Aspectos imunológicos do sistema enzimático fenoloxidase de Schistosoma mansoni

Ribeiro-Paes, João Tadeu; Costa, Paulo Inácio da; Silva, Duarte da; Barth, Léo Roberto; Rodrigues, Vanderlei

doi:10.1590/S0036-46651994000500002

Resumos

O sistema enzimático fenoloxidase (EC 1.10.3.1, EC 1.10.3.2) está amplamente distribuido entre os seres vivos, tendo sido descrito em diferentes espécies do reino animal e vegetal. Apesar de desempenhar um papel fundamental na formação da cápsula ou parede dos ovos de trematódeos, o sistema enzimático fenoloxidase (PO) tem sido pouco estudado nesses organismos. No presente trabalho são apresentados os resultados iniciais de imunizações de coelhos contra PO de fêmeas adultas de S. mansoni e tirosinase de cogumelo (Sigma). As análises imunológicas, realizadas através de imunodifusão dupla (teste de Ouchterlony) e imunoeletroforese, revelaram identidade imunitária parcial entre a PO de machos e fêmeas. Não se observou reação cruzada entre os antissoros de coelhos imunizados contra PO e aqueles com tirosinase, indicando que, embora os sítios catalíticos de ambas as enzimas devam ser semelhantes, já que atuam sobre os mesmos substratos, os determinantes antigênicos devem ser diferentes. Os resultados descritos no presente trabalho representam um primeiro passo no sentido da purificação das isoenzimas da fenoloxidase e sua posterior utilização ao estudo dos mecanismos moleculares envolvidos na esclerotização da parede dos ovos de S. mansoni.

Schistosoma mansoni; Fenoloxidase; Parede dos ovos

The phenol oxidase enzymatic system (EC 1.10.3.1, EC 1.10.3.2) is widespread in different species of the animal and vegetal kingdom. Despite its importance in the eggshell formation of the trematodes phenol oxidase (PO) has been little studied in these organisms, mainly in S. mansoni. This report presents the initial results concerning the immunization of rabbits with PO of S. mansoni and mushroom tyrosinase. The immunological analysis done by means of double immu-nodifusion (Ouchterlony) and immunoelectrophoresis techniques revealed some immunological identity between the PO of males and females. It was not seen cross reaction between the antisera against PO and tyrosinase, what suggests that the antigenic determinants of both enzymes are different in spite of their catalytic sites being similar, since they act over the same substrate. The results reported here represent a first step in way to obtain the PO isoenzymes in their pure form and should open new insights for further studies on the molecular mechanisms involved in the sclerotization process of the S. mansoni eggshell.

SEROLOGIA

Aspectos imunológicos do sistema enzimático fenoloxidase de Schistosoma mansoni

Immunological aspects of the phenol oxidase enzymatic system of Schistosoma mansoni

João Tadeu Ribeiro-Paes^I; Paulo Inácio da Costa^I; Duarte da Silva^I; Léo Roberto Barth^II; Vanderlei Rodrigues^I

^IDepto. de Parasitologia, Microbiologia e Imunologia - Faculdade de Medicina de Ribeirão Preto - Universidade de São Paulo

^IIDepto. de Pediatria - Faculdade de Medicina de Ribeirão Preto - Univ. de São Paulo

^{Endereço para correspondência} Endereço para correspondência: Dr. Vanderlei Rodrigues Depto. de Parasitologia,Microbiologia e Imunologia - Faculdade de Medicina de Ribeirão Preto 14049-900 Ribeirão Preto, São Paulo, SP, Brasil

