Characterization of filarial phosphoglycerate kinase

Abstract

Phosphoglycerate kinase (PGK) is a key enzyme of glycolysis which also acts as a mediator of DNA replication and repair in the nucleus. We have cloned and expressed PGK in Brugia malayi. The rBmPGK was found to be 415 amino acid residues long having 45 kDa subunit molecular weight. This enzyme was also identified in different life stages of bovine filarial parasite Setaria cervi. The enzyme activity was highest in microfilarial stage followed by adult female and male as also shown by real time PCR in the present study. Further using BmPGK primers the cDNA prepared from S. cervi was amplified and sequenced which showed 100% homology with Brugia malayi PGK. B. malayi and S. cervi, PGK consists of conserved calmodulin binding domain (CaMBD) having 21 amino acids. In the present study we have shown the CaMBD binds to calcium-calmodulin and regulates its activity. The binding of calmodulin (CaM) with CaMBD was confirmed using calmodulin agarose binding pull down assay, which showed that the rBmPGK binds to CaM agarose-calcium dependent manner. The effect of CaM-Ca²⁺on the activity of rBmPGK was studied at different concentration of CaM (0.01-5.0 μM) and calcium chloride (0.01-100 μM). The rBmPGK was activated up to 85% in the presence of CaM at 1 μM and 10 μM concentration of CaCl₂. Interestingly this activation was abrogated by metal chelator EDTA. Similar results were shown in case of Setaria cervi PGK. A significant increase (90 ± 10) % in ScPGK activity was observed in the presence of CaM and CaCl₂ at 1.0 μM and 1.0 mM respectively, further increase in the conc. of CaCl₂, the activity of ScPGK was found to be decreased like rBmPGK. Bioinformatics studies have also confirmed the interaction between CaMBD and CaM which showed CaM interacted to Phe 206, Gln 220, Arg 223 and Asn 224 of rBmPGK CaM binding domain. On the basis of these findings, it has been suggested that the activity of filarial PGK could be regulated in cells by Ca²⁺-CaM depending upon the concentration of calcium. To the best of our knowledge this is first report in filarial parasite.

Keywords: Brugia malayi; Calmodulin; Filariasis; Phosphoglycerate kinase; Setaria cervi.