title: Molecular and biochemical characterisation of abomasal nematode parasites Teladorsagia circumcincta and Haemonchus contortus phosphofructokinases and their recognition by the immune host.
abstract: 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 coli strain BL21, purified and characterised. The recombinant TcPFK and HcPFK had very similar kinetic properties: the pH optima were pH 7.0, Km 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 Vmax 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 Vmax for TcPFK and HcPFK (890 ± 10 and 860 ± 12 nM min(-1 )mg(-1) protein) but the substrate affinity doubled and Km 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.
authors: AgResearch Ltd, Private Bag 11-008, Palmerston North, New Zealand. Electronic address: saleh.umair@agresearch.co.nz.
authors: S
authors: S
authors: Umair
authors: AgroSup Dijon, 26 Boulevard Petitjean, BP 87999 21079-Dijon cedex, France.
authors: E
authors: E
authors: Dagnicourt
authors: AgResearch Ltd, Private Bag 11-008, Palmerston North, New Zealand.
authors: J S
authors: JS
authors: Knight
authors: Institute of Veterinary, Animal and Biomedical Sciences, Massey University, Private Bag 11-222, Palmerston North, New Zealand.
authors: H V
authors: HV
authors: Simpson
authors: AgResearch Ltd, Private Bag 11-008, Palmerston North, New Zealand.
authors: A
authors: A
authors: Pernthaner
mesh_terms: D000018:Abomasum / Q000469:parasitology*; D000595:Amino Acid Sequence; D000818:Animals; D000909:Antibodies, Helminth / Q000032:analysis / Q000097:blood / Q000276:immunology; D001483:Base Sequence; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000737:chemistry; D018509:DNA, Helminth / Q000737:chemistry; D006188:Haemonchiasis / Q000276:immunology / Q000469:parasitology / Q000662:veterinary; D006189:Haemonchus / Q000145:classification / Q000201:enzymology / Q000235:genetics / Q000276:immunology; D007700:Kinetics; D008969:Molecular Sequence Data; D025221:Phosphofructokinases / Q000737:chemistry* / Q000145:classification / Q000235:genetics / Q000276:immunology; D010802:Phylogeny; D011994:Recombinant Proteins / Q000737:chemistry / Q000145:classification / Q000235:genetics / Q000276:immunology; D012463:Saliva / Q000276:immunology; D016415:Sequence Alignment; D012756:Sheep; D012757:Sheep Diseases / Q000276:immunology / Q000469:parasitology*; D014251:Trichostrongyloidea / Q000145:classification / Q000201:enzymology* / Q000235:genetics / Q000276:immunology; D014252:Trichostrongyloidiasis / Q000276:immunology / Q000469:parasitology / Q000662:veterinary*
chemical_list: D000909:Antibodies, Helminth; D018076:DNA, Complementary; D018509:DNA, Helminth; D011994:Recombinant Proteins; D025221:Phosphofructokinases
keywords: ATP; Cloning; Expression; Haemonchus contortus; Kinetic properties; Phosphofructokinase; Teladorsagia circumcincta