Molecular and biochemical characterisation and recognition by the immune host of the glyceraldehyde 3-phosphate dehydrogenase (GAPDH) of the abomasal nematode parasite Teladorsagia circumcincta

Abstract A 1023 bp full length cDNA encoding Teladorsagia circumcincta GAPDH ( Teci GAPDH) was cloned, expressed in Escherichia coli and the recombinant protein purified and its kinetic properties determined. A phylogenetic tree was constructed using helminth GAPDH sequences. The predicted protein consisted of 341 amino acids and was present as a single band of about 38 kDa on SDS-PAGE. Multiple alignments of the protein sequence of Teci GAPDH with homologues from other helminths showed that the greatest similarity (93%) to the GAPDH 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 °C, the optimum pH for Teci GAPDH activity was pH 8, the V max was 1052 ± 23 nmol min −1  mg −1 protein and the apparent K m for the substrate glyceraldehyde-3-phosphate was 0.02 ± 0.01 mM (both mean ± SD, n = 2). Antibodies in both serum and saliva from field-immune, but not nematode-naive, sheep recognised recombinant Teci GAPDH in enzyme-linked immunosorbent assays. The recognition of the recombinant protein by antibodies generated by exposure of sheep to native GAPDH indicates similar antigenicity of the two proteins.