Abstract
A 1095 bp full length cDNA encoding Teladorsagia circumcincta aldolase (TciALDO-1) was cloned and expressed in Escherichia coli. Recombinant TciALDO-1 was purified, and its kinetic properties determined. 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-1 with homologues from other helminths showed 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 30 °C, the optimum pH for TciALDO-1 activity was pH 7.5, the Vmax was 432 ± 23 nmol × min−1 × mg−1 protein, and the apparent Km for the substrate fructose 1,6-bisphosphate was 0.24 ± 0.01 µM (mean ± SEM, n = 3). Recombinant TciALDO-1 was recognized by antibodies in both serum and saliva from field-immune sheep in ELISA, however, that was not the case with nematode-naïve sheep. Teladorsagia circumcincta fructose 1,6-bisphosphate aldolase appears to have potential as a vaccine candidate to control this common sheep parasite.
Highlights
Fructose 1,6-bisphosphate aldolase (FBA) (EC 4.1.2.13) catalyses the reversible reaction that splits fructose 1,6-bisphosphate into the 3-phosphate dihydroxyacetone phosphate (DHAP) and glyceraldehyde 3-phosphate (G3P)
The genes encoding FBAs have been sequenced from the free-living nematode Caenorhabditis elegans [5], the animal-parasitic Haemonchus contortus [6], the plant-parasitic Heterodera glycines and Globodera rostochiensis [7], as well as helminths, including Schistosoma mansoni
The 365 amino acid TciALDO-1 protein expressed in E. coli was typical of aldolase monomers of many species, and had 77–94%
Summary
Fructose 1,6-bisphosphate aldolase (FBA) (EC 4.1.2.13) catalyses the reversible reaction that splits fructose 1,6-bisphosphate into the 3-phosphate dihydroxyacetone phosphate (DHAP) and glyceraldehyde 3-phosphate (G3P). FBA enzymes belong to two classes depending on the mechanism of the reaction: class I, which form covalent Schiff-base conjugates with a conserved lysine, are present mainly in higher eukaryotes and a few bacteria, whereas class II require a divalent metal ion as cofactor for enzymatic activity and are found principally in bacteria, algae and fungi [1,2,3,4]. There are three isoforms of vertebrate FBA: aldolase A which is principally expressed in muscle, aldolase B in liver and aldolase C in brain. As nematode FBAs were shown to have some structural properties similar to vertebrate FBAA, but catalytic properties more like those of FBAC, aldolases
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