RESUMO

O sistema enzimático fenoloxidase (EC 1.10.3.1, EC 1.10.3.2) está amplamente distribuido entre os seres vivos, tendo sido descrito em diferentes espécies do reino animal e vegetal. Apesar de desempenhar um papel fundamental na formação da cápsula ou parede dos ovos de trematódeos, o sistema enzimático fenoloxidase (PO) tem sido pouco estudado nesses organismos. No presente trabalho são apresentados os resultados iniciais de imunizações de coelhos contra PO de fêmeas adultas de S. mansoni e tirosinase de cogumelo (Sigma). As análises imunológicas, realizadas através de imunodifusão dupla (teste de Ouchterlony) e imunoeletroforese, revelaram identidade imunitária parcial entre a PO de machos e fêmeas. Não se observou reação cruzada entre os antissoros de coelhos imunizados contra PO e aqueles com tirosinase, indicando que, embora os sítios catalíticos de ambas as enzimas devam ser semelhantes, já que atuam sobre os mesmos substratos, os determinantes antigênicos devem ser diferentes. Os resultados descritos no presente trabalho representam um primeiro passo no sentido da purificação das isoenzimas da fenoloxidase e sua posterior utilização ao estudo dos mecanismos moleculares envolvidos na esclerotização da parede dos ovos de S. mansoni.

Unitermos:Schistosoma mansoni; Fenoloxidase; Parede dos ovos.

SUMMARY

The phenol oxidase enzymatic system (EC 1.10.3.1, EC 1.10.3.2) is widespread in different species of the animal and vegetal kingdom. Despite its importance in the eggshell formation of the trematodes phenol oxidase (PO) has been little studied in these organisms, mainly in S. mansoni. This report presents the initial results concerning the immunization of rabbits with PO of S. mansoni and mushroom tyrosinase. The immunological analysis done by means of double immu-nodifusion (Ouchterlony) and immunoelectrophoresis techniques revealed some immunological identity between the PO of males and females. It was not seen cross reaction between the antisera against PO and tyrosinase, what suggests that the antigenic determinants of both enzymes are different in spite of their catalytic sites being similar, since they act over the same substrate. The results reported here represent a first step in way to obtain the PO isoenzymes in their pure form and should open new insights for further studies on the molecular mechanisms involved in the sclerotization process of the S. mansoni eggshell.

Texto completo disponível apenas em PDF.

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AGRADECIMENTOS

Agradecemos a Olinda Mara B. Trevilato e Vânia Fernandez pela contribuição técnica e ao Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) pelo suporte financeiro.

Recebido para publicação em 20/12/1993.

Aceito para publicação em 08/07/1994.

