, S., Frangione, B., Goni, F. and Berasain, P. (2004). Purification, characterization, and immunolocalization of paramyosin from the adult stage of Fasciola hepatica. Parasitology Research 92, 441448.
Cancela, M., Acosta, D., Rinaldi, G., Silva, E., Duran, R., Roche, L., Zaha, A., Carmona, C. and Tort, J. F. (2008). A distinctive repertoire of cathepsins is expressed by juvenile invasive Fasciola hepatica. Biochimie 90, 14611475.
Cancela, M., Ruetalo, N., Dell'Oca, N., da Silva, E., Smircich, P., Rinaldi, G., Roche, L., Carmona, C., Alvarez-Valin, F., Zaha, A. and Tort, J. F. (2010). Survey of transcripts expressed by the invasive juvenile stage of the liver fluke Fasciola hepatica. BMC Genomics 11, 227.
Canevari, J., Ceballos, L., Sanabria, R., Romero, J., Olaechea, F., Ortiz, P., Cabrera, M., Gayo, V., Fairweather, I., Lanusse, C. and Alvarez, L. (2013). Testing albendazole resistance in Fasciola hepatica: validation of an egg hatch test with isolates from South America and the United Kingdom. Journal of Helminthology 88, 286292.
Carmona, C. and Tort, J. F. (2017). Fasciolosis in South America: epidemiology and control challenges. Journal of Helminthology 91, 99109.
Carmona, C., Dowd, A. J., Smith, A. M. and Dalton, J. P. (1993). Cathepsin L proteinase secreted by Fasciola hepatica in vitro prevents antibody-mediated eosinophil attachment to newly excysted juveniles. Molecular and Biochemical Parasitology 62, 917.
Chen, L., He, B., Hou, W. and He, L. (2017). Cysteine protease inhibitor of Schistosoma japonicum – a parasite-derived negative immunoregulatory factor. Parasitology Research 116, 901908.
Collins, P. R., Stack, C. M., O'Neill, S. M., Doyle, S., Ryan, T., Brennan, G. P., Mousley, A., Stewart, M., Maule, A. G., Dalton, J. P. and Donnelly, S. (2004). Cathepsin L1, the major protease involved in liver fluke (Fasciola hepatica) virulence: propetide cleavage sites and autoactivation of the zymogen secreted from gastrodermal cells. Journal of Biological Chemistry 279, 1703817046.
Coronado, S., Barrios, L., Zakzuk, J., Regino, R., Ahumada, V., Franco, L., Ocampo, Y. and Caraballo, L. (2017). A recombinant cystatin from Ascaris lumbricoides attenuates inflammation of DSS-induced colitis. Parasite Immunology 39, e12425.
Corvo, I., Cancela, M., Cappetta, M., Pi-Denis, N., Tort, J. F. and Roche, L. (2009). The major cathepsin L secreted by the invasive juvenile Fasciola hepatica prefers proline in the S2 subsite and can cleave collagen. Molecular and Biochemical Parasitology 167, 4147.
Cuesta-Astroz, Y., Scholte, L. L., Pais, F. S., Oliveira, G. and Nahum, L. A. (2014). Evolutionary analysis of the cystatin family in three Schistosoma species. Frontiers in Genetics 5, 206.
Cwiklinski, K., de la Torre-Escudero, E., Trelis, M., Bernal, D., Dufresne, P. J., Brennan, G. P., O'Neill, S., Tort, J., Paterson, S., Marcilla, A., Dalton, J. P. and Robinson, M. W. (2015). The extracellular vesicles of the helminth pathogen, Fasciola hepatica: biogenesis pathways and cargo molecules involved in parasite pathogenesis. Molecular and Cellular Proteomics 14, 32583273.
Dainichi, T., Maekawa, Y., Ishii, K., Zhang, T., Nashed, B. F., Sakai, T., Takashima, M. and Himeno, K. (2001). Nippocystatin, a cysteine protease inhibitor from Nippostrongylus brasiliensis, inhibits antigen processing and modulates antigen-specific immune response. Infection and Immunology 69, 73807386.
Dalton, J. P. and Heffernan, M. (1989). Thiol proteases released in vitro by Fasciola hepatica . Molecular and Biochemical Parasitology 35, 161166.
