Bioinformatic characterization and expression of a P-glycoprotein from the parasitic cestode Echinococcus sp

NICOLAO, MARÍA CELESTE; GUILLERMO M. DENEGRI; CUMINO, ANDREA C.

Buenos Aires

Congreso; I Congreso Internacional de Zoonosis y Enfermedades Emergentes y VII Congreso Argentino de Zoonosis; 2011

Asociación Argentina de Zoonosis

Echinococcosis is a near-cosmopoitan zoonosis caused by helminthic parasites belonging to the genus Echinococcus sp. Expression and up-regulation of the memberane-bound efflux pump P-glycoprotein (Pgp) is associated with the phenomenon of lack of therapeutic efficiency and/or multidrug-resistance in pathogenic organisms, including helminth parasites.We have sequenced and characterized an Echinococcus granulosus cDNA, termed Eg-mrd1, encoding the Eg-Pgp. The isolated 591bp long cDNA contains a partial ORF of 197 amino acids and shows an amino acid identity of 57% with the nucleotide-binding domain (NBD) domain of ATP-binding cassette transporters, and specially 62% and 59% with this domain of nematode and human Pgps, respectively. The localization of Pgp transporter was analyzed by in situ and in vitro immuno-detection in protoscoleces and by confocal microscopy. This protein showed in western blots a molecular mass of aprox. 130 kDa, it is localized in the tegumental region, particularly around suckers of protoscoleces. Calcium channel blockers inhibited calcein-AM export with IC50 values in the low micromolar range and verapamil acts as a model substrate in concordance with mammalian members of the Pgp transporters family.TBlastN search in Echinococcus full-length genome database (http://www.sanger.ac.uk), detected the E. multilocularis EMU scaffold10 with 95% identity (amino acid 454-651) to Eg-mrd1. Additional bioinformatics analysis of Eg-Pgp and putative Em-Pgp, confirmed the result obtained by similarity searches and showed the presence of characteristic domains (ATP binding sites; Walker A and B-loops; Q, D and H-loops), consensus sequences for glycosylation and phosphorylation, and highly similar profile with known member of ABC-membrane and P-loop NTPases superfamilies. The Em-mrd1 gene contained one ORF organized in 10 exons and 9 introns (between 37 and 2565 ntd) distributed over 11.7 kilobase pairs of DNA with encodes an 1199 amino acids polypeptide (molecular weight of 131,74 KDa and 8.37 of isolectric point). The presence of an in frame TGA stop codon, 6 bp upstream of the first ATG codon of this ORF confirmed that the full-length ORF has been determined. A transmembrane domain (TMD) and NBD domain were predicted by the Conserved domain Search service (CD NCBI) with an E value of 1.16 e-102. To know about of the sequences, the molecular structure and expression analysis of Eg-Pgp or Em-Pgp pave the way for functional studies of possible Pgp interfering agents, and also may be a valid pharmacological approach to improve the activity and extend the lifespan of the antiechinococcal drugs.