Optimization of Western blotting analysis for the isolation and detection of membrane xenobiotic transporter ABCG2

Małgorzata Szczygieł; Marcin Markiewicz; Milena Julia Szafraniec; Roxana Zuziak; Krystyna Urbańska; Leszek Fiedor

doi:10.18388/abp.2017_2299

Małgorzata Szczygieł Faculty of Biochemistry, Biophysics and Biotechnology, Jagiellonian University
Marcin Markiewicz Faculty of Biochemistry, Biophysics and Biotechnology, Jagiellonian University
Milena Julia Szafraniec Faculty of Biochemistry, Biophysics and Biotechnology, Jagiellonian University
Roxana Zuziak Faculty of Biochemistry, Biophysics and Biotechnology, Jagiellonian University
Krystyna Urbańska Faculty of Biochemistry, Biophysics and Biotechnology, Jagiellonian University
Leszek Fiedor Faculty of Biochemistry, Biophysics and Biotechnology, Jagiellonian University

DOI: https://doi.org/10.18388/abp.2017_2299

Keywords: Xenobiotic transporter, ABCG2, BCRP, Western blotting

Abstract

All organisms are exposed to numerous stress factors, which include harmful xenobiotics. The diversity of these compounds is enormous, thus in the course of evolution diverse biological defense mechanisms at various levels of organization have developed. One of them engages an evolutionarily conserved family of transporters from the ABC superfamily, found in most species - from bacteria to humans. An important example of such a transporter is the breast cancer resistance protein (BCRP/ABCG2), a typical integral membrane protein. It plays a key role in the absorption, distribution and elimination of a wide variety of xenobiotics, including drugs used in chemotherapy, and is involved in multidrug resistance. It also protects against phototoxic chlorophyll derivatives of dietary origin. BCRP is a hemitransporter which consists of one transmembrane domain, made of six alpha-helices forming a characteristic pore structure, and one ATP‑binding domain, which provides the energy from ATP hydrolysis, required for active transport of the substrates. The isolation of BCRP is still not an easy task, because its insolubility in water and the presence of membrane rafts pose serious methodological and technical challenges during the purification.

The aim of this study was to optimize the methods for detection and isolation of BCRP‑enriched fractions obtained either from the cell cultures grown in vitro or animal tissue samples. In this report we describe an optimization of isolation of a BCRP‑enriched membrane fraction, which is suitable for further protein quantitative and qualitative analysis using the molecular biology tools.

References

Azzouzi, A.-R., Barret, E., Moore, C. M., Villers, A., Allen, C., Scherz, A., Emberton, M. (2013). TOOKAD: Soluble vascular-targeted photodynamic (VTP) therapy: Determination of optimal treatment conditions and assessment of effects in patients with localised prostate cancer. BJU Int., 112(6), 766–774.

Bass, J. J., Wilkinson, D. J., Rankin, D., Phillips, B. E., Szewczyk, N. J., Smith, K., Atherton, P. J. (2017). An overview of technical considerations for Western blotting applications to physiological research. Scand. J. Med. Sci. Sports, 27, 4–25.

Bradford, M. (1976). A rapid and sensitive method for the quantification of microgram quantities of protein utilizing the principle of protein– dye binding. Anal. Biochem., 72, 248–254.

Diestra, J. E., Scheffer, G. L., Català, I., Maliepaard, M., Schellens, J. H. M., Scheper, R. J., Izquierdo, M. A. (2002). Frequent expression of the multi-drug resistance-associated protein BCRP/MXR/ABCP/ABCG2 in human tumours detected by the BXP-21 monoclonal antibody in paraffin-embedded material. J. Pathol., 198, 213–219.

Doyle, L. A., & Ross, D. D. (2003). Multidrug resistance mediated by the breast cancer resistance protein BCRP (ABCG2). Oncogene, 22, 7340–7358.

Eldashera, L. M., Wena, X., Littlea, M. S., Bircsaka, K. M., Yacovinoa, L. L., & Aleksunesa, L. M. (2013). Hepatic and renal Bcrp transporter expression in mice treated with perfluorooctanoic acid. Toxicol. Appl. Pharmacol., 306, 108–113.