Dalton, J. P., Neill, S. O., Stack, C., Collins, P., Walshe, A., Sekiya, M., Doyle, S., Mulcahy, G., Hoyle, D., Khaznadji, E., Moire, N., Brennan, G., Mousley, A., Kreshchenko, N., Maule, A. G. and Donnelly, S. M. (2003). Fasciola hepatica cathepsin L-like proteases: biology, function, and potential in the development of first generation liver fluke vaccines. International Journal of Parasitology 33, 11731181.
Danilowicz-Luebert, E., Steinfelder, S., Kuhl, A. A., Drozdenko, G., Lucius, R., Worm, M., Hamelmann, E. and Hartmann, S. (2013). A nematode immunomodulator suppresses grass pollen-specific allergic responses by controlling excessive Th2 inflammation. International Journal of Parasitology 43, 201210.
Darawshe, S., Tsafadyah, Y. and Daniel, E. (1987). Quaternary structure of erythrocruorin from the nematode Ascaris suum. Evidence for unsaturated haem-binding sites. Biochemical Journal 242, 689694.
Di Maggio, L. S., Tirloni, L., Pinto, A. F., Diedrich, J. K., Yates, J. R. III, Benavides, U., Carmona, C., da Silva Vaz, I. Jr. and Berasain, P. (2016). Across intra-mammalian stages of the liver fluke Fasciola hepatica: a proteomic study. Scientific Reports 6, 32796.
Geadkaew, A., Kosa, N., Siricoon, S., Grams, S. V. and Grams, R. (2014). A 170 kDa multi-domain cystatin of Fasciola gigantica is active in the male reproductive system. Molecular and Biochemical Parasitology 196, 100107.
Guindon, S., Dufayard, J. F., Lefort, V., Anisimova, M., Hordijk, W. and Gascuel, O. (2010). New algorithms and methods to estimate maximum-likelihood phylogenies: assessing the performance of PhyML 3.0. Systematic Biology 59, 307321.
Guo, A. (2015). Comparative analysis of cystatin superfamily in platyhelminths. PLoS ONE 10, e0124683.
Hartmann, S. and Lucius, R. (2003). Modulation of host immune responses by nematode cystatins. International Journal of Parasitology 33, 12911302.
He, B., Cai, G., Ni, Y., Li, Y., Zong, H. and He, L. (2011). Characterization and expression of a novel cystatin gene from Schistosoma japonicum . Molecular and Cellular Probes 25, 186193.
Henskens, Y. M., Veerman, E. C. and Nieuw Amerongen, A. V. (1996). Cystatins in health and disease. Biological Chemistry Hoppe Seyler 377, 7186.
Kang, J. M., Lee, K. H., Sohn, W. M. and Na, B. K. (2011). Identification and functional characterization of CsStefin-1, a cysteine protease inhibitor of Clonorchis sinensis . Molecular and Biochemical Parasitology 177, 126134.
Kang, J. M., Ju, H. L., Lee, K. H., Kim, T. S., Pak, J. H., Sohn, W. M. and Na, B. K. (2014). Identification and characterization of the second cysteine protease inhibitor of Clonorchis sinensis (CsStefin-2). Parasitology Research 113, 4758.
Katoh, K. and Standley, D. M. (2013). MAFFT multiple sequence alignment software version 7: improvements in performance and usability. Molecular Biology and Evolution 30, 772780.
Kelley, J. M., Elliott, T. P., Beddoe, T., Anderson, G., Skuce, P. and Spithill, T. W. (2016). Current threat of triclabendazole resistance in Fasciola hepatica . Trends in Parasitology 32, 458469.
Khaznadji, E., Collins, P., Dalton, J. P., Bigot, Y. and Moire, N. (2005). A new multi-domain member of the cystatin superfamily expressed by Fasciola hepatica . International Journal of Parasitology 35, 11151125.
Klotz, C., Ziegler, T., Danilowicz-Luebert, E. and Hartmann, S. (2011 a). Cystatins of parasitic organisms. Advances in Experimental Medicine and Biology 712, 208221.
Klotz, C., Ziegler, T., Figueiredo, A. S., Rausch, S., Hepworth, M. R., Obsivac, N., Sers, C., Lang, R., Hammerstein, P., Lucius, R. and Hartmann, S. (2011 b). A helminth immunomodulator exploits host signaling events to regulate cytokine production in macrophages. PLoS Pathogens 7, e1001248.
