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Identification and characterization of an asparaginyl proteinase (legumain) from the parasitic nematode, Haemonchus contortus

Published online by Cambridge University Press:  02 May 2006

E. M. OLIVER ,
P. J. SKUCE ,
C. M. McNAIR  and
D. P. KNOX
E. M. OLIVER
Affiliation:
Moredun Research Institute, Pentlands Science Park, Bush Loan, Penicuik, Midlothian EH26 0PZ, UK
P. J. SKUCE
Affiliation:
Moredun Research Institute, Pentlands Science Park, Bush Loan, Penicuik, Midlothian EH26 0PZ, UK
C. M. McNAIR
Affiliation:
Moredun Research Institute, Pentlands Science Park, Bush Loan, Penicuik, Midlothian EH26 0PZ, UK
D. P. KNOX
Affiliation:
Moredun Research Institute, Pentlands Science Park, Bush Loan, Penicuik, Midlothian EH26 0PZ, UK
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Abstract

Asparaginyl proteinases (or legumains) are a recently identified, novel class of cysteine proteinase which specifically hydrolyse peptide bonds after asparagine residues. Legumains have been implicated in the activation of cysteine proteases, particularly cathepsin B-like proteinases which are thought to help degrade the bloodmeal in blood-feeding helminths such as schistosomes, hookworms and other nematode species. An EST sequence representing a full-length legumain was identified from the Haemonchus contortus datasetNucleotide sequence data described in this paper have been deposited in the EMBL database under the Accession number AM177177.. This encoded a protein with a predicted Mr of 49 kDa, the amino acid sequence of which showed good homology (34–40% identity) to legumains from Schistosoma mansoni, human and rat and contained a legumain-like active site. RT-PCR indicated that the legumain transcript was expressed from the L4 life-cycle stage onwards. The coding sequence was expressed in E. coli and antibodies to the resultant recombinant protein indicated that the enzyme was expressed in the microvillar surface of the intestinal cells. Legumain activity was detected in extracts of the adult parasite but not the host protective Thiol-Sepharose-binding fraction, although it was detectable in the latter by immunoblot. Activity was relatively insensitive to E64, an inhibitor of cysteine proteinases and completely inhibited by the alkylating agent, N-ethylmaleimide, consistent with inhibitor effects on previously characterized legumains.

Keywords

Haemonchus contortuslegumainenzyme activitylocalization
Type
Research Article
Information
Parasitology , Volume 133 , Issue 2 , August 2006 , pp. 237 - 244
Copyright
2006 Cambridge University Press

