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

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 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.

Blastocystis Legumain Is Localized on the Cell Surface, and Specific Inhibition of Its Activity Implicates a Pro-survival Role for the Enzyme

“…We postulate that Blastocystis legumain possesses multiple functions and plays important roles at the cell surface and within acidic compartments. Legumains are generally located within lysosomes of mammalian cells and vacuoles of plant cells but have been reported to localize to the cell surface of metastatic tumors (17) and on the microvillar surfaces of helminth intestinal cells (25). Surface legumains were suggested to activate local zymogens that may aid tumor invasion or participate in helminth alimentary digestion of host proteins.…”

Characterization of asparaginyl endopeptidase, legumain induced by blood feeding in the ixodid tick Haemaphysalis longicornis

“…However, the fact that HlLgm expression was most abundant in the midgut, and furthermore that endogenous HlLgm expression was up regulated in partially fed ticks clearly suggest some critical relationship between HlLgm and blood-meal digestion. Legumains in the parasitic nematode Haemonchus contortus (Oliver et al, 2006) andS. mansoni (El Meanawy et al, 1990) have been reported to be expressed in the gut epithelial cells of the parasites.…”

“…The pH optima of the legumains of blood-feeding helminth parasites S. mansoni and H. contortus are pH 6.8 and pH 7, respectively (Dalton, et al, 1995;Oliver et al, 2006). In contrast, mammalian and plant legumains have been shown to be active in acidic environment and are very labile at neutral pH (Hara-Nishimura et al, 1991;Ishii, 1994;Chen et al, 1997, Chen et al, 1998a.…”

Identification and functional characterization of legumain in amphioxus Branchiostoma belcheri

“…22.34), also known as asparaginyl endopeptidase, is a novel class of cysteine protease, which was identified originally in leguminous plants, hence the name legumain [1], and later first identified in animals in the human blood fluke Schistosoma mansoni [2]. It has since been found in mammals [3,4], in the parasitic protozoan Trichomonas vaginalis [5], in the parasitic nematodes Toxocara canis and Brugia malayi [6], and Haemonchus contortus [7], and in the parasitic arthropods Ixodes ricinus [8], Fasciola gigantica [9] and Haemaphysalis longicornis [10]. However, little information regarding legumains is available in the lower chordates.…”