Parasites, Hosts and Diseases

The Korea Society for Parasitology and Tropical Medicine (대한기생충학·열대의학회)
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Molecular and biochemical characterization of hemoglobinase, a cysteine proteinase, in Paragonimus westermani

  • Choi Joon-Hyuck (Department of Parasitology, Kyungpook National University School of Medicine) ;
  • Lee Jae-Hyuk (Department of Parasitology, Kyungpook National University School of Medicine) ;
  • Yu Hak-Sun (Department of Parasitology, Pusan National University School of Medicine) ;
  • Jeong Hae-Jin (Department of Parasitology, Pusan National University School of Medicine) ;
  • Kim Jin (Department of Parasitology, College of Medicine, Seonam University) ;
  • Hong Yeon-Chul (Department of Parasitology, Kyungpook National University School of Medicine) ;
  • Kong Hyun-Hee (Department of Parasitology, Kyungpook National University School of Medicine) ;
  • Chung Dong-Il (Department of Parasitology, Kyungpook National University School of Medicine)
  • Published : 2006.09.01
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Abstract

The mammalian trematode Paragonimus westermani is a typical digenetic parasite, which can cause paragonimiasis in humans. Host tissues and blood cells are important sources of nutrients for development, growth and reproduction of P. westermani. In this study, a cDNA clone encoding a 47 kDa hemoglobinase of P. westermani was characterized by sequencing analysis, and its localization was investigated immunohistochemically. The phylogenetic tree prepared based on the hemoglobinase gene showed high homology with hemoglobinases of Fasciola hepatica and Schistosoma spp. Moreover, recombinant P. westermani hemoglobinase degradaded human hemoglobin at acidic pH (from 3.0 to 5.5) and its activity was almost completely inhibited by E-64, a cysteine proteinase inhibitor. Immunohistochemical studies showed that P. westermani hemoglobinase was localized in the epithelium of the adult worm intestine implying that the protein has a specific function. These observations suggest that hemoglobinase may act as a digestive enzyme for acquisition of nutrients from host hemoglobin. Further investigations may provide insights into hemoglobin catabolism in P. westermani.

