title: Molecular and biochemical characterisation and recognition by the immune host of the enolase of the abomasal nematode parasite Teladorsagia circumcincta.
abstract: A 1299 bp full length cDNA encoding Teladorsagia circumcincta enolase (TeciENO) was cloned, expressed in Escherichia coli and the recombinant protein purified and its kinetic properties determined. Helminth enolase sequences were used to construct a phylogenetic tree. The predicted protein consisted of 433 amino acids and was present as a single band of about 50 kDa on SDS-PAGE. Multiple alignments of the protein sequence of TeciENO with homologues from other helminths showed 98% similarity with Haemonchus contortus enolase, 78-95% similarity to other nematode sequences and 72-75% similarity to cestode and trematode enolases. Substrate binding sites and conserved regions were identified and were completely conserved in other homologues. The optimum pH for TeciENO activity at 25 °C was pH 7, the Kmfor 2-phophoglycerate 0.09 ± 0.04 mM and the Vmaxwas 604 ± 6 nmol min-1mg-1protein (both mean ± SD, n = 2). TeciENO activity was inhibited by 11.5% by 1 mM citrate (p < 0.001). Antibodies in both serum and saliva from field-immune, but not nematode-naïve, sheep recognised recombinant TeciENO in enzyme-linked immunosorbent assays. The recognition of the recombinant protein by antibodies generated by exposure of sheep to native enolase indicates similar antigenicity of the two proteins.
authors.affiliation: AgResearch Ltd, Private Bag 11-008, Palmerston North, New Zealand. Electronic address: saleh.umair@agresearch.co.nz.
authors.forename: S
authors.initials: S
authors.lastname: Umair
authors.affiliation: AgResearch Ltd, Private Bag 11-008, Palmerston North, New Zealand.
authors.forename: C L G
authors.initials: CL
authors.lastname: Bouchet
authors.affiliation: AgResearch Ltd, Private Bag 11-008, Palmerston North, New Zealand.
authors.forename: J S
authors.initials: JS
authors.lastname: Knight
authors.affiliation: AgResearch Ltd, Private Bag 11-008, Palmerston North, New Zealand.
authors.forename: A
authors.initials: A
authors.lastname: Pernthaner
authors.affiliation: Institute of Veterinary, Animal and Biomedical Sciences, Massey University, Private Bag 11-222, Palmerston North, New Zealand.
authors.forename: H V
authors.initials: HV
authors.lastname: Simpson
mesh_terms: D000018:Abomasum / Q000469:parasitology; D000595:Amino Acid Sequence; D000818:Animals; D000909:Antibodies, Helminth / Q000097:blood / Q000276:immunology*; D003001:Cloning, Molecular; D018076:DNA, Complementary; D018509:DNA, Helminth / Q000235:genetics; D004797:Enzyme-Linked Immunosorbent Assay; D015801:Helminth Proteins / Q000737:chemistry / Q000235:genetics / Q000276:immunology / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D010751:Phosphopyruvate Hydratase / Q000737:chemistry / Q000235:genetics / Q000276:immunology* / Q000378:metabolism*; D010802:Phylogeny; D011994:Recombinant Proteins; D012463:Saliva / Q000276:immunology; D012756:Sheep; D012757:Sheep Diseases / Q000276:immunology; D014251:Trichostrongyloidea / Q000201:enzymology* / Q000276:immunology*; D014252:Trichostrongyloidiasis / Q000276:immunology / Q000469:parasitology / Q000662:veterinary*
chemical_list: D000909:Antibodies, Helminth; D018076:DNA, Complementary; D018509:DNA, Helminth; D015801:Helminth Proteins; D011994:Recombinant Proteins; D010751:Phosphopyruvate Hydratase
keywords: Antigenicity; Enolase; Gene sequence; Kinetic properties; Teladorsagia circumcincta