Molecular and biochemical characterisation of abomasal nematode parasites Teladorsagia circumcincta and Haemonchus contortus phosphofructokinases and their recognition by the immune host

Full length cDNAs encoding phosphofructokinase (PFK) were cloned from Teladorsagia circumcincta (TcPFK) and Haemonchus contortus (HcPFK). TcPFK (2361 bp) and HcPFK (2367 bp) cDNA encoded 787 and 789 amino acid proteins respectively. The predicted amino acid sequences showed 98% similarity with each other and 70% with a Caenorhabditis elegans PFK. Substrate binding sites were completely conserved in both proteins. Soluble N-terminal His-tagged PFK proteins were expressed in Escherichia call strain BL21, purified and characterised. The recombinant TcPFK and HcPFK had very similar kinetic properties: the pH optima were pH 7.0, K-m for fructose 6-phosphate was 0.50 +/- 0.01 and 0.55 +/- 0.01 mM respectively when higher (inhibiting concentration, 0.3 mM) ATP concentration was used and the curve was sigmoidal. The V-max for TcPFK and HcPFK were 1110 16 and 910 10 nM min(-1) mg(-1) protein respectively. Lower ATP concentration (non-inhibiting, 0.01 mM) did not change the V-max for TcPFK and HcPFK (890 +/- 10 and 860 12 nM min-1 me protein) but the substrate affinity doubled and K-m for fructose 6-phosphate were 0.20 +/- 0.05 and 0.25 +/- 0.01 mM respectively. Recognition of TcPFK and HcPFK by mucosal and serum antibodies in nematode exposed animals demonstrates antigenicity and suggests involvement in the host response to nematode infection. (C) 2015 Elsevier Inc. All rights reserved.