Immunological identification of a cAMP-dependent protein kinase regulatory subunit-like protein from the Trypanosoma equiperdum TeAp-N/D1 isolate

Abstract

ABSTRACT Polyclonal immunoglobulin Y (IgY) antibodies were produced in chicken eggs against the purified RII-subunit of the cAMP-dependent protein kinase (PKA) from pig heart, which corresponds to the Sus scrofa RIIα isoform. In order to evaluate whether Trypanosoma equiperdum possessed PKA R-like proteins, parasites from the Venezuelan TeAp-N/D1 strain were examined using the generated anti-RII IgY antibodies. Western blot experiments revealed a 57-kDa polypeptide band that was distinctively recognized by these antibodies. Likewise, polyclonal antibodies raised in mice ascites against the recombinant T. equiperdum PKA R-like protein recognized the PKA RII-subunit purified from porcine heart and the recombinant human PKA RIβ-subunit by immunoblotting. However, a partially purified fraction of the parasite PKA R-like protein was not capable of binding cAMP, implying that this protein is not a direct downstream cAMP effector in T. equiperdum. Although the function of the S. scrofa PKA RIIα and the T. equiperdum PKA R-like protein appear to be different, their cross-reactivity together with results obtained by bioinformatics techniques corroborated the high level of homology exhibited by both proteins. Moreover, its presence in other trypanosomatids suggests an important cellular role of PKA R-like proteins in parasite physiology.