Human–Mouse Chimeras with Normal Expression and Function Reveal That Major Domain Swapping Is Tolerated by P‑Glycoprotein (ABCB1)

The efflux transporter P-glycoprotein (P-gp) plays a vital role in the transport of molecules across cell membranes and has been shown to interact with a panoply of functionally and structurally unrelated compounds. How human P-gp interacts with this large number of drugs has not been well understood, although structural flexibility has been implicated. To gain insight into this transporter’s broad substrate specificity and to assess its ability to accommodate a variety of molecular and structural changes, we generated human–mouse P-gp chimeras by the exchange of homologous transmembrane and nucleotide-binding domains. High-level expression of these chimeras by BacMam- and baculovirus-mediated transduction in mammalian (HeLa) and insect cells, respectively, was achieved. There were no detectable differences between wild-type and chimeric P-gp in terms of cell surface expression, ability to efflux the P-gp substrates rhodamine 123, calcein-AM, and JC-1, or to be inhibited by the substrate cyclosporine A and the inhibitors tariquidar and elacridar. Additionally, expression of chimeric P-gp was able to confer a paclitaxel-resistant phenotype to HeLa cells characteristic of P-gp-mediated drug resistance. P-gp ATPase assays and photo-cross-linking with [¹²⁵I]­iodoarylazidoprazosin confirmed that transport and biochemical properties of P-gp chimeras were similar to those of wild-type P-gp, although differences in drug binding were detected when human and mouse transmembrane domains were combined. Overall, chimeras with one or two mouse P-gp domains were deemed functionally equivalent to human wild-type P-gp, demonstrating the ability of human P-gp to tolerate major structural changes

Human–Mouse Chimeras with Normal Expression and Function Reveal That Major Domain Swapping Is Tolerated by P‑Glycoprotein (ABCB1)

The efflux transporter P-glycoprotein (P-gp) plays a vital role in the transport of molecules across cell membranes and has been shown to interact with a panoply of functionally and structurally unrelated compounds. How human P-gp interacts with this large number of drugs has not been well understood, although structural flexibility has been implicated. To gain insight into this transporter’s broad substrate specificity and to assess its ability to accommodate a variety of molecular and structural changes, we generated human–mouse P-gp chimeras by the exchange of homologous transmembrane and nucleotide-binding domains. High-level expression of these chimeras by BacMam- and baculovirus-mediated transduction in mammalian (HeLa) and insect cells, respectively, was achieved. There were no detectable differences between wild-type and chimeric P-gp in terms of cell surface expression, ability to efflux the P-gp substrates rhodamine 123, calcein-AM, and JC-1, or to be inhibited by the substrate cyclosporine A and the inhibitors tariquidar and elacridar. Additionally, expression of chimeric P-gp was able to confer a paclitaxel-resistant phenotype to HeLa cells characteristic of P-gp-mediated drug resistance. P-gp ATPase assays and photo-cross-linking with [¹²⁵I]­iodoarylazidoprazosin confirmed that transport and biochemical properties of P-gp chimeras were similar to those of wild-type P-gp, although differences in drug binding were detected when human and mouse transmembrane domains were combined. Overall, chimeras with one or two mouse P-gp domains were deemed functionally equivalent to human wild-type P-gp, demonstrating the ability of human P-gp to tolerate major structural changes

Identification of a Cryptic Bacterial Promoter in Mouse (<i>mdr1a</i>) P-Glycoprotein cDNA

<div><p>The efflux transporter P-glycoprotein (P-gp) is an important mediator of various pharmacokinetic parameters, being expressed at numerous physiological barriers and also in multidrug-resistant cancer cells. Molecular cloning of homologous cDNAs is an important tool for the characterization of functional differences in P-gp between species. However, plasmids containing mouse <i>mdr1a</i> cDNA display significant genetic instability during cloning in bacteria, indicating that <i>mdr1a</i> cDNA may be somehow toxic to bacteria, allowing only clones containing mutations that abrogate this toxicity to survive transformation. We demonstrate here the presence of a cryptic promoter in mouse <i>mdr1a</i> cDNA that causes mouse P-gp expression in bacteria. This expression may account for the observed toxicity of <i>mdr1a</i> DNA to bacteria. Sigma 70 binding site analysis and GFP reporter plasmids were used to identify sequences in the first 321 bps of <i>mdr1a</i> cDNA capable of initiating bacterial protein expression. An <i>mdr1a</i> M107L cDNA containing a single residue mutation at the proposed translational start site was shown to allow sub-cloning of <i>mdr1a</i> in <i>E</i>. <i>coli</i> while retaining transport properties similar to wild-type P-gp. This mutant <i>mdr1a</i> cDNA may prove useful for efficient cloning of <i>mdr1a</i> in <i>E</i>. <i>coli</i>.</p></div

Mutations and their position in <i>mdr1a</i> cDNA after transformation into <i>E</i>. <i>coli</i>.