1. AMIRI, P.; LOCKSLEY, R.M.; PARSLOV, T.G. et al. - Tumour necrosis factor restores granulomas and induces egg-laying in schistosome-infected SCID mice. Nature, 356: 604-607, 1992.
2. ANDRADE, Z. & GRIMAUD, J.A. - Morfology of chronic collagen resorption: a study on the late stages of Schistosoma granuloma involution. Amer. J. Path., 132: 389-399, 1988.
3. BENNETT, J.L.; SEED, J.L. & BOFF, M. - Fluorescent histochemical localization of phenol oxidase in female Schistosoma mansoni. J. Parasit., 64: 941-944, 1978.
4. BYRAM, J.E. & SENFT, A.W. - Structure of the Schistosome eggshell - amino acid analysis and incorporation of labelled amino acids. Amer. J. trop. Med. Hyg., 28: 539-547, 1979.
5. CANICATTI, C. & SEYMOUR, J. - Evidence for phenoloxidase activity in Holothuria tubulosa (Echinodermata) brown bodies and cells. Parasit. Res., 77: 50-53, 1991.
6. CLAUSEN, J. - Immunochemical techniques for the identification and estimation of macromolecules. In: WORK, T.S. & WORK, E., ed. - Laboratory techniques in biochemistry and molecular biology. Amsterdan, North Holland Publ., 1972. v. 1, p. 397-572.
7. CORDINGLEY, J.S. - Trematode eggshells: novel protein biopolymers. Parasit. today, 3: 341-344, 1987.
8. DURRANT, H.J.; RATCLIFFE, N.A.; HIPKIN, C.R.; ASPAN, A. & SODERHALL, K. - Purification of the pro-phenol oxidase enzyme from haemocytes of the cockroach Blaberus discoidalis. Biochem. J., 289: 87-91, 1993
9. GILLESPIE, J.P.; BIDOCHKA, M.J. & KHACHATOURIANS, G.G. - Separation and characterization of grasshopper hemolymph phenoloxidases by Sodium Dodecyl Sulfate - polyacrilamide gel electrophoresis. Comp. Biochem. Physiol., 98C: 351-358, 1991.
10. HAGAN, P. & WILKINS, H.A. - Concomitant immunity in schistosomiasis. Parasit. today, 9: 3-6, 1993.
11. HARTREE, E.F. - Determination of protein: a modification of the Lowry method that gives a linear photometric response. Analyt. Biochem., 48: 422-427, 1972.
12. JOHNSON, K.S.; TAYLOR, D.W. & CORDINGLEY, J.S. - Possible eggshell protein gene from Schistosoma mansoni. Molec. Biochem. Parasit., 22: 89-100, 1987.
13. MA, L. - Trace elements and polyphenoloxidase in Clonorchis sinensis. J. Parasit., 49: 197-203, 1963.
14. MANSOUR, T.E. - Effect of serotonin on phenol oxidase from the liver fluke Fasciola hepatica and from others sources. Biochim. biophys. Acta, 30: 492-500, 1958.
15. MITCHELL, G.F.; TIU, W.U. & GARCIA, E.G. - Infection characteristics of Schistosoma japonicum in mice and relevance to the assesment to Schistosomes vaccines. Advanc. Parasit., 30: 167-200, 1991.
16. NELLAIAPPAN, K. & RAMALINGAM, K. - Specificity of the enzyme phenoloxidase and possible pathway of sclerotization in Parapleurus sauridae. J. Parasit., 66: 217-219, 1980.
17. NELLAIAPPAN, K.; DEVASUNDRAI, A.F. & CHAN-DAYUTHAPANI, S. - Properties of phenol oxidase in Fasciola gigantica. Parasitology, 99: 403-407, 1989.
18. RIBEIRO-PAES, J.T. - Caracterizaçăo do sistema enzimático fenoloxidase em Schistosoma mansoni. Ribeirăo Preto, 1993. (Tese de Doutoramento - Faculdade de Medicina de Ribeirăo Preto da Universidade de Săo Paulo).
