Relative protective properties of three membrane glycoprotein fractions from Haemonchus contortus

Abstract

Jacalin lectin was used as a ligand to isolate a fraction containing two distinct protective antigens from detergent extracts of membranes from Haemonchus contortus. The first antigen was identified as a complex which appeared very similar to Haemonchus galactose-containing glycoprotein (H-gal-GP), which is a previously described protective protease complex, except that it was substantially depleted of one of the main H-gal-GP components, a 230 kDa metallopeptidase-containing band. The new complex was termed Haemonchus sialylated galactose-containing glycoprotein (H-sialgal-GP), because it bound to jacalin but not to peanut lectin and only jacalin will bind the sialylated form of galactosyl (beta-1, 3) N-acetylgalactosamine. Two protection trials with sheep showed that H-sialgal-GP and H-gal-GP were equally efficacious, reducing numbers of Haemonchus eggs by between 86% and 93% and worms by between 52% and 75%, respectively. The second jacalin-binding protective antigen fraction was separated from H-sialgal-GP by ion exchange and gel filtration chromatography. It was greatly enriched for two proteins termed p46 and p52 according to their apparent molecular weights. Immunization of sheep with these proteins gave protection values of 78% for eggs and 33% for worms, which are significantly lower than those obtained with either H-gal-GP or H-sialgal-GP. N-terminal amino acid sequence data from p46 and p52 showed that both proteins were closely related to a previously described 45 kDa Haemonchus membrane protein, which had conferred protection against Haemonchus in guinea-pigs.

Identification of Secreted Cysteine Proteases from the Parasitic Nematode Haemonchus contortus Detected by Biotinylated Inhibitors

“…The estimated molecular masses of spots 1 and 2 (32.4 and 32.5 kDa, respectively) correspond to the 31-kDa size reported for the C-terminal domain (22). Similarly, MEP3 (with a predicted size of 95.5 kDa) was shown to resolve in Nand C-terminal domains of 41 and 47 kDa under reducing conditions (24), and the size observed for MEP3 in spot 2 (32.5 kDa) indicates further processing. Previous proteomic analysis of H. contortus ES identified the presence of the N-and Cterminal domains obtained after cleavage of other metalloproteases (MEP1, MEP1B, and MEP2) and serine proteases (28).…”

“…Both are components of a galactose-containing glycoprotein complex (H-gal-GP) with immunoprotective properties located on the luminal surface of the intestine (22)(23)(24). PEP2 is cleaved into N-and C-terminal domains that are held together by disulfide bonds which are broken under the reducing conditions used for gel electrophoresis in the second dimension (28).…”

Proteomic identification of Galectin-11 and 14 ligands from Haemonchus contortus

“…Zinc metallopeptidases have been reported as the major protein fraction of host protective glycoprotein complex H-gal-GP (Haemonchus galactose containing glycoprotein). Several studies isolated zinc metallopeptidases from crude extracts of H. contortus using lectins that have a binding preference to β-D-galactose and, following vaccination of sheep, led to reduced worm burdens following challenge infection (Dicker et al 2014;Newlands GFJ 2006;Smith et al 1999;Smith et al 2000).…”

Affinity-Based Methods for the Separation of Parasite Proteins

“…However, the reports on this subject demonstrate that this technique is useful for the retrieval of putative virulence factors or potential protective immunogens from a large array of parasites, including apicomplexan, trypanosomatids and nematodes (e.g., Fauquenoy et al, 2008, Gardiner et al, 1996, Smith et al, 2000. In addition to its utility in the isolation of parasite factors, lectinbased affinity chromatography is also a valuable resource for characterization of the structure of carbohydrates bound to proteins from these organisms due to the distinct specificities of the lectins that are available for this type of analysis.…”