Keywords

References

  1. Auriault C, Pierce R, Cesari IM, Capron A (1982) Neutral protease activities at different developmental stages of Schistosoma mansoni in mammalian hosts. Comp Biochem Physiol B 72: 377-384 https://doi.org/10.1016/0305-0491(82)90215-2
  2. Baig S, Damian RT, Peterson DS (2002) A novel cathepsin B active site motif is shared by helminth bloodfeeders. Exp Parasitol 101: 83-89 https://doi.org/10.1016/S0014-4894(02)00105-4
  3. Becker MM, Harrop SA, Dalton JP, Kalinna BH, McManus DP, Brindley PJ (1995) Cloning and characterization of the Schistosoma japonicum aspartic proteinase involved in hemoglobin degradation. J Biol Chem 270: 24496-24501 https://doi.org/10.1074/jbc.270.41.24496
  4. Berry C, Humphreys MJ, Matharu P, Granger R, Horrocks P, Moon RP, Certa U, Ridley RG, Bur D, Kay J (1999) A distinct member of the aspartic proteinase gene family from the human malaria parasite Plasmodium falciparum. FEBS Letter 447: 149-154 https://doi.org/10.1016/S0014-5793(99)00276-8
  5. Brady CP, Dowd AJ, Brindley PJ, Ryan T, Day SR, Dalton JP (1999) Recombinant expression and localization of Schistosoma mansoni cathepsin L1 support its role in the degradation of host hemoglobin. Infect Immun 67: 368- 374
  6. Brown A, Burleigh JM, Billett EE, Pritchard DI (1995) An initial characterization of the proteolytic enzymes secreted by the adult stage of the human hookworm Necator americanus. Parasitology 110: 555-563 https://doi.org/10.1017/S0031182000065276
  7. Caffrey CR, McKerrow JH, Salter JP, Sajid M (2004) Blood 'n' guts: an update on schistosome digestive peptidases. Trends Parasitol 20: 241-248 https://doi.org/10.1016/j.pt.2004.03.004
  8. Carmona C, Dowd AJ, Smith AM, Dalton JP (1993) Cathepsin L proteinase secreted by Fasciola hepatica in vitro prevents antibody-mediated eosinophil attachment to newly excysted juveniles. Mol Biochem Parasitol 62: 9-17 https://doi.org/10.1016/0166-6851(93)90172-T
  9. Chung YB, Kong Y, Joo IJ, Cho SY, Kang SY (1995) Excystment of Paragonimus westermani metacercariae by endogenous cysteine protease. J Parasitol 81: 137-142 https://doi.org/10.2307/3283911
  10. Chung YB, Yang HJ, Kang SY, Kong Y, Cho SY (1997) Activities of different cysteine proteases of Paragonimus westermani in cleaving human IgG. Korean J Parasitol 35: 139-142 https://doi.org/10.3347/kjp.1997.35.2.139
  11. Dahl EL, Rosenthal PJ (2005) Biosynthesis, localization, and processing of falcipain cysteine proteases of Plasmodium falciparum. Mol Biochem Parasitol 139: 205-212 https://doi.org/10.1016/j.molbiopara.2004.11.009
  12. Dalton JP, Hola-Jamriska L, Brindley PJ (1995) Asparaginyl endopeptidase activity in adult Schistosoma mansoni. Parasitology 111: 575-580 https://doi.org/10.1017/S0031182000077052
  13. Gallego SG, Slade RW, Brindley PJ (1998) A cDNA encoding a pepsinogen-like, aspartic protease from the human roundworm parasite Strongyloides stercoralis. Acta Trop 71: 17-26 https://doi.org/10.1016/S0001-706X(98)00050-3
  14. Goldberg DE, Slater AF, Cerami A, Henderson GB (1990) Hemoglobin degradation in the malaria parasite Plasmodium falciparum: an ordered process in a unique organelle. Proc Natl Acad Sci USA 87: 2931-2935
  15. Gotz B, Klinkert MQ (1993) Expression and partial characterization of cathepsin B-like enzyme(Sm31) and a proposed 'haemoglobinase' (Sm32) from Schistosoma mansoni. Biochem J 290: 801-806 https://doi.org/10.1042/bj2900801
  16. Hamajima F, Yamakami K, Fujino T (1985) Localization of a thiol protease in metacercarial lung fluke. Jpn J Parasitol 34: 507-508
  17. Harrop SA, Prociv P, Brindley PJ (1996) Acasp, a gene encoding a cathepsin D-like aspartic protease from the hookworm Ancylostoma caninum. Biochem Biophys Res Commun 227: 294-302 https://doi.org/10.1006/bbrc.1996.1503
  18. Hata H, Yokogawa M, Kobayashi M, Niimura M, Kojima S (1987) In vitro cultivation of Paragonimus miyazakii and P. ohirai. J Parasitol 73: 792-796 https://doi.org/10.2307/3282416
  19. Hawthorne SJ, Halton DW, Walker B (1994) Identification and characterization of the cysteine proteinase and serine proteinases of the trematode, Haplometra cylindracea and determination of their haemoglobinase activity. Parasitology 108: 595-601 https://doi.org/10.1017/S0031182000077465
  20. Kim TS, De-Guzman JV, Kong HH, Chung DI (2006) Comparison of gene representation between diploid and triploid Paragonimus westermani by expressed sequence analyses. J Parasitol 92: (in press)
  21. Longbottom D, Redmond DL, Russell M, Liddell S, Smith WD, Knox DP (1997) Molecular cloning and characterization of a putative aspartate proteinase associated with a gut membrane protein complex from adult Haemonchus contortus. Mol Biochem Parasitol 88: 63-72 https://doi.org/10.1016/S0166-6851(97)00074-1
  22. Loukas A, Bethony JM, Williamson AL, Goud GN, Mendez S, Zhan B, Hawdon JM, Elena Bottazzi M, Brindley PJ, Hotez PJ (2004) Vaccination of dogs with a recombinant cysteine protease from the intestine of canine hookworms diminishes the fecundity and growth of worms. J Infect Dis 189: 1952-1961 https://doi.org/10.1086/386346
  23. McKerrow JH (1989) Parasite protease. Exp Parasitol 68: 111- 115 https://doi.org/10.1016/0014-4894(89)90016-7