Kopitar-Jerala, N. (2015). Innate immune response in brain, NF-Kappa B signaling and cystatins. Frontiers in Molecular Neuroscience 8, 73.
Krogh, A., Larsson, B., von Heijne, G. and Sonnhammer, E. L. (2001). Predicting transmembrane protein topology with a hidden Markov model: application to complete genomes. Journal of Molecular Biology 305, 567580.
Kumar, S., Stecher, G. and Tamura, K. (2016). MEGA7: molecular evolutionary genetics analysis version 7.0 for bigger datasets. Molecular Biology and Evolution 33, 18701874.
Laemmli, U. K. (1970). Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227, 680685.
Law, R. H., Smooker, P. M., Irving, J. A., Piedrafita, D., Ponting, R., Kennedy, N. J., Whisstock, J. C., Pike, R. N. and Spithill, T. W. (2003). Cloning and expression of the major secreted cathepsin B-like protein from juvenile Fasciola hepatica and analysis of immunogenicity following liver fluke infection. Infection and Immunology 71, 69216932.
Liu, F., Cheng, W., Pappoe, F., Hu, X., Wen, H., Luo, Q., Wang, S., Deng, F., Xie, Y., Xu, Y. and Shen, J. (2016). Schistosoma japonicum cystatin attenuates murine collagen-induced arthritis. Parasitology Research 115, 37953806.
Mas-Coma, S., Valero, M. A. and Bargues, M. D. (2014). Fascioliasis. Advances in Experimental Medicine and Biology 766, 77114.
McGonigle, L., Mousley, A., Marks, N. J., Brennan, G. P., Dalton, J. P., Spithill, T. W., Day, T. A. and Maule, A. G. (2008). The silencing of cysteine proteases in Fasciola hepatica newly excysted juveniles using RNA interference reduces gut penetration. International Journal of Parasitology 38, 149155.
Morales, F. C., Furtado, D. R. and Rumjanek, F. D. (2004). The N-terminus moiety of the cystatin SmCys from Schistosoma mansoni regulates its inhibitory activity in vitro and in vivo. Molecular and Biochemical Parasitology 134, 6573.
Morrison, J. F. (1969). Kinetics of the reversible inhibition of enzyme-catalysed reactions by tight-binding inhibitors. Biochimica et Biophysica Acta 185, 269286.
Overend, D. J. and Bowen, F. L. (1995). Resistance of Fasciola hepatica to triclabendazole. Australian Veterinary Journal 72, 275276.
Petersen, T. N., Brunak, S., von Heijne, G. and Nielsen, H. (2011). SignalP 4.0: discriminating signal peptides from transmembrane regions. Nature Methods 8, 785786.
Rawlings, N. D., Barrett, A. J. and Bateman, A. (2012). MEROPS: the database of proteolytic enzymes, their substrates and inhibitors. Nucleic Acids Research 40, D343D350.
Rawlings, N. D., Barrett, A. J. and Bateman, A. (2014). Using the MEROPS database for proteolytic enzymes and their inhibitors and substrates. Current Protocols in Bioinformatics 48, 1 25, 2133.
Reolon, L. A., Martello, C. L., Schrank, I. S. and Ferreira, H. B. (2014). Survey of surface proteins from the pathogenic Mycoplasma hyopneumoniae strain 7448 using a biotin cell surface labeling approach. PLoS ONE 9, e112596.
Rinaldi, G., Morales, M. E., Cancela, M., Castillo, E., Brindley, P. J. and Tort, J. F. (2008). Development of functional genomic tools in trematodes: RNA interference and luciferase reporter gene activity in Fasciola hepatica . PLoS Neglected Tropical Diseases 2, e260.
Robinson, M. W., Dalton, J. P. and Donnelly, S. (2008 a). Helminth pathogen cathepsin proteases: it's a family affair. Trends in Biochemical Sciences 33, 601608.
Robinson, M. W., Tort, J. F., Lowther, J., Donnelly, S. M., Wong, E., Xu, W., Stack, C. M., Padula, M., Herbert, B. and Dalton, J. P. (2008 b). Proteomics and phylogenetic analysis of the cathepsin L protease family of the helminth pathogen Fasciola hepatica: expansion of a repertoire of virulence-associated factors. Molecular and Cellular Proteomics 7, 11111123.
Schnoeller, C., Rausch, S., Pillai, S., Avagyan, A., Wittig, B. M., Loddenkemper, C., Hamann, A., Hamelmann, E., Lucius, R. and Hartmann, S. (2008). A helminth immunomodulator reduces allergic and inflammatory responses by induction of IL-10-producing macrophages. Journal of Immunology 180, 42654272.
Schuijs, M. J., Hartmann, S., Selkirk, M. E., Roberts, L. B., Openshaw, P. J. and Schnoeller, C. (2016). The helminth-derived immunomodulator AvCystatin reduces virus enhanced inflammation by induction of regulatory IL-10+ T cells. PLoS ONE 11, e0161885.
Siricoon, S., Grams, S. V. and Grams, R. (2012). Efficient inhibition of cathepsin B by a secreted type 1 cystatin of Fasciola gigantica . Molecular and Biochemical Parasitology 186, 126133.
Spithill, T. M., Smooker, P. M. and Copeman, D. B. (1999). Fasciola gigantica: epidemiology, control, immunology and molecular biology. In Fasciolosis (ed. Dalton, J. P.), pp. 465525. CABI Publishing, Oxin, UK.
Stoka, V., Turk, V. and Turk, B. (2016). Lysosomal cathepsins and their regulation in aging and neurodegeneration. Ageing Research Reviews 32, 2237.
Sun, Y., Liu, G., Li, Z., Chen, Y., Liu, Y., Liu, B. and Su, Z. (2013). Modulation of dendritic cell function and immune response by cysteine protease inhibitor from murine nematode parasite Heligmosomoides polygyrus . Immunology 138, 370381.
Tarasuk, M., Vichasri Grams, S., Viyanant, V. and Grams, R. (2009). Type I cystatin (stefin) is a major component of Fasciola gigantica excretion/secretion product. Molecular and Biochemical Parasitology 167, 6071.
Turk, V., Stoka, V. and Turk, D. (2008). Cystatins: biochemical and structural properties, and medical relevance. Frontiers in Bioscience 13, 54065420.
Wang, S., Xie, Y., Yang, X., Wang, X., Yan, K., Zhong, Z., Xu, Y., Zhang, Y., Liu, F. and Shen, J. (2016). Therapeutic potential of recombinant cystatin from Schistosoma japonicum in TNBS-induced experimental colitis of mice. Parasites & Vectors 9, 6.
Whelan, R. A., Rausch, S., Ebner, F., Gunzel, D., Richter, J. F., Hering, N. A., Schulzke, J. D., Kuhl, A. A., Keles, A., Janczyk, P., Nockler, K., Wieler, L. H. and Hartmann, S. (2014). A transgenic probiotic secreting a parasite immunomodulator for site-directed treatment of gut inflammation. Molecular Therapy 22, 17301740.
WHO (2007). Report of the WHO Informal Meeting on Use of Triclabendazole in Fascioliasis Control. World Health Organization, Geneva, Switzerland.
Yang, X., Liu, J., Yue, Y., Chen, W., Song, M., Zhan, X. and Wu, Z. (2014). Cloning, expression and characterisation of a type II cystatin from Schistosoma japonicum, which could regulate macrophage activation. Parasitology Research 113, 39853992.
Zawistowska-Deniziak, A., Wasyl, K., Norbury, L. J., Wesolowska, A., Bien, J., Grodzik, M., Wisniewski, M., Baska, P. and Wedrychowicz, H. (2013). Characterization and differential expression of cathepsin L3 alleles from Fasciola hepatica . Molecular and Biochemical Parasitology 190, 2737.

Keywords

Type Description Title
UNKNOWN
Supplementary materials

Cancela supplementary material
Figure S1

 Unknown (2.6 MB)
2.6 MB
PDF
Supplementary materials

Cancela supplementary material
Figure S2

 PDF (312 KB)
312 KB
WORD
Supplementary materials

Cancela supplementary material
Table S1

 Word (14 KB)
14 KB

Functional characterization of single-domain cystatin-like cysteine proteinase inhibitors expressed by the trematode Fasciola hepatica

  • MARTÍN CANCELA (a1), ILEANA CORVO (a2) (a3), EDILEUZA DA SILVA (a1), ALINE TEICHMANN (a1), LEDA ROCHE (a2), ALVARO DÍAZ (a4), JOSÉ FRANSISCO TORT (a2), HENRIQUE B. FERREIRA (a1) and ARNALDO ZAHA (a1)...

Metrics

Altmetric attention score

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