Identification and bioinformatic characterization of a multidrug resistance associated protein (ABCC) gene in Plasmodium berghei

María González-Pons,Adelfa E Serrano,Ricardo González-Méndez,Ada C Szeto

doi:10.1186/1475-2875-8-1

María González-Pons, Adelfa E Serrano + Show 2 more

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https://doi.org/10.1186/1475-2875-8-1

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Abstract

BackgroundThe ATP-binding cassette (ABC) superfamily is one of the largest evolutionarily conserved families of proteins. ABC proteins play key roles in cellular detoxification of endobiotics and xenobiotics. Overexpression of certain ABC proteins, among them the multidrug resistance associated protein (MRP), contributes to drug resistance in organisms ranging from human neoplastic cells to parasitic protozoa. In the present study, the Plasmodium berghei mrp gene (pbmrp) was partially characterized and the predicted protein was classified using bioinformatics in order to explore its putative involvement in drug resistance.MethodsThe pbmrp gene from the P. berghei drug sensitive, N clone, was sequenced using a PCR strategy. Classification and domain organization of pbMRP were determined with bioinformatics. The Plasmodium spp. MRPs were aligned and analysed to study their conserved motifs and organization. Gene copy number and organization were determined via Southern blot analysis in both N clone and the chloroquine selected line, RC. Chromosomal Southern blots and RNase protection assays were employed to determine the chromosomal location and expression levels of pbmrp in blood stages.ResultsThe pbmrp gene is a single copy, intronless gene with a predicted open reading frame spanning 5820 nucleotides. Bioinformatic analyses show that this protein has distinctive features characteristic of the ABCC sub-family. Multiple sequence alignments reveal a high degree of conservation in the nucleotide binding and transmembrane domains within the MRPs from the Plasmodium spp. analysed. Expression of pbmrp was detected in asexual blood stages. Gene organization, copy number and mRNA expression was similar in both lines studied. A chromosomal translocation was observed in the chloroquine selected RC line, from chromosome 13/14 to chromosome 8, when compared to the drug sensitive N clone.ConclusionIn this study, the pbmrp gene was sequenced and classified as a member of the ABCC sub-family. Multiple sequence alignments reveal that this gene is homologous to the Plasmodium y. yoelii and Plasmodium knowlesi mrp, and the Plasmodium vivax and Plasmodium falciparum mrp2 genes. There were no differences in gene organization, copy number, or mRNA expression between N clone and the RC line, but a chromosomal translocation of pbmrp from chromosome 13/14 to chromosome 8 was detected in RC.

Highlights

The ATP-binding cassette (ABC) superfamily is one of the largest evolutionarily conserved families of proteins
Identification and characterization of an mrp homologue in P. berghei An mrp homologue was identified in P. berghei using primers designed to PCR-amplify the pbmrp gene in the drug sensitive N clone
Transmembrane helix predictions by both the DAS and TMHMM transmembrane prediction servers predict that pbMRP has 12 transmembrane helices organized into two transmembrane domains (TMD) and that both, the N-terminal and C-terminal, are intracellular

Summary

Introduction

The ATP-binding cassette (ABC) superfamily is one of the largest evolutionarily conserved families of proteins. Overexpression of certain ABC proteins, among them the multidrug resistance protein (MDR) and the multidrug resistance associated proteins (MRPs), contribute to drug resistance in a variety organisms ranging from parasitic protozoa to human neoplastic cells Membrane transporters, such as the Plasmodium falciparum chloroquine resistant transporter (pfcrt) and the Plasmodium falciparum mdr, which is a member of the ABC superfamily, have been identified as key contributors in decreasing susceptibility to several anti-malarial drugs [14]. The ABC transporter superfamily is comprised of eight subfamilies in eukaryotes: ABCA, ABCB, ABCC, ABCD, ABCE, ABCF, ABCG, and ABCH Proteins within this superfamily were classified based on the sequence and organization of their conserved nucleotide binding domains (NBD). Genes are organized either as full transporters containing two of each domain or half transporters with one of each (Figure 1c)

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