Teladorsagia circumcincta 1,6 bisphosphate aldolase: molecular and biochemical characterisation, structure analysis and recognition by immune hosts.

Abstract

A 1095 bp full length cDNA encoding Teladorsagia circumcincta aldolase (TciALDO) was cloned, expressed in Escherichia coli, the recombinant protein purified and its kinetic properties determined. A phylogenetic tree was constructed using helminth aldolase sequences. The predicted protein consisted of 365 amino acids and was present as a single band of about 44 kDa on SDS-PAGE. Multiple alignments of the protein sequence of TciALDO with homologues from other helminths showed that the greatest similarity (93%) to the aldolases of Haemonchus contortus and Dictyocaulus viviparus, 82-86% similarity to the other nematode sequences and 68-71% similarity to cestode and trematode enzymes. Substrate binding sites and conserved regions were identified and were completely conserved in other homologues. At 25 {degrees}C, the optimum pH for TciALDO activity was pH 7.5, the Vmax was 432 {+/-} 23 nmoles.min-1.mg-1 protein and the apparent Km for the substrate fructose 1,6-bisphosphate was 0.24 {+/-} 0.01 M (mean {+/-} SEM, n = 3). Antibodies in both serum and saliva from field-immune, but not nematode-naive, sheep recognised recombinant TciALDO in enzyme-linked immunosorbent assays. The recognition of the recombinant protein by antibodies generated by exposure of sheep to native aldolase indicates similar antigenicity of the two proteins.