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

• Authors: Małgorzata Szczygieł , Marcin Markiewicz , Milena Szafraniec , Roxana Zuziak , Krystyna Urbańska , Leszek Fiedor
• Languages of publication: EN

Abstracts

EN

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 from 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.

Keywords

EN

Xenobiotic transporter; ABCG2; BCRP; Western blotting

• Publisher: Polish Biochemical Society
• Journal: Acta Biochimica Polonica
• Year: 2017
• Volume: 64
• Issue: 3
• Pages: 437-443

Dates

published

2017

received

2017-05-05

revised

2017-06-22

accepted

2017-08-11

(unknown)

2017-09-06

Contributors

author

Małgorzata Szczygieł

• Department of Biophysics, Faculty of Biochemistry, Biophysics and Biotechnology, Jagiellonian University, Kraków, Poland

author

Marcin Markiewicz

• Department of Biophysics, Faculty of Biochemistry, Biophysics and Biotechnology, Jagiellonian University, Kraków, Poland

author

Milena Szafraniec

• Department of Biophysics, Faculty of Biochemistry, Biophysics and Biotechnology, Jagiellonian University, Kraków, Poland

author

Roxana Zuziak

• Department of Biophysics, Faculty of Biochemistry, Biophysics and Biotechnology, Jagiellonian University, Kraków, Poland

author

Krystyna Urbańska

• Department of Biophysics, Faculty of Biochemistry, Biophysics and Biotechnology, Jagiellonian University, Kraków, Poland

author

Leszek Fiedor

• Department of Biophysics, Faculty of Biochemistry, Biophysics and Biotechnology, Jagiellonian University, Kraków, Poland

References

• Azzouzi AR, Barret E, Moore CM, 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: 766-774. doi: 10.1111/bju.12265.
• Bass JJ, Wilkinson DJ, Rankin D, Phillips BE, Szewczyk NJ, Smith K, Atherton PJ (2017) An overview of technical considerations for Western blotting applications to physiological research. Scand J Med Sci Sports 27: 4-25. doi: 10.1111/sms.12702.
• 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. doi: 10.1016/0003-2697(76)90527-3.
• Diestra JE, Scheffer GL, Català I, Maliepaard M, Schellens JH, Scheper RJ, Izquierdo MA (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. doi: 10.1002/path.1203.
• Doyle LA, Ross DD (2003) Multidrug resistance mediated by the breast cancer resistance protein BCRP (ABCG2) Oncogene 22: 7340-7358. doi: 10.1038/sj.onc.1206938.
• Eldasher LM, Wen X, Little MS, Bircsak KM, Yacovino LL, Aleksunes LM (2013) Hepatic and renal Bcrp transporter expression in mice treated with perfluorooctanoic acid. Toxicology 306: 108-113. doi: 10.1016/j.tox.2013.02.009.
• Fetsch PA, Abati A, Litman T, Morisaki K, Honjo Y, Mittal K, Bates SE (2006) Localization of the ABCG2 mitoxantrone resistance-associated protein in normal tissues. Cancer Lett 235: 84-92. doi: 10.1016/j.canlet.2005.04.024.
• Gorr TA, Vogel J (2015) Western blotting revisited: Critical perusal of underappreciated technical issues. Proteomics Clin Appl 9: 396-405. doi: 10.1002/prca.201400118.
• 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: 695-699. doi: 10.1016/j.bcp.2005.05.031.
• 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. doi: 10.1016/bs.acr.2014.10.004.
• 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: 266-277. doi: 10.1016/j.pdpdt.2012.12.004.
• Jonker JW, Buitelaar M, Wagenaar E, Van Der Valk MA, Scheffer GL, Scheper RJ, Schinkel AH (2002) The breast cancer resistance protein protects against a major chlorophyll-derived dietary phototoxin and protoporphyria. Proc Natl Acad Sci USA 99: 15649-15654. doi: 10.1073/pnas.202607599.
• 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: 626-630. doi: 10.1002/ijc.10100.
• Kotkowiak M, Dudkowiak A, Fiedor L (2017) Intrinsic photoprotective mechanisms in chlorophylls. Angew Chem Int Ed Engl 56: 10457-10461. doi: 10.1002/anie.201705357.
• Kubicek J, Block H, Maertens B, Spriestersbach A, Labahn J (2014) Expression and purification of membrane proteins. Methods Enzymol 541: 117-140. doi: 10.1016/B978-0-12-420119-4.00010-0.
• Kupper H, Kupper CF (2006) [Heavy metal]-chlorophylls formed in vivo during heavy metal stress and degradation products formed during digestion extraction and storage of plant material. Chlorophylls and Bacteriochlorophylls 25: 67-77
• Lehner I, Niehof M, Borlak J (2003) An optimized method for the isolation and identification of membrane proteins. Electrophoresis 24: 1795-1808. doi: 10.1002/elps.200305387.
• Mahmood T, Yang PC (2012) Western blot: Technique, theory, and trouble shooting. N Am J Med Sci 4: 429-434. doi: 10.4103/1947-2714.100998.
• Maliepaard M, Scheffer GL, Faneyte IF, van Gastelen MA, Pijnenborg AC, Schinkel AH, van De Vijver MJ, Scheper RJ, Schellens JH (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 172: 85-93. doi: 10.1016/j.jsb.2010.03.021.
• Mao Q, Conseil G, Gupta A, Cole SP, Unadkat JD (2004) Functional expression of the human breast cancer resistance protein in Pichia pastoris. Biochem Biophys Res Commun 320: 730-737. doi: 10.1016/j.bbrc.2004.06.012.
• Mayer P (1891) Uber das Farben mit Hamatoxylin. Mitt Zool Stat Neapel.
• Mitchell BL, Yasui Y, Li CI, Fitzpatrick AL, Lampe PD (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 MF, Mao Q (2010) Structure and function of the human breast cancer resistance protein (BCRP/ABCG2) Curr Drug Metab 11: 603-617. doi: 10.2174/138920010792927325.
• 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. doi: 10.1006/bbrc.2001.5130.
• Robey RW, Steadman K, Polgar O, Morisaki K, Blayney M, Mistry P, Bates SE (2004) Pheophorbide a is a specific probe for ABCG2 function and inhibition. Cancer Res 64: 1242-1246. doi: 10.1158/0008-5472.CAN-03-3298.
• Scharff-Poulsen P, Pedersen PA (2013) Saccharomyces cerevisiae-based platform for rapid production and evaluation of eukaryotic nutrient transporters and transceptors for biochemical studies and crystallography. PLoS One 8. doi: 10.1371/journal.pone.0076851.
• Staroń