Identification and characterization of an asparaginyl proteinase (legumain) from the parasitic nematode, Haemonchus contortus.
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 dataset. 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.
Oliver
E M
EM
Moredun Research Institute, Pentlands Science Park, Bush Loan, Penicuik, Midlothian EH26 0PZ, UK.
Skuce
P J
PJ
McNair
C M
CM
Knox
D P
DP
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D015153:Blotting, Western; D003001:Cloning, Molecular; D003546:Cysteine Endopeptidases / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification*; D015971:Gene Expression Regulation, Enzymologic; D006189:Haemonchus / Q000145:classification / Q000201:enzymology*; D006801:Humans; D008018:Life Cycle Stages; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010802:Phylogeny; D051381:Rats; D020133:Reverse Transcriptase Polymerase Chain Reaction; D017386:Sequence Homology, Amino Acid; D012756:Sheep; D000099240:Asparaginyl Endopeptidase
D003546:Cysteine Endopeptidases; D000099240:Asparaginyl Endopeptidase