International Journal of Molecular Sciences, Vol.14, No.10, 20692-20703, 2013
Molecular Cloning and Characterization of the Calcineurin Subunit A from Plutella xylostella
Calcineurin (or PP2B) has been reported to be involved in an array of physiological process in insects, and the calcineurin subunit A (CNA) plays a central role in calcineurin activity. We cloned the CNA gene from Plutella xylostella (PxCNA). This gene contains an ORF of 1488 bp that encodes a 495 amino acid protein, showing 98%, and 80% identities to the CNA of Bombyx mori, and humans respectively. The full-length of PxCNA and its catalytic domain (CNA(1-341), defined as PxCN) were both expressed in Escherichia coli. Purified recombinant PxCNA displayed no phosphatase activity, whereas recombinant PxCN showed high phosphatase activity with a Km of 4.6 mM and a kcat of 0.66 S-1 against pNPP. It could be activated at different degrees by Mn2+, Ni2+, Mg2+, and Ca2+. The optimum reaction pH was about 7.5 and the optimum reaction temperature was around 45 degrees C. An in vitro inhibition assay showed that okadaic acid (OA) and cantharidin (CTD) competitively inhibited recombinant PxCN activity with the IC50 values of 8.95 M and 77.64 M, respectively. However, unlike previous reports, pyrethroid insecticides were unable to inhibit recombinant PxCN, indicating that the P. xylostella calcineurin appears not to be sensitive to class II pyrethroid insecticides.