Absolute immunoquantification of the expression of ABC transporters P-glycoprotein, breast cancer resistance protein and multidrug resistance-associated protein 2 in human liver and duodenum
Graphical abstract
Introduction
The ATP-binding cassette (ABC) transporters breast cancer resistance protein (BCRP, ABCG2), multidrug resistance-associated protein 2 (MRP2, ABCC2), and P-glycoprotein (Pgp, ABCB1) use ATP hydrolysis to drive the export of a huge range of structurally diverse compounds from cells and are important in the absorption, distribution, metabolism and elimination of xenobiotics and endogenous chemicals [1], [2], [3], [4]. Expression of BCRP, MRP2, and Pgp at the apical side of enterocytes and hepatocytes means that they are ideally situated to restrict absorption of dietary toxins and drugs from the gut, and to eliminate compounds from the liver into the bile [5], [6]. Expression levels of these transporters have a profound effect on individual response to drugs and other xenobiotics.
Computer-based methods predicting pharmacokinetics, pharmacodynamics and drug–drug interactions are important tools supporting drug development [7], [8]. Their viability is dependent, among other factors, on the availability of high quality data on the expression, distribution, variability of drug metabolizing enzymes, transporters, etc. In contrast to drug metabolizing enzymes such as cytochromes P450 [9], [10] and sulfotransferases [11], there is little information on the protein expression levels of ABC transporters. This is mainly because transporters, being integral membrane proteins, are extremely difficult to purify. Purification and reconstitution into proteoliposomes has been achieved (e.g. [12]), but this is expensive and technically challenging. Thus studies investigating transporter protein expression levels in solid tissues in particular have been comparative at best. Proteomic approaches [13] have been used to determine protein expression levels of trypsin-digested BCRP and MRP2 in tissue samples [14], [15], [16], however these methods have a number of limitations, including the reproducibility of extraction and sample digestion. Other approaches to quantification, for example flow cytometry, have been taken using cells such as bone marrow (e.g. [17], [18]), but these are not applicable to solid tissues. A straightforward method for the production and quantification of full-length protein standards would therefore be an important contribution to the study of ABC transporters.
Here we present the application of a novel approach to this problem recently devised in our laboratory for quantification of UDP-glucuronosyltransferases [19] using the S-tag/S-protein system [20] to generate transporter protein standards for immunoquantification. Cloning and expression of cDNAs encoding these transporters as fusion proteins with the 15 amino acid S•tag™ peptide allowed the recombinant transporters to be quantified using the fact that S•tag™ and S-protein combine with high affinity and efficiency to reconstitute a functional ribonuclease enzyme whose activity can be determined using a sensitive spectrophotometric assay. The amount of S•tag™ present can thus be quantified which in turn infers the quantity of S-tagged transporter in the preparation. The quantified recombinant S-tagged transporters were then used to generate standard curves for immunoquantification in human duodenum and liver. This represents the first comprehensive analysis of protein expression levels of these transporters in human tissue using an immunochemical approach.
Section snippets
Cloning and expression of S-tagged transporters
S-tagged BCRP, MRP2, and Pgp were cloned by PCR, using pET32b (Novagen/Merck, Darmstadt, Germany), IMAGE clone 100004938 (Source BioScience, Nottingham, UK), pcDNA3.1 harbouring MRP2 (kindly provided by Professor D. Keppler, German Cancer Research Centre, Heidelberg, Germany), and IMAGE clone 40146455 (Source BioScience) as templates for S-tag, BCRP (GI:123982985), MRP2 (GI:188595701), and Pgp (GI:120660210), respectively. The sequence encoding the 15 amino acid S-tag was cloned at either the
Characterisation of recombinant transporter proteins
Recombinant BCRP, MRP2, and Pgp generated with no tag, or with the S-tag placed at the N- or C-terminus were characterised using a combination of immunofluorescence microscopy, immunoblotting, and transport assays (full details will be published elsewhere). We found C-terminally tagged BCRP to be mislocalised to the endoplasmic reticulum with no expression at the plasma membrane, however with the S-tag at the N-terminus BCRP was found predominantly at the plasma membrane and was able to extrude
Discussion
Due to the lack of quantified protein standards, studies determining transporter protein levels have generally been comparative rather than absolute. Here we have applied a novel method to quantify the expression of three important transporter proteins in human liver and intestine using the S-tag/S-protein system. The method is robust and easily applicable to any protein for which good antibodies are available. The principal drawbacks of this method include the dependence on high quality,
Acknowledgements
TGHAT was supported by a Biotechnology and Biological Sciences Research Council CASE studentship in collaboration with Pfizer (BBS/S/N/2004/11530, awarded to MWHC). Part of this work was performed under the auspices of a Royal Society International Joint Grant and a European Associated Laboratory between CNRS-UHP Nancy I and the University of Dundee (both to MWHC and SF-G).
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Present address: DMPK In Vitro Screening & Profiling, AstraZeneca, Alderley Park, Macclesfield SK10 4TG, England, UK.