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Acta tropica

Biochemical and molecular characterization of the calcineurin in Echinococcus granulosus larval stages.


PMID 25818323

Abstract

Calcineurin (CaN) is a Ca(2+)-calmodulin activated serine-threonine protein phosphatase that couples the local or global calcium signals, thus controlling important cellular functions in physiological and developmental processes. The aim of this study was to characterize CaN in Echinococcus granulosus (Eg-CaN), a human cestode parasite of clinical importance, both functionally and molecularly. We found that the catalytic subunit isoforms have predicted sequences of 613 and 557 amino acids and are substantially similar to those of the human counterpart, except for the C-terminal end. We also found that the regulatory subunit consists of 169 amino acids which are 87% identical to the human ortholog. We cloned a cDNA encoding for one of the two catalytic subunit isoforms of CaN (Eg-can-A1) as well as the only copy of the Eg-can-B gene, both constitutively transcribed in all Echinococcus larval stages and responsible for generating a functionally active heterodimer. Eg-CaN native enzyme has phosphatase activity, which is enhanced by Ca(2+)/Ni(2+) and reduced by cyclosporine A and Ca(2+) chelators. Participation of Eg-CaN in exocytosis was demonstrated using the FM4-64 probe and Eg-CaN-A was immunolocalized in the cytoplasm of tegumental cells, suckers and excretory bladder of protoscoleces. We also showed that the Eg-can-B transcripts were down-regulated in response to low Ca(2+) intracellular level, in agreement with decreased enzyme activity. Confocal microscopy revealed a striking pattern of Eg-CaN-A in discrete fluorescent spots in the protoscolex posterior bladder and vesicularized protoscoleces beginning the vesicular differentiation. In contrast, Eg-CaN-A was undetectable during the pre-microcyst closing stage while a high DDX-like RNA helicase expression was evidenced. Finally, we identified and analyzed the expression of CaN-related endogenous regulators.