Identification and Biochemical Characterization of Protein Phosphatase 5 from the Cantharidin-Producing Blister Beetle, Epicauta chinensis

Abstract

Protein phosphatase 5 (PP5) is a unique member of serine/threonine phosphatases which has been recognized in regulation of diverse cellular processes. A cDNA fragment encoding PP5 (EcPP5) was cloned and characterized from the cantharidin-producing blister beetle, E. chinensis. EcPP5 contains an open reading frame of 1500 bp that encodes a protein of 56.89 kDa. The deduced amino acid sequence shares 88% and 68% identities to the PP5 of Tribolium castaneum and humans, respectively. Analysis of the primary sequence shows that EcPP5 has three TPR (tetratricopeptide repeat) motifs at its N-terminal region and contains a highly conserved C-terminal catalytic domain. RT-PCR reveals that EcPP5 is expressed in all developmental stages and in different tissues. The recombinant EcPP5 (rEcPP5) was produced in Escherichia coli and purified to homogeneity. The purified protein exhibited phosphatase activity towards pNPP (p-nitrophenyl phosphate) and phosphopeptides, and its activity can be enhanced by arachidonic acid. In vitro inhibition study revealed that protein phosphatase inhibitors, okadaic acid, cantharidin, norcantharidin and endothall, inhibited its activity. Further, protein phosphatase activity of total soluble protein extract from E. chinensis adults could be impeded by these inhibitors suggesting there might be some mechanism to protect this beetle from being damaged by its self-produced cantharidin.

Characterization of Protein Phosphatase 5 from Three Lepidopteran Insects: Helicoverpa armigera, Mythimna separata and Plutella xylostella

“…In Epicauta chinensis , PP5 was also constitutively expressed in all developmental stages, but most highly expressed in the adult stage [16]. Similar to the two reports, we found three lepidopteran PP5 genes were expressed in all tested tissues and life stages.…”

“…The IC 50 values of okadaic acid on lepidopteran PP5 proteins (17.57 nM on MsPP5, 25.01 nM on PxPP5, and 41.82 nM on HaPP5) are significantly higher than those reported for mammalian PP5 (IC50 = 3.5 nM), but are much lower than in E. chinensis PP5 (IC 50  = 190.0 nM) [16]. Cantharidin impeded the activities of three lepidopteran PP5 proteins to a similar degree with IC 50 values of 0.73–2.38 µM which are consistent with previous reports on other sources of PP5 [12], [16], [22]. The IC 50 values of endothall were 6.84 µM for PxPP5, 12.64 µM for MsPP5, and 19.33 µM for HaPP5, respectively.…”

“…Our results indicated that the enzyme activities of three recombinant lepidopteran PP5 proteins were also modulated by the TPR domain. Previous work reported that the maximum activation of arachidonic acid on E. coli expressed PP5 from T. brucei , P. falciparum and E. chinensis were 2.6-, 2.0-, and 2.3-fold, respectively [16] , [19] , [20] . Whereas, the activity of both E. coli -expressed and native mammalian PP5 was low and could be enhanced by arachidonic acid up to much higher degrees [4] , [18] .…”

“…In D. melanogaster , PP5 was expressed across the life cycle, but more highly expressed in the embryonic than later developmental stages [15] . In Epicauta chinensis , PP5 was also constitutively expressed in all developmental stages, but most highly expressed in the adult stage [16] . Similar to the two reports, we found three lepidopteran PP5 genes were expressed in all tested tissues and life stages.…”

“…Successful expression of active recombinant PP5 in E. coli has been reported in mammals [4] , Plasmodium falciparum [19] , and Trypanosoma brucei [20] . Recently, we obtained active E. coli -yielded recombinant PP5 from P. xylostella and the cantharidin-producing beetle, E. chinensis [12] , [16] . No other studies have focused on recombinant PP5 in other insects.…”