Characterisation of Macrophage Inhibitory Factor-2 (MIF-2) in Haemonchus contortus and Teladorsagia circumcincta

Abstract

Full-length cDNAs encoding macrophage inhibitory factor-2 (MIF-2) were cloned from Teladorsagia circumcincta (TcMIF-2) and Haemonchus contortus (HcMIF-2). TcMIF-2 and HcMIF-2 cDNA (342 bp) encoded proteins of 114 amino acids, each of which was present as a single band of about 16 kDa on SDS-PAGE. Multiple alignments of the protein sequences showed homology of 84% between TcMIF-2 and HcMIF-2, 54–76% with MIF-2s of seven nematodes, but low homology with other MIF sequences. The predicted three-dimensional structures revealed an overall structural homology of TcMIF-2 and HcMIF-2, highly conserved binding and catalytic sites and minor differences in the tautomerase binding site residues in other nematode MIF-2 homologues. A phylogenetic tree was constructed using helminth and mammalian MIF-1 and MIF-2 sequences. Soluble C-terminal MIF-2 proteins were cloned in arabinose inducible promotor AY2.4, expressed in Escherichia coli strain AY2.4 and purified. Recombinant TcMIF-2 and HcMIF-2 had similar enzyme activities in a standard tautomerase assay. Recombinant HcMIF-2 activity was approximately halved by storage at 4 °C, −20 °C or −70 °C. Antibodies in serum and saliva from field-immune, but not nematode-naïve, sheep recognised recombinant HcMIF-2 and TcMIF-2 in enzyme-linked immunosorbent assays. Recognition of the recombinant proteins by antibodies generated by exposure of sheep to the native enzyme indicates similar antigenicity of the two proteins.