Molecular evolutionary analysis of ABCB5: the ancestral gene is a full transporter with potentially deleterious single nucleotide polymorphisms

Abstract

Background: ABCB5 is a member of the ABC protein superfamily, which includes the transporters ABCB1, ABCC1 and ABCG2 responsible for causing drug resistance in cancer patients and also several other transporters that have been linked to human disease. The ABCB5 full transporter (ABCB5.ts) is expressed in human testis and its functional significance is presently unknown. Another variant of this transporter, ABCB5 beta possess a "half-transporter-like" structure and is expressed in melanoma stem cells, normal melanocytes, and other types of pigment cells. ABCB5 beta has important clinical implications, as it may be involved with multidrug resistance in melanoma stem cells, allowing these stem cells to survive chemotherapeutic regimes.

Methodology/principal findings: We constructed and examined in detail topological structures of the human ABCB5 protein and determined in-silico the cSNPs (coding single nucleotide polymorphisms) that may affect its function. Evolutionary analysis of ABCB5 indicated that ABCB5, ABCB1, ABCB4, and ABCB11 share a common ancestor, which began duplicating early in the evolutionary history of chordates. This suggests that ABCB5 has evolved as a full transporter throughout its evolutionary history.

Conclusions/significance: From our in-silco analysis of cSNPs we found that a large number of non-synonymous cSNPs map to important functional regions of the protein suggesting that these SNPs if present in human populations may play a role in diseases associated with ABCB5. From phylogenetic analyses, we have shown that ABCB5 evolved as a full transporter throughout its evolutionary history with an absence of any major shifts in selection between the various lineages suggesting that the function of ABCB5 has been maintained during mammalian evolution. This finding would suggest that ABCB5 beta may have evolved to play a specific role in human pigment cells and/or melanoma cells where it is predominantly expressed.

Figure 1. Predicted topological structures of ABCB5.

A. Topological structure of the ABCB5 full transporter. The figure depicts 12 transmembrane alpha helices and 2 nucleotide-binding domains. The 2 ABC_membrane motifs, amino acids 48–339 and 692–968, are highlighted in orange and the 2 ABC_transporter_2 motifs, amino acids 386–622 and 1015–1253, are highlighted in purple. The 2 N-glycosylation sites, amino acids 85–88 and 91–94, which may be important for targeting of the transporter to the membrane, are highlighted in blue. The topological structure was determined using HMMTOP and displayed using the TOPO2 software. Functional motifs were determined using MOTIFSCAN. B. Topology of ABCB5 beta. Topology was determined with HMMTOP and displayed using TOPO2. Diagram depicts 6 transmembrane alpha helices and 2 nucleotide-binding domains. One ABC_membrane domain, amino acids 247–523, is shown in orange and the 2 ABC_transporter_2 domains, amino acids 2–177 and 570–808, are depicted in purple.

Figure 2. Coiled-coil regions of ABCB5 beta.

Coiled-coil regions were calculated using the program COILS. These regions may indicate potential dimerization motifs in ABCB5 beta. Each alpha helix in a coiled coil is amphipathic, and the pattern of hydrophilic and hydrophobic amino acids repeats every 7 residues. Since the coiled-coil is a 7 residue heptad the sliding windows are set at multiples of 7, ie 14, 21 and 28 to enable the structure prediction algorithm to optimally detect the heptads.

Figure 3. Genomic location (Chromosome 7p21) and multiple isoforms of the ABCB5.

The figure depicts eleven ABCB5 splice variants (from Ensemble).

Figure 4. Non-synonymous cSNPs in ABCB5.

A. Topological model of predicted ABCB5 beta protein structure. Diagram showing the locations of the 10 non-synonymous cSNPs. Black highlighted circles indicate ABCB5 non-synonymous cSNPs. TOPO2 was used to display annotated results. B. Topological model of predicted ABCB5.ts (full length) protein structure The model incorporates 23 ABCB5 non-synonymous cSNPs highlighted in black circles.

Figure 5. Phylogeny of the ABCB full transporters.

The tree is a maximum likelihood phylogram, with Bayesian posterior probabilities (expressed as percentages). Support for each node is 100% unless otherwise indicated. Abbreviated species names are as follows; Hsap  =  Homo sapiens, Ptro  =  Pan troglodytes, Ppyg  =  Pongo pygmaeus, Mmul  =  Macaca mulatta, Mmur  =  Microcebus murinus, Ogar  =  Otolemur garnettii, Tbel  =  Tupaia belangeri, Stri  =  Spermophilus tridecemlineatus, Mmus  =  Mus musculus, Rnor  =  Rattus norvegicus, Cpor  =  Cavia porcellus, Opri  =  Ochotona princeps, Ocun  =  Oryctolagus cuniculus, Btau  =  Bos taurus, Ecab  =  Equus caballus, Cfam  =  Canis familiaris, Fcat  =  Felis catus, Mluc  =  Myotis lucifugus, Eeur  =  Erinaceus europaeus, Sara  =  Sorex araneus, Lafr  =  Loxodonta africanus, Etel  =  Echinops telfairi, Dnov  =  Dasypus novemcinctus, Mdom  =  Monodelphis domestica, Oana  =  Ornithorhynchus anatinus, Ggal  =  Gallus gallus, Xtro  =  Xenopus tropicalis, Drer  =  Danio rerio, Trub  =  Takifugu rubripes, Tnig  =  Tetraodon nigroviridis, Gacu  =  Gasterosteus aculeatus, Olat  =  Oryzias latipes, Csar  =  Ciona savignyi, Cint  =  Ciona intestinalis. Letters after the species name refer to the gene sequence, with an additional “l” indicating that the gene is not a direct ortholog, e.g., XtroB1l is a B1 “like” gene.

Figure 6. The level of selection operating on each site of the ABCB5 sequence.

Selecton output showing the level of selection on human ABCB5. Orange residues indicate an ω value greater than 1, suggesting positive selection, while dark purple residues have an ω value significantly lower than 1, indicating strong purifying selection.