  24. el Meanawy MA, Aji T, Phillips NF, Davis RE, Salata RA, Malhotra I, McClain D, Aikawa M, Davis AH (1990) Definition of the complete Schistosoma mansoni hemoglobinase mRNA sequence and gene expression in developing parasites. Am J Trop Med Hyg 43: 67-78 https://doi.org/10.4269/ajtmh.1990.43.67
  25. Merckelbach A, Hasse S, Dell R, Eschlbeck A, Ruppel A (1994) cDNA sequences of Schistosoma japonicum coding for two cathepsin B-like proteins and Sj32. Trop Med Parasitol 45: 193-198
  26. Moon RP, Tyas L, Certa U, Rupp K, Bur D, Jacquet C, Matile H, Loetscher H, Grueninger-Leitch F, Kay J, Dunn BH, Berry C, Ridley RG (1997) Expression and characterisation of plasmepsin I from Plasmodium falciparum. Eur J Biochem 244: 552-560 https://doi.org/10.1111/j.1432-1033.1997.00552.x
  27. Na BK, Kim SH, Lee EG, Kim TS, Bae YA, Kang I, Yu JR, Sohn WM, Cho SY, Kong Y (2006) Critical roles for excretory-secretory cysteine proteases during tissue invasion of Paragonimus westermani newly excysted metacercariae. Cell Microbiol 8: 1034-1046 https://doi.org/10.1111/j.1462-5822.2006.00685.x
  28. Otto HH, Schirmeister T (1997) Cysteine proteinase and their inhibitors. Chem Rev 97: 133-172 https://doi.org/10.1021/cr950025u
  29. Park GM, Lee KJ, Im KI, Park H, Young TS (2001) Occurence of a diploid type and a new first intermediate host of a human lung fluke, Paragonimus westermani, in Korea. Exp Parasitol 99: 206-212 https://doi.org/10.1006/expr.2001.4668
  30. Park H, Hong KM, Sakanari JA, Choi JH, Park SK, Kim KY, Hwang HA, Paik MK, Yun KJ, Shin CH, Lee JB, Ryu JS, Min DY (2001) Paragonimus westermani: cloning of a cathepsin F-like cysteine proteinase from the adult worm. Exp Parasitol 98: 223-227 https://doi.org/10.1006/expr.2001.4634
  31. Piekarski G (1989) Medical Parasitology. Springer, Berlin, p. 154
  32. Ray C, McKerrow JH (1992) Gut-specific and developmental expression of a Caenorhabditis elegans cysteine protease gene. Mol Biochem Parasitol 51: 239-249 https://doi.org/10.1016/0166-6851(92)90074-T
  33. Rosenthal PJ, Mckerrow JH, Aikawa M, Nagasawa H, Leech JH (1988) A malarial cysteine proteinase is necessary for hemoglobin degradation by Plasmodium falciparum. J Clin Invest 82: 1560-1566 https://doi.org/10.1172/JCI113766
  34. Sakanari JA, Staunton CE, Eakin AE, Craik CS, McKerrow JH (1989) Serine proteases from nematode and protozoan parasites isolation of sequence homologs using generic molecular probes. Proc Natl Acad Sci USA 86: 4863-4867
  35. Serrano-Luna JJ, Negrete E, Reyes M, de la Garza M (1998) Entamoeba histolytica HM1: IMSS: hemoglobin-degrading neutral cysteine proteases. Exp Parasitol 89: 71-77 https://doi.org/10.1006/expr.1998.4258
  36. Shim YS, Cho SY, Han YC (1991) Pulmonary paragonimiasis: a Korean perspective. Semin Respir Med 12: 35-45 https://doi.org/10.1055/s-2007-1006223
  37. Silva AM, Lee AY, Gulnik SV, Maier P, Collins J, Bhat TN, Collins PJ, Cachau RE, Luker KE, Gluzman IY, Francis SE, Oksman A, Goldberg DE, Erickson JW (1996) Structure and inhibition of plasmepsin II, a hemoglobin degrading enzyme from Plasmodium falciparum. Proc Natl Acad Sci USA 93: 10034-10039
  38. Silva FP Jr, Ribeiro F, Katz N, Giovanni-De-Simone S (2002) Exploring the subsite specificity of Schistosoma mansoni aspartyl hemoglobinase through comparative molecular modelling. FEBS Letter 514: 141-148 https://doi.org/10.1016/S0014-5793(02)02270-6
  39. Sijwali PS, Shenai BR, Gut J, Singh A, Rosenthal PJ (2001) Expression and characterization of the Plasmodium falciparum haemoglobinase falcipain-3. Biochem J 360: 481-489 https://doi.org/10.1042/0264-6021:3600481
  40. Tamashiro WK, Rao M, Scott AL (1987) Proteolytic cleavage of IgG and other protein substrates by Dirofilaria immitis microfilarial enzymes. J Parasitol 73: 149-154 https://doi.org/10.2307/3282360
  41. Toscano C, Yu SH, Nunn P, Mott KE (1995) Paragonimiasis and tubeculosis, diagnostic confusion: a review of the literature. Trop Dis Bull 92: R1-R26
  42. Williamson AL, Brindley PJ, Abbenante G, Datu BJ, Prociv P, Berry C, Girdwood K, Pritchard DI, Fairlie DP, Hotez PJ, Zhan B, Loukas A (2003) Hookworm aspartic protease, Na-APR-2, cleaves human hemoglobin and serum proteins in a host-specific fashion. J Infect Dis 187: 484-494 https://doi.org/10.1086/367708
  43. Williamson AL, Brindley PJ, Abbenante G, Prociv P, Berry C, Girdwood K, Pritchard DI, Fairlie DP, Hotez PJ, Dalton JP, Loukas A (2002) Cleavage of hemoglobin by hookworm cathepsin D aspartic proteases and its potential contribution to host specificity. FASEB J 16: 1458-1460 https://doi.org/10.1096/fj.02-0181fje
  44. Yang SH, Park JO, Lee JH, Jeon BH, Kim WS, Kim SI, Yun KJ, Jeong ET, Lee KW, Kim YM, Lee MH, Park H (2004) Cloning and characterization of a new cysteine proteinase secreted by Paragonimus westermani adult worms. Am J Trop Med Hyg 71: 87-92
  45. Yun DH, Chung JY, Chung YB, Bahk YY, Kang SY, Kong Y, Cho SY (2000) Structural and immunological characteristics of a 28-kilodalton cruzipain-like cysteine protease of Paragonimus westermani expressed in the definitive host stage. Clin Diagn Lab Immunol 7: 932-939 https://doi.org/10.1128/CDLI.7.6.932-939.2000