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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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