19. ROCH, P.; CANICATTI, P. & SEYMOUR, S. - Tetrameric structure of the active phenoloxidase evidenced in the coelomocytes of the echinoderm Holothuria tubulosa. Comp. Biochem. Physiol., 102B: 349-355, 1992.
20. RODRIGUES, V.; CHAUDRI, M.; KNIGHT, M. et al. - Predicted structure of a major Schistosoma mansoni eggshell protein. Molec. Biochem. Parasit., 32: 7-14, 1989.
21. ROLLE, R.S.; MARSHALL, M.R.; WEI, C.I. & CHEN, J.S. - Phenoloxidase forms of the florida spiny lobster: immunological and spectropolarimetric characterization. Comp. Biochem. Physiol., 97B: 483-489, 1990.
22. SCHAEFFER, J.; KRAMER, K.J.; GARBOW, J.R. et al. - Aromatic cross-links in insect cuticle: detection by solid-state ¹³Cand ¹⁵NNMR. Science, 235: 1200-1204, 1987.
23. SCHEIDEGGER, J.J. - Une micro-méthode de l'immuno-electrophorese. Intern. Arch. Allergy, 7: 103-110, 1955.
24. SEED, J.L.; BOFF, M. & BENNETT, J.L. - Phenol oxidase activity: induction by in vitro incubation. J. Parasit., 64: 283-289, 1978.
25. SEED, J.L. & BENNETT, J.L. - Schistosoma mansoni: phenol oxidases role in eggshell formation. Exp. Parasit., 49: 430-441, 1980.
26. SEED, J.L.; KILTS, C.D. & BENNETT, J.L. - Schistosoma mansoni: tyrosine, a putative in vivo substrate of phenol oxidase. Exp. Parasit., 50: 33-44, 1980.
27. SHER, A. - Schistosomiasis. Parasiting the cytokine system. Nature, 36: 565-566, 1992.
28. SMITHERS, S R. & TERRY, R.J. - Infection of laboratory hosts with cercariae of Schistosoma mansoni and the recovery of adult worms. Parasitology, 55: 695-700, 1965.
29. SMITHERS, S.R. & TERRY, R.J. - Immunity in schistosomiasis. Ann. N. Y. Acad. Sci., 160: 826-840, 1969.
30. SMYTH, J.D. & CLEGG, J.A. - Egg-shell formation in Treamatodes and Cestodes. Exp. Parasit., 8: 286-323, 1959.
31. STADECKER, M.J. - The role of T-cell anergy in the immu-nomodulation of schistosomiasis. Parasit today, 8: 199-204, 1992.
32. VON LICHTENBERG, F. - Consequences of infections with Schistosomes. In: ROLLINSON, D. & SIMPSON, A.J.G., ed. - The biology of Schistosomes: from genes to latrines. London, Academic Press, 1987. p. 185-232.
33. WAITE, J.H. & RICE-FICHT, A.C. - Presclerotized eggshell protein from the liver fluke Fasciola hepatica. Biochemistry, 26: 7819-7825, 1987.
34. WAITE, J.H. - The phylogeny and chemical diversity of quinone-tanned glues and varnishes. Comp. Biochem. Physiol., 97B: 19-29, 1990.
35. WARREN, K.S.; DOMINGO, E.O. & COWAN, R.T.B. - Granuloma formation around schistosome eggs as a manifestation of delayed hipersensitivity. Amer. J. Path., 51: 735-745, 1967.
36. WARREN, K.S. - Pathophysiology and pathogenesis of hepatosplenic schistosomiasis mansoni. Bull. N. Y. Acad. Med. 44: 280-294, 1968.
37. WELLS, K. & CORDINGLEY, J.S. - Schistosoma mansoni: eggshell formation is regulated by pH and calcium. Exp. Parasit., 73: 295-310, 1991.
38. WRIGHT, T.R.F. - The genetics of biogenic amine metabolism, sclerotization and melanization in Drosophila melanogaster. Advanc. Genet., 24: 127-222, 1987.
39. WYLER, D.J. - Why does liver fibrosis occurs in schistosomiasis? Parasit. today, 8: 277-279, 1992.