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References

REFERENCES

Altschul, S. F., Gish, W., Miller, W., Meyers, E. W. and Lipman, D. J. ( 1990). Basic local alignment search tool. Journal of Molecular Biology 215, 403–410.CrossRefGoogle Scholar
Baig, S., Damian, R. T. and Peterson, D. S. ( 2002). A novel cathepsin B active site motif is shared by helminth blood feeders. Experimental Parasitology 101, 83–89.CrossRefGoogle Scholar
Brindley, P. J. and Dalton, J. P. ( 2004). Schistosome legumain. In Handbook of Proteolytic Enzymes, 2nd Edn ( ed. Barrett, A. J., Rawlings, N. D. and Woessner, J. F.), pp. 1305–1310. Academic Press, London, UK.
Caffrey, C. R., Mathieu, M. A., Gaffney, A. M., Salter, J. P., Sajid, M., Lucas, K. D., Franklin, C., Bogyo, M. and McKerrow, J. H. ( 2000). Identification of a cDNA encoding an active asparaginyl endopeptidase of Schistosoma mansoni and its expression in Pichia pastoris. FEBS Letters 466, 244–248.CrossRefGoogle Scholar
Chen, J.-M., Dando, P. M., Rawlings, N. D., Brown, M. A., Young, N. E., Stevens, R. A., Hewitt, E., Watts, C. and Barrett, A. J. ( 1997). Cloning, isolation, and characterization of mammalian legumain, an asparaginyl endopeptidase. Journal of Biological Chemistry 272, 8090–8098.CrossRefGoogle Scholar
Chen, J.-M., Rawlings, N. D., Stevens, R. and Barrett, A. J. ( 1998). Identification of the active site of legumain links it to caspases, clostripain and gingipains in a new clan of cysteine endopeptidases. FEBS Letters 441, 361–365.CrossRefGoogle Scholar
Dalton, J. P., Hola-Jamriska, L. and Brindley, P. J. ( 1995). Asparaginyl endopeptidase activity in adult Schistosoma mansoni. Parasitology 111, 575–580.CrossRefGoogle Scholar
Dalton, J. P. and Brindley, P. J. ( 1996). Schistosome asparaginyl endopeptidase Sm32 in haemoglobin digestion. Parasitology Today 12, 125.CrossRefGoogle Scholar
Dalton, J. P., Brindley, P. J., Knox, D. P., Brady, C. P., Hotez, P. J., Donnelly, S., O'Neill, S. M., Mulcahy, G. and Loukas, A. ( 2003). Helminth vaccines: from mining genomic information for vaccine targets to systems used for protein expression. International Journal for Parasitology 33, 621–640.CrossRefGoogle Scholar
Knox, D. P., Redmond, D. L. and Jones, D. G. ( 1993). Characterization of proteinases in extracts of adult Haemonchus contortus, the ovine abomasal nematode. Parasitology 106, 395–404.CrossRefGoogle Scholar
Knox, D. P., Smith, S. K. and Smith, W. D. ( 1999). Immunisation with an affinity purified protein extract from the adult parasite protects lambs against infection with Haemonchus contortus. Parasite Immunology 21, 201–210.CrossRefGoogle Scholar
Kumar, S., Tamura, K. and Nei, M. ( 2004). MEGA3: Integrated software for molecular evolutionary genetics analysis and sequence alignment. Briefings in Bioinformatics 5, 150–163.CrossRefGoogle Scholar
Leon-Felix, J., Ortega-Lopez, J., Orozco-Solis, R. and Arroyo, R. ( 2004). Two novel asparaginyl endopeptidase-like cysteine proteinases from the protist Trichomonas vaginalis: their evolutionary relationship within the clan CD cysteine proteinases. Gene 335, 25–35.CrossRefGoogle Scholar
Maizels, R. M., Tetteh, K. K. A. and Loukas, A. ( 2000). Toxocara canis: genes expressed by the arrested infective larval stage of a parasitic nematode. International Journal for Parasitology 30, 495–508.CrossRefGoogle Scholar
Munn, E. A., Smith, T. S., Graham, M., Tavernor, A. S. and Greenwood, C. A. ( 1993). The potential value of integral membrane proteins in the vaccination of lambs against Haemonchus contortus. International Journal for Parasitology 23, 261–269.CrossRefGoogle Scholar
Newton, S. E. and Munn, E. A. ( 1999). The development of vaccines against gastrointestinal nematode parasites, particularly Haemonchus contortus. Parasitology Today 15, 116–122.CrossRefGoogle Scholar
Parkinson, J., Whitton, C., Schmid, R., Thomson, M. and Blaxter, M. ( 2004). NEMBASE: a resource for parasitic nematodes ESTs. Nucleic Acids Research, Database issue D427-D430.CrossRef
Redmond, D. L., Knox, D. P., Newlands, G. and Smith, W. D. ( 1997). Molecular cloning and characterisation of a developmentally regulated putative metallopeptidase present in a host protective extract of Haemonchus contortus. Molecular and Biochemical Parasitology 85, 77–87.CrossRefGoogle Scholar
Redmond, D. L. and Knox, D. P. ( 2004). Protection studies in sheep using affinity purified and recombinant cysteine proteinases of adult Haemonchus contortus. Vaccine 22, 4252–4261.CrossRefGoogle Scholar
Sajid, M. and McKerrow, J. H. ( 2002). Cysteine proteases of parasitic organisms. Molecular and Biochemical Parasitology 120, 1–21.CrossRefGoogle Scholar
Sajid, M., McKerrow, J. H., Hansell, E., Mathieu, M. A., Lucas, K. D., Hsieh, I., Greenbaum, D., Bogyo, M., Salter, J. P., Lim, K. C., Franklin, C., Kim, J. and Caffrey, C. R. ( 2003). Functional expression and characterisation of Schisosoma mansoni cathepsin B and its trans-activation by an endogenous asparaginyl endopeptidase. Molecular and Biochemical Parasitolgy 131, 65–75.CrossRefGoogle Scholar
Sharp, P. J., Smith, D. R. J., Bach, W., Wagland, B. M. and Cobon, G. S. ( 1991). Purified Glutathione S-transferases from parasites as candidate protective antigens. International Journal for Parasitology 21, 839–846.CrossRefGoogle Scholar
Skuce, P. J., Redmond, D. L., Liddell, S., Stewart, E. M., Newlands, G. F. J., Smith, W. D. and Knox, D. P. ( 1999). Molecular cloning and characterization of gut derived cysteine proteinases associated with a host protective extract from Haemonchus contortus. Parasitology 119, 405–412.CrossRefGoogle Scholar
Smith, S. K., Pettit, D., Newlands, G. F., Redmond, D. L., Skuce, P. J., Knox, D. P. and Smith, W. D. ( 1999). Further immunization and biochemical studies with a protective antigen complex from the microvillar membrane of the intestine of Haemonchus contortus. Parastie Immunology 21, 187–199.CrossRefGoogle Scholar
Smith, W. D., Smith, S. K. and Murray, J. M. ( 1994). Protection studies with integral membrane fractions of Haemonchus contortus. Parasite immunology 16, 231–241.CrossRefGoogle Scholar
Torodova, V. K., Knox, D. P. and Kennedy, M. W. ( 1995). Proteinases in the excretory/secretory products (ES) of adult Trichinella spiralis. Parasitology 111, 201–208.CrossRefGoogle Scholar
Tort, J., Brindley, P. J., Knox, D., Wolfe, K. H. and Dalton, J. P. ( 1999). Proteinases and associated genes of parasitic helminths. Advances in Parasitology 43, 162–266.CrossRefGoogle Scholar
Williamson, A. L., Brindley, P. J., Knox, D. P., Hotez, P. J. and Loukas, A. ( 2003). Digestive proteases of blood-feeding nematodes. Trends in Parasitology 19, 417–423.CrossRefGoogle Scholar
Williamson, A. L., Lecchi, P., Turk, B. E., Choe, Y., Hotez, P. J., McKerrow, J. H., Cantley, L. C., Sajid, M., Craik, C. S. and Loukas, A. ( 2004). A multi-enzyme cascade of hemoglobin proteolysis in the intestine of blood-feeding hookworms. Journal of Biological Chemistry 279, 35950–35957.CrossRefGoogle Scholar