<p>The pCR-Blunt-II-TOPO vector containing <i>mdr1a</i> cDNA was used to transform <i>E</i>.<i>coli</i> and 20 colonies were selected for small-scale bacterial growth, plasmid purification, and analysis of plasmid DNA by agarose gel electrophoresis. Sixteen of the colonies selected for analysis contained large insertion or deletion mutations (not shown), and the remaining four colonies containing potentially correct <i>mdr1a</i> cDNA were sequenced. Each of the four sequenced plasmids contained mutated <i>mdr1a</i> cDNA, indicating a 100% mutational rate. Mutations observed were classified as point, insertion, or deletion mutations. Wild-type <i>mdr1a</i> cDNA (top) is compared to sequenced, “mutant” <i>mdr1a</i> cDNA (bottom). Insertion and point mutations are indicated in red in the mutant cDNA. Deleted base pairs are highlighted in red in wild-type cDNA, and the resulting mutated cDNA sequence is shown below. A) A point mutation (C→T) at location 1027 bp resulting in an introduced stop codon into <i>mdr1a</i> cDNA. B) A single adenine base pair insertion at location 1033 bp. C) Two examples of deletion mutations. All mutations resulted either directly (point mutations) or indirectly (insertion or deletion mutations) in the introduction of a stop codon into <i>mdr1a</i> cDNA. Locations of the introduced stop codons are indicated.</p

Identification of putative transcriptional and translational start sites in <i>mdr1a</i> cDNA.

<p>An <i>in silico</i> sigma 70 binding site analysis was used to screen for a putative cryptic bacterial promoter in <i>mdr1a</i> cDNA. (A) Sequence of <i>mdr1a</i> cDNA shown with predicted -35 and extended -10 elements (red). The location in the cDNA for each element is noted above the sequence. (B) Predicted Shine-Dalgarno and ATG of a methionine that is in-frame with regards to the <i>mdr1a</i> ORF.</p

Mouse P-gp model in apo (open) conformation with methionine residue at position 107 highlighted.

<p>(A) Mouse P-gp is shown with the methionine to leucine mutation highlighted in TM1-ECL1 by the presence of a space filled leucine residue. The region of the drug-binding pocket (DBP) is indicated. (B) Enlarged view of M107L mutation. Figures were generated using PyMOL from PDB deposit 4M1M [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0136396#pone.0136396.ref016" target="_blank">16</a>].</p

Schematic of GFP fusion constructs.

<p>GFP reporter constructs were generated to assess the ability of sequences of <i>mdr1a</i> cDNA to drive protein expression. Black lines represent DNA sequences fused upstream of GFP. GFP cDNA is represented in green and the presence of introduced RBSs are shown in blue where applicable. The p300-GFP plasmid contains the first 300 bps of <i>mdr1a</i> fused to GFP immediately preceded by a strong RBS. The p321-GFP plasmid contains the first 321 bps of <i>mdr1a</i> fused to GFP without the presence of an introduced RBS. pGFP and λpR-GFP were used as positive and negative control for GFP expression, respectively. p321-GFP and p-321-M107L-GFP are equivalent with the exception of the methionine to leucine mutation at position 107 in the amino acid sequence.</p

Fluorescence of <i>E</i>. <i>coli</i> transformed with GFP fusion plasmids.

<p>(A) Confocal images of <i>E</i>. <i>coli</i> transformed with GFP fusion plasmids. From left to right, columns represent fluorescence, bright field, and a merged image. Yellow bars indicate 20 μm. (B) Fluorescence of <i>E</i>. <i>coli</i> transformed with GFP fusion plasmids. Fluorescence spectroscopy was used to determine levels of GFP fluorescence in bacteria transformed with GFP plasmids. Confocal images and fluorescence spectroscopy values for bacteria transformed with λpR-GFP plasmids were omitted due to the signal saturation resulting from high GFP fluorescence. Data represent the mean and standard deviation of five individual colonies from one transformation event. Multiple groups were analyzed using one-way ANOVA where significance was defined as p < 0.05 in GraphPad Prism.</p