Endereço para correspondência:

Dr. Vanderlei Rodrigues

Depto. de Parasitologia,Microbiologia e Imunologia - Faculdade de Medicina de Ribeirão Preto

14049-900 Ribeirão Preto, São Paulo, SP, Brasil

Datas de Publicação

Publicação nesta coleção
21 Set 2006
Data do Fascículo
Out 1994

Histórico

Recebido
20 Dez 1993
Aceito
08 Jul 1994

This work is licensed under a Creative Commons Attribution-NonCommercial 4.0 International License.

[1] 1. AMIRI, P.; LOCKSLEY, R.M.; PARSLOV, T.G. et al. - Tumour necrosis factor restores granulomas and induces egg-laying in schistosome-infected SCID mice. Nature, 356: 604-607, 1992.

[2] 2. ANDRADE, Z. & GRIMAUD, J.A. - Morfology of chronic collagen resorption: a study on the late stages of Schistosoma granuloma involution. Amer. J. Path., 132: 389-399, 1988.

[3] 3. BENNETT, J.L.; SEED, J.L. & BOFF, M. - Fluorescent histochemical localization of phenol oxidase in female Schistosoma mansoni. J. Parasit., 64: 941-944, 1978.

[4] 4. BYRAM, J.E. & SENFT, A.W. - Structure of the Schistosome eggshell - amino acid analysis and incorporation of labelled amino acids. Amer. J. trop. Med. Hyg., 28: 539-547, 1979.

[5] 5. CANICATTI, C. & SEYMOUR, J. - Evidence for phenoloxidase activity in Holothuria tubulosa (Echinodermata) brown bodies and cells. Parasit. Res., 77: 50-53, 1991.

[6] 6. CLAUSEN, J. - Immunochemical techniques for the identification and estimation of macromolecules. In: WORK, T.S. & WORK, E., ed. - Laboratory techniques in biochemistry and molecular biology. Amsterdan, North Holland Publ., 1972. v. 1, p. 397-572.

[7] 7. CORDINGLEY, J.S. - Trematode eggshells: novel protein biopolymers. Parasit. today, 3: 341-344, 1987.

[8] 8. DURRANT, H.J.; RATCLIFFE, N.A.; HIPKIN, C.R.; ASPAN, A. & SODERHALL, K. - Purification of the pro-phenol oxidase enzyme from haemocytes of the cockroach Blaberus discoidalis. Biochem. J., 289: 87-91, 1993

[9] 9. GILLESPIE, J.P.; BIDOCHKA, M.J. & KHACHATOURIANS, G.G. - Separation and characterization of grasshopper hemolymph phenoloxidases by Sodium Dodecyl Sulfate - polyacrilamide gel electrophoresis. Comp. Biochem. Physiol., 98C: 351-358, 1991.

[10] 10. HAGAN, P. & WILKINS, H.A. - Concomitant immunity in schistosomiasis. Parasit. today, 9: 3-6, 1993.

[11] 11. HARTREE, E.F. - Determination of protein: a modification of the Lowry method that gives a linear photometric response. Analyt. Biochem., 48: 422-427, 1972.

[12] 12. JOHNSON, K.S.; TAYLOR, D.W. & CORDINGLEY, J.S. - Possible eggshell protein gene from Schistosoma mansoni. Molec. Biochem. Parasit., 22: 89-100, 1987.

[13] 13. MA, L. - Trace elements and polyphenoloxidase in Clonorchis sinensis. J. Parasit., 49: 197-203, 1963.

[14] 14. MANSOUR, T.E. - Effect of serotonin on phenol oxidase from the liver fluke Fasciola hepatica and from others sources. Biochim. biophys. Acta, 30: 492-500, 1958.

[15] 15. MITCHELL, G.F.; TIU, W.U. & GARCIA, E.G. - Infection characteristics of Schistosoma japonicum in mice and relevance to the assesment to Schistosomes vaccines. Advanc. Parasit., 30: 167-200, 1991.

[16] 16. NELLAIAPPAN, K. & RAMALINGAM, K. - Specificity of the enzyme phenoloxidase and possible pathway of sclerotization in Parapleurus sauridae. J. Parasit., 66: 217-219, 1980.

[17] 17. NELLAIAPPAN, K.; DEVASUNDRAI, A.F. & CHAN-DAYUTHAPANI, S. - Properties of phenol oxidase in Fasciola gigantica. Parasitology, 99: 403-407, 1989.

[18] 18. RIBEIRO-PAES, J.T. - Caracterizaçăo do sistema enzimático fenoloxidase em Schistosoma mansoni. Ribeirăo Preto, 1993. (Tese de Doutoramento - Faculdade de Medicina de Ribeirăo Preto da Universidade de Săo Paulo).

[19] 19. ROCH, P.; CANICATTI, P. & SEYMOUR, S. - Tetrameric structure of the active phenoloxidase evidenced in the coelomocytes of the echinoderm Holothuria tubulosa. Comp. Biochem. Physiol., 102B: 349-355, 1992.

[20] 20. RODRIGUES, V.; CHAUDRI, M.; KNIGHT, M. et al. - Predicted structure of a major Schistosoma mansoni eggshell protein. Molec. Biochem. Parasit., 32: 7-14, 1989.

