Calabokis M1González Y1,2Merchán A1,2Escalona JL1,3Araujo NA1,3Sanz-Rodríguez CE1,4,5Cywiak C1,4Spencer LM1,6Martínez JC7Bubis J12016. J Immunoassay Immunochem. 37(5):485-514. doi: 10.1080/15321819.2016.1162799.

1
Departamento de Biología Celular , Universidad Simón Bolívar , Caracas , Venezuela.
2
Licenciatura en Química , Universidad Simón Bolívar , Caracas , Venezuela.
3
Postgrado en Química , Universidad Simón Bolívar , Caracas , Venezuela.
4
Postgrado en Ciencias Biológicas , Universidad Simón Bolívar , Caracas , Venezuela.
5
Laboratorio de Dinámica Estocástica, Centro de Física , Instituto Venezolano de Investigaciones Científicas , Caracas , Venezuela.
6
Departamento de Biología , Universidad de Yachay Tech , Hacienda San José , Urcuquí , Ecuador.
7
Dirección de Salud , Fundación Instituto de Estudios Avanzados IDEA , Caracas , Venezuela.

 

Abstract

Polyclonal immunoglobulin Y (IgY) antibodies were produced in chicken eggs against the purified R(II)-subunit of the cAMP-dependent protein kinase (PKA) from pig heart, which corresponds to the Sus scrofa R(II)α 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-R(II) 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 R(II)-subunit purified from porcine heart and the recombinant human PKA R(I)β-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 R(II)α 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.

KEYWORDS:

Trypanosoma equiperdum; cAMP; immunoglobulin Y antibodies; immunological cross-reactivity; regulatory subunits of the cAMP-dependent protein kinase

PMID:
 
26983367
 
DOI:
 
10.1080/15321819.2016.1162799