Fetsch, P. A., Abati, A., Litman, T., Morisaki, K., Honjo, Y., Mittal, K., & Bates, S. E. (2006). Localization of the ABCG2 mitoxantrone resistance-associated protein in normal tissues. Cancer Lett., 235(1), 84–92.

Gorr, T. A., & Vogel, J. (2015). Western blotting revisited: Critical perusal of underappreciated technical issues. Proteomics - Clin. Appl., 9(3–4), 396–405.

Gutmann, H., Hruz, P., Zimmermann, C., Beglinger, C., & Drewe, J. (2005). Distribution of breast cancer resistance protein (BCRP/ABCG2) mRNA expression along the human GI tract. Biochem. Pharmacol., 70(5), 695–9.

Hegedus, C., Telbisz, A., Hegedus, T., Sarkadi, B., & Ozvegy-Laczka, C. (2015). Lipid Regulation of the ABCB1 and ABCG2 Multidrug Transporters. Adv. Cancer Res., 125, 97–137.

Jakubowska, M., Szczygieł, M., Michalczyk-Wetula, D., Susz, A., Stochel, G., Elas, M., Urbanska, K. (2013). Zinc-pheophorbide a-highly efficient low-cost photosensitizer against human adenocarcinoma in cellular and animal models. Photodiagnosis Photodyn. Ther., 10(3), 266–77.

Jonker, J. W., Buitelaar, M., Wagenaar, E., Valk, M. A. van der, Scheffer, G. L., Scheper, R. J., Schinkel, A. H. (2002). The breast cancer resistance protein protects against a major chlorophyll-derived dietary phototoxin and protoporphyria. Proc. Natl. Acad. Sci. USA, 99(24), 15649–15654.

Kage, K., Tsukahara, S., Sugiyama, T., Asada, S., Ishikawa, E., Tsuruo, T., & Sugimoto, Y. (2002). Dominant-negative inhibition of breast cancer resistance protein as drug efflux pump through the inhibition of S-S dependent homodimerization. Int. J. Cancer, 97(5), 626–630.

Kotkowiak, M., Dudkowiak, A., & Fiedor, L. (2017). Intrinsic Photoprotective Mechanisms in Chlorophylls. Angew. Chemie Int. Ed., 56. http://doi.wiley.com/10.1002/anie.201705357

Kubicek, J., Block, H., Maertens, B., Spriestersbach, A., & Labahn, J. (2014). Expression and purification of membrane proteins. Methods Enzymol.

Kupper, H., & Kupper C., F. (2006). Chlorophylls and Bacteriochlorophylls. In Chlorophylls and Bacteriochlorophylls (Vol. 25, pp. 67–77).

Lehner, I., Niehof, M., & Borlak, J. (2003). An optimized method for the isolation and identification of membrane proteins. Electrophoresis, 24, 1795–1808.

Mahmood, T., & Yang, P. C. (2012). Western blot: Technique, theory, and trouble shooting. N. Am. J. Med. Sci., 4(9), 429–434.

Maliepaard, M., Scheffer, G. L., Faneyte, I. F., Gastelen, M. A. van, Pijnenborg, A. C. L. M., Schinkel, A. H., Schellens, J. H. M. (2001). Subcellular Localization and Distribution of the Breast Cancer Resistance Protein Transporter in Normal Human Tissues. Cancer Res., 61, 3458–3464.

Mancia, F., & Love, J. (2010). High-throughput expression and purification of membrane proteins. J. Struct. Biol.

Mao, Q., Conseil, G., Gupta, A., Cole, S. P. C., & Unadkat, J. D. (2004). Functional expression of the human breast cancer resistance protein in Pichia pastoris. Biochem. Biophys. Res. Commun., 320(3), 730–737.

Mayer, P. (1891). Uber das Farben mit Hamatoxylin. Mitt Zool Stat Neapel.