[21] 21. ROLLE, R.S.; MARSHALL, M.R.; WEI, C.I. & CHEN, J.S. - Phenoloxidase forms of the florida spiny lobster: immunological and spectropolarimetric characterization. Comp. Biochem. Physiol., 97B: 483-489, 1990.

[22] 22. SCHAEFFER, J.; KRAMER, K.J.; GARBOW, J.R. et al. - Aromatic cross-links in insect cuticle: detection by solid-state ¹³Cand ¹⁵NNMR. Science, 235: 1200-1204, 1987.

[23] 23. SCHEIDEGGER, J.J. - Une micro-méthode de l'immuno-electrophorese. Intern. Arch. Allergy, 7: 103-110, 1955.

[24] 24. SEED, J.L.; BOFF, M. & BENNETT, J.L. - Phenol oxidase activity: induction by in vitro incubation. J. Parasit., 64: 283-289, 1978.

[25] 25. SEED, J.L. & BENNETT, J.L. - Schistosoma mansoni: phenol oxidases role in eggshell formation. Exp. Parasit., 49: 430-441, 1980.

[26] 26. SEED, J.L.; KILTS, C.D. & BENNETT, J.L. - Schistosoma mansoni: tyrosine, a putative in vivo substrate of phenol oxidase. Exp. Parasit., 50: 33-44, 1980.

[27] 27. SHER, A. - Schistosomiasis. Parasiting the cytokine system. Nature, 36: 565-566, 1992.

[28] 28. SMITHERS, S R. & TERRY, R.J. - Infection of laboratory hosts with cercariae of Schistosoma mansoni and the recovery of adult worms. Parasitology, 55: 695-700, 1965.

[29] 29. SMITHERS, S.R. & TERRY, R.J. - Immunity in schistosomiasis. Ann. N. Y. Acad. Sci., 160: 826-840, 1969.

[30] 30. SMYTH, J.D. & CLEGG, J.A. - Egg-shell formation in Treamatodes and Cestodes. Exp. Parasit., 8: 286-323, 1959.

[31] 31. STADECKER, M.J. - The role of T-cell anergy in the immu-nomodulation of schistosomiasis. Parasit today, 8: 199-204, 1992.

[32] 32. VON LICHTENBERG, F. - Consequences of infections with Schistosomes. In: ROLLINSON, D. & SIMPSON, A.J.G., ed. - The biology of Schistosomes: from genes to latrines. London, Academic Press, 1987. p. 185-232.

[33] 33. WAITE, J.H. & RICE-FICHT, A.C. - Presclerotized eggshell protein from the liver fluke Fasciola hepatica. Biochemistry, 26: 7819-7825, 1987.

[34] 34. WAITE, J.H. - The phylogeny and chemical diversity of quinone-tanned glues and varnishes. Comp. Biochem. Physiol., 97B: 19-29, 1990.

[35] 35. WARREN, K.S.; DOMINGO, E.O. & COWAN, R.T.B. - Granuloma formation around schistosome eggs as a manifestation of delayed hipersensitivity. Amer. J. Path., 51: 735-745, 1967.

[36] 36. WARREN, K.S. - Pathophysiology and pathogenesis of hepatosplenic schistosomiasis mansoni. Bull. N. Y. Acad. Med. 44: 280-294, 1968.

[37] 37. WELLS, K. & CORDINGLEY, J.S. - Schistosoma mansoni: eggshell formation is regulated by pH and calcium. Exp. Parasit., 73: 295-310, 1991.

[38] 38. WRIGHT, T.R.F. - The genetics of biogenic amine metabolism, sclerotization and melanization in Drosophila melanogaster. Advanc. Genet., 24: 127-222, 1987.

[39] 39. WYLER, D.J. - Why does liver fibrosis occurs in schistosomiasis? Parasit. today, 8: 277-279, 1992.

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