Advertisement

Medical Microbiology and Immunology

, Volume 207, Issue 2, pp 117–128 | Cite as

Trypanosoma cruzi serinecarboxipeptidase is a sulfated glycoprotein and a minor antigen in human Chagas disease infection

  • Luciana L. Soprano
  • Juliana E. Parente
  • Malena Landoni
  • Alicia S. Couto
  • Vilma G. Duschak
Original Investigation

Abstract

In this work, the presence of sulfated N-glycans was studied in a high-mannose-type glycoprotein of Trypanosoma cruzi with serinecarboxipeptidase (TcSCP) activity. The immune cross-reactivity between purified SCP and Cruzipain (Cz) was evidenced using rabbit sera specific for both glycoproteins. Taking advantage that SCP co-purifies with Cz from Concanavalin-A affinity columns, the Cz–SCP mixture was desulfated, ascribing the cross-reactivity to the presence of sulfate groups in both molecules. Therefore, knowing that Cz is a sulfated glycoprotein, with antigenic sulfated epitopes (sulfotopes), SCP was excised from SDS-PAGE and the N-glycosydic chains were analyzed by UV–MALDI–TOF-MS, confirming the presence of short-sulfated high-mannose-type oligosaccharidic chains. Besides, the presence of sulfotopes was analyzed in lysates of the different parasite stages demonstrating that a band with apparent molecular weight similar to SCP was highly recognized in trypomastigotes. In addition, SCP was confronted with sera of infected people with different degrees of cardiac dysfunction. Although most sera recognized it in different groups, no statistical association was found between sera antibodies specific for SCP and the severity of the disease. In summary, our findings demonstrate (1) the presence of sulfate groups in the N-glycosidic short chains of native TcSCP, (2) the existence of immune cross-reactivity between Cz and SCP, purified from epimastigotes, (3) the presence of common sulfotopes between both parasite glycoproteins, and (4) the enhanced presence of sulfotopes in trypomastigotes, probably involved in parasite–host relationship and/or infection. Interestingly, we show for the first time that SCP is a minor antigen recognized by most of chronic Chagas disease patient’s sera.

Keywords

Trypanosoma cruzi Serinecarboxypeptidase Glycomics Sulfate groups Sulfotopes Chagas’ disease Antigenicity 

Notes

Acknowledgements

The authors thank to the Consejo Nacional de Investigaciones Científicas y Técnicas, CONICET, (PIP 11220110100660; PIP 07912012-2014), Universidad de Buenos Aires, UBA, (20020130100476BA) and Agencia Nacional de Promoción Científica y Tecnológica (ANPCyT) PICT-2013-0736 grants as well as to the Instituto Nacional de Parasitología “Dr M. Fatala Chaben”, ANLIS-Malbrán, Ministerio de Salud de la Nación, Argentina. The Ultraflex II (Bruker) TOF/TOF mass spectrometer was supported by the ANPCyT, (Grant PME 125, CEQUIBIEM).

References

  1. 1.
    World Health Organization (2014) WHO Fact Sheet No 340. http://www.who.int/mediacentre/factsheets/fs340/en/. Accessed 29 May 2014
  2. 2.
    Gascon J, Bern, Pinazo MJ (2010) Chagas disease in Spain, the United States and other non-endemic countries. Acta Trop 115(1–2):22–27CrossRefPubMedGoogle Scholar
  3. 3.
    Pérez-Molina JA, Norman, López-Vélez R (2012) Chagas disease in non-endemic countries: epidemiology, clinical presentation and treatment. Curr Infect Dis Rep 14(3):263–274CrossRefPubMedGoogle Scholar
  4. 4.
    Cazzulo JJ (2002) Proteinases of Trypanosoma cruzi: potential targets for the chemotherapy of Chagas disease. Curr Topics Med Chem 2:1257–1267CrossRefGoogle Scholar
  5. 5.
    Duschak VG (2011) A decade of targets and patented drugs for chemotherapy of Chagas disease. Recent Pat Antiinfect Drug Discov 6:216–259CrossRefPubMedGoogle Scholar
  6. 6.
    Duschak VG (2016) Targets and patented drugs for chemotherapy of chagas disease in the last 15 years-period, review article. Recent Pat Antiinfect Drug Discov 11:74–173CrossRefPubMedGoogle Scholar
  7. 7.
    El Sayed NM, Myler PJ, Bartholomeu DC et al (2005) The genome sequence of Trypanosoma cruzi, etiologic agent of Chagas disease. Science 309(5733):409–415CrossRefPubMedGoogle Scholar
  8. 8.
    Duschak VG, Couto AS (2009) Cruzipain, the major cysteine protease of Trypanosoma cruzi: a sulfated glycoprotein antigen as relevant candidate for vaccine development and drug target. A review. Curr Med Chem 16:3174–3202CrossRefPubMedGoogle Scholar
  9. 9.
    Burleigh BA, Andrews NW (1995) 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 270(10):5172–5180CrossRefPubMedGoogle Scholar
  10. 10.
    Burleigh BA, Caler EV, Webster I, Andrews NW (1997) A cytosolic serine endopeptidase from Trypanosoma cruzi is required for the generation of Ca2+ signaling in mammalian cells. J Cell Biol 136(3):609–620CrossRefPubMedCentralPubMedGoogle Scholar
  11. 11.
    Santana JM, Grellier P, Schrevel J, Teixeira A (1997) A Trypanosoma cruzi-secreted 80 kDa proteinase with specificity for human collagen types I and IV. Biochem J 325(1):129–137CrossRefPubMedCentralPubMedGoogle Scholar
  12. 12.
    Bastos IM, Grellier P, Martins NF, Cadavid-Restrepo G, de Souza-Ault MR, Augustyns K, Teixeira AR, Schrevel J, Maigret B, da Silveira JF, Santana JM (2005) Molecular, functional and structural properties of the prolyl oligopeptidase of Trypanosoma cruzi (POP Tc80), which is required for parasite entry into mammalian cells. Biochem J 388:29–38CrossRefPubMedCentralPubMedGoogle Scholar
  13. 13.
    Parussini F, García M, Mucci J, Agüero F, Sánchez D, Hellman U, Aslund L. Cazzulo JJ (2003) Characterization of a lysosomal serine carboxypeptidase from Trypanosoma cruzi. Mol Biochem Parasitol 131(1):11–23CrossRefPubMedGoogle Scholar
  14. 14.
    da Silva-Lopez RE, Morgado-Díaz JA, dos Santos PT, Giovanni-De-Simone S (2008) Purification and subcellular localization of a secreted 75 kDa Trypanosoma cruzi serine oligopeptidase. Acta Trop 107(2):159–167CrossRefPubMedGoogle Scholar
  15. 15.
    Remington SJ, Breddam K (1994) Carboxypeptidases C and D. Meth Enzymol 244:231–248CrossRefPubMedGoogle Scholar
  16. 16.
    Breddam K (1986) Serine carboxypeptidases. A review. Carlsberg Res Commun 51:83–128CrossRefGoogle Scholar
  17. 17.
    Barrett A, Rawlings N, Woessner J (2004) Handbook of proteolytic enzymes. Elsevier Academic Press, LondonGoogle Scholar
  18. 18.
    Vendrell J, Avilés FX (1999) Carboxypeptidases. In: Turk V (ed) Proteases: new perspectives, vol 2. Birkhauser, Basel, pp 13–34CrossRefGoogle Scholar
  19. 19.
    Ferreira KA, Fajardo EF, Baptista RP, Macedo AM, Lages-Silva E, Ramírez LE, Pedrosa AL (2014) Species-specific markers for the differential diagnosis of Trypanosoma cruzi and Trypanosoma rangeli and polymorphisms detection in Trypanosoma rangeli. Parasitol Res 113(6):2199–2207CrossRefPubMedGoogle Scholar
  20. 20.
    Kovensky J (2009) Sulfated oligosaccharides: new targets for drug development? Curr Med Chem 16:2338–2344CrossRefPubMedGoogle Scholar
  21. 21.
    Kawasaki N, Ohta M, Hyuga S, Hyuga M, Hayakawa T (2000) Application of liquid chromatography mass spectrometry and liquid chromatography with tandem mass spectrometry to the analysis of the site specific carbohydrate heterogeneity in erythropoietin. Anal Biochem 285:82–91CrossRefPubMedGoogle Scholar
  22. 22.
    Honke K, Taniguchi N (2002) Sulfotransferases and sulfated oligosaccharides. Med Res Rev 22:637–654CrossRefPubMedGoogle Scholar
  23. 23.
    Klaassen CD, Boles JW (1997) Sulfation and sulfotransferases 5: the importance of 3ʹ-phosphoadenosine 5ʹ-phosphosulfate (PAPS) in the regulation of sulfation. FASEB J 11(6):404–418CrossRefPubMedGoogle Scholar
  24. 24.
    Fukuda M, Hiraoka N, Akama TO, Fukuda MN (2001) Carbohydrate-modifyingsulfotransferases: structure, function, and pathophysiology. J Biol Chem 276:47747–47750CrossRefPubMedGoogle Scholar
  25. 25.
    Esko JD, Lindahl U (2001) Molecular diversity of heparan sulfate. J Clin Invest 108(2):169–173CrossRefPubMedCentralPubMedGoogle Scholar
  26. 26.
    Baeuerle PA, Huttner WB (1986) Chlorate. A potent inhibitor of protein sulfation in intact cells. Biochem Biophys Res Commun 141(2):870–877CrossRefPubMedGoogle Scholar
  27. 27.
    Ferrero MR, Soprano LL, Acosta DM, García GA, Esteva MI, Couto AS, Duschak VG (2014) Effects of chlorate on the sulfation process of Trypanosoma cruzi glycoconjugates. Implication of parasite sulfates in cellular invasion. Acta Trop 137:161–173CrossRefPubMedGoogle Scholar
  28. 28.
    Barboza M, Duschak VG, Fukuyama Y, Nonami H, Erra-Balsells R, Cazzulo JJ, Couto AS (2005) Structural analysis of the N-glycans of the major cysteine proteinase of Trypanosoma cruzi. Identification of sulfated high-mannose type oligosaccharides. FEBS J 272(15):3803–3815CrossRefPubMedGoogle Scholar
  29. 29.
    Acosta DM, Arnaiz MR, Esteva MI, Barboza M, Stivale D, Orlando UD, Torres S, Laucella SA, Couto AS, Duschak VG (2008) Sulfates are main targets of immune responses to cruzipain and are involved in heart damage in BALB/c immunized mice. Int Immunol 20(4):461–470CrossRefPubMedGoogle Scholar
  30. 30.
    Cazzulo JJ, Franke de Cazzulo BM, Engel JC, Cannata JJ (1985) End products and enzyme levels of aerobic glucose fermentation in trypanosomatids. Mol Biochem Parasitol 16:329–343CrossRefPubMedGoogle Scholar
  31. 31.
    Andrews NW, Colli W (1982) Adhesion and interiorization of Trypanosoma cruzi in mammalian cells. J Protozool 29(2):264–269CrossRefPubMedGoogle Scholar
  32. 32.
    Labriola C, Sousa M, Cazzulo JJ (1993) Purification of the major cysteine proteinase (cruzipain) from Trypanosoma cruzi by affinity chromatography. Biol Res 26(1–2):101–107PubMedGoogle Scholar
  33. 33.
    Bradford J (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein–dye binding. Anal Biochem 72:248–254CrossRefPubMedGoogle Scholar
  34. 34.
    Freeze HH, Mierendorf RC, Wunderlich R, Dimond RL (1984) Sulfated oligosaccharides block antibodies to many Dictyostelium discoideum acid hydrolases. J Biol Chem 259(16):10641–10643PubMedGoogle Scholar
  35. 35.
    Ey PL, Prowse SJ, Jenkin CR (1978) Isolation of pure IgG1, IgG2a and IgG2b immunoglobulins from mouse serum using protein A-Sepharose. Immunochem 15:429–436CrossRefGoogle Scholar
  36. 36.
    Acosta DM, Soprano LL, Ferrero M, Landoni M, Esteva MI, Couto AS, Duschak VG (2011) A striking common O-linked N-acetylglucosaminyl moiety between cruzipain and myosin. Parasite Immunol 33(7):363–370CrossRefPubMedGoogle Scholar
  37. 37.
    World Health Organization (1991) Control of Chagas disease. Report of a WHO Expert Committee. World Health Organ Tech Rep Ser 811:1–95Google Scholar
  38. 38.
    Kuschnir E, Sgammini H, Castro R, Evequoz C, Ledesma R, Brunetto J (1985) Evaluation of cardiac function by radioisotopic angiography, in patients with chronic Chagas cardiopathy. Arq Bras Cardiol 45:249–256PubMedGoogle Scholar
  39. 39.
    Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227(5259):680–685CrossRefPubMedGoogle Scholar
  40. 40.
    Duschak VG, Couto AS (2009) Cruzipain, the major cysteine protease of Trypanosoma cruzi: a sulfated glycoprotein antigen as relevant candidate for vaccine development and drug target. A review. Curr Med Chem 16(24):3174–3202CrossRefPubMedGoogle Scholar
  41. 41.
    Duschak VG, Riarte A, Segura EL, Laucella SA (2001) Humoral immune response to cruzipain and cardiac dysfunction in chronic Chagas disease. Immunol Lett 78(3):135–142CrossRefPubMedGoogle Scholar
  42. 42.
    Rapraeger AC, Krufka A, Olwin BB (1991) Requirement of heparan sulfate for ßFGF-mediated fibroblast growth and myoblast differentiation. Science 252:1705–1708CrossRefPubMedGoogle Scholar
  43. 43.
    Petry K, Nudelman E, Eisen H, Hakomori S (1988) Sulfated lipids represent common antigens on the surface of Trypanosoma cruzi and mammalian tissues. Mol Biochem Parasitol 30:113–121CrossRefPubMedGoogle Scholar
  44. 44.
    Uhrig ML, Couto AS, Colli W, Lederkremer RM (1996) Characterization of inositolphospholipids in Trypanosoma cruzi trypomastigote forms. Biochim Biophys Acta 1300:233–239CrossRefPubMedGoogle Scholar
  45. 45.
    Acosta DM, Soprano LL, Ferrero MR, Esteva MI, Riarte A, Couto AS, Duschak VG (2012) Structural and immunological characterization of sulfatides: relevance of sulfate moieties in Trypanosoma cruzi glycoconjugates. Parasite Immunol 34(11):499–510CrossRefPubMedGoogle Scholar
  46. 46.
    Sant’Anna C, Parussini F, Lourenço D, de Souza W, Cazzulo JJ, Cunha-e-Silva NL (2008) All Trypanosoma cruzi developmental forms present lysosome-related organelles. Histochem Cell Biol 130(6):1187–1198CrossRefPubMedGoogle Scholar
  47. 47.
    Liu F, Tachibana S, Taira T, Ishihara M, Kato F, Yasuda M (2004) Purification and characterization of a high molecular mass serine carboxypeptidase from Monascus pilosus. J Ind Microbiol Biotechnol 31:572–580CrossRefPubMedGoogle Scholar
  48. 48.
    Larriba E, Martín-Nieto J, Lopez-Llorca LV (2012) Gene cloning, molecular modeling, and phylogenetics of serine protease P32 and serine carboxypeptidase SCP1 from nematophagous fungi Pochonia rubescens and Pochonia chlamydosporia. Can J Microbiol 58(7):815–827CrossRefPubMedGoogle Scholar
  49. 49.
    Garrido VV, Dulgerian LR, Stempin CC, Cerbán FM (2011) The increase in mannose receptor recycling favors arginase induction and Trypanosoma cruzi survival in macrophages. Int J Biol Sci 7(9):1257–1272CrossRefPubMedCentralPubMedGoogle Scholar

Copyright information

© Springer-Verlag GmbH Germany, part of Springer Nature 2017

Authors and Affiliations

  • Luciana L. Soprano
    • 1
  • Juliana E. Parente
    • 2
  • Malena Landoni
    • 2
  • Alicia S. Couto
    • 2
  • Vilma G. Duschak
    • 1
  1. 1.Area de Bioquímica de Proteínas y Glicobiología de Parásitos, Consejo Nacional de Investigaciones Científicas y Tecnicas (CONICET), Departamento de InvestigaciónInstituto Nacional de Parasitología “Dr. Mario Fatala Chaben”, ANLIS-Malbrán, Ministerio de Salud de la Nación, CONICETBuenos AiresArgentina
  2. 2.Centro de Investigación en Hidratos de Carbono (CIHIDECAR), Departamento de Química Orgánica-Consejo Nacional de Investigaciones Científicas y Técnicas, Facultad de Ciencias Exactas y NaturalesUniversidad de Buenos Aires, Ciudad UniversitariaBuenos AiresArgentina

Personalised recommendations