Mitchell, B. L., Yasui, Y., Li, C. I., Fitzpatrick, A. L., & Lampe, P. D. (2005). Impact of Freeze-thaw Cycles and Storage Time on Plasma Samples Used in Mass Spectrometry Based Biomarker Discovery Projects. Cancer Inform., 1, 98–104.

Ni, Z., Bikadi, Z., Rosenberg, M. F., & Mao, Q. (2010). Structure and function of the human breast cancer resistance protein (BCRP/ABCG2). Curr. Drug Metabol., 11, 603–617.

Ozvegy, C., Litman, T., Szakács, G., Nagy, Z., Bates, S., Váradi, A., & Sarkadi, B. (2001). Functional Characterization of the Human Multidrug Transporter, ABCG2, Expressed in Insect Cells. Biochem. Biophys. Res. Commun., 285, 111–117.

Robey, R. W., Steadman, K., Polgar, O., Morisaki, K., Blayney, M., Mistry, P., & Bates, S. E. (2004). Pheophorbide a is a specific probe for ABCG2 function and inhibition. Cancer Res., 64, 1242–1246.

Scharff-Poulsen, P., & Pedersen, P. A. (2013). Saccharomyces cerevisiae-Based Platform for Rapid Production and Evaluation of Eukaryotic Nutrient Transporters and Transceptors for Biochemical Studies and Crystallography. PLoS One, 8(10).

Staron, J., Boron, B., Karcz, D., Szczygiel, M., & Fiedor, L. (2015). Recent Progress in Chemical Modifications of Chlorophylls and Bacteriochlorophylls for the Applications in Photodynamic Therapy. Curr. Med. Chem., 22(26), 3054–3074.

Storch, C. H., Ehehalt, R., Haefeli, W. E., & Weiss, J. (2007). Localization of the Human Breast Cancer Resistance Protein (BCRP / ABCG2 ) in Lipid Rafts / Caveolae and Modulation of Its Activity by Cholesterol in Vitro. Pharmacology, 323(1), 257–264.

Szafraniec, M. J., Szczygieł, M., Urbanska, K., & Fiedor, L. (2014). Determinants of the activity and substrate recognition of breast cancer resistance protein (ABCG2). Drug Metab Rev, 46(4), 459–474.

Szczygieł, M., Urbańska, K., Jurecka, P., Stawoska, I., Stochel, G., & Fiedor, L. (2008). Central metal determines pharmacokinetics of chlorophyll-derived xenobiotics. J. Med. Chem., 51, 4412–4418.

Takano, M., Yumoto, R., & Murakami, T. (2006). Expression and function of efflux drug transporters in the intestine. Pharmacol. Ther., 109(1–2), 137–161.

Taylor, S. C., & Posch, A. (2014). The design of a quantitative western blot experiment. Biomed Res. Int., 2014.

Telbisz, A., Müller, M., Özvegy-Laczka, C., Homolya, L., Szente, L., Váradi, A., & Sarkadi, B. (2007). Membrane cholesterol selectively modulates the activity of the human ABCG2 multidrug transporter. Biochim. Biophys. Acta, 1768, 2698–2713.

Telbisz, A., Ozvegy-Laczka, C., Heged, T., Aradi, A., & Sarkadi, B. (2013). Effects of the lipid environment, cholesterol and bile acids on the function of the purified and reconstituted human ABCG2 protein. Biochem. J, 450, 387–395.

Théou, N., Gil, S., Devocelle, A., Julié, C., Lavergne-Slove, A., Beauchet, A., Emile, J. F. (2005). Multidrug resistance proteins in gastrointestinal stromal tumors: Site-dependent expression and initial response to imatinib. Clin. Cancer Res., 11(21), 7593–7598.

Watson, D. H. (2001). Food chemical safety. CRC Press.

Xu, J., Peng, H., Chen, Q., Liu, Y., Dong, Z., & Zhang, J.-T. (2007). Oligomerization domain of the multidrug resistance-associated transporter ABCG2 and its dominant inhibitory activity. Cancer Res., 67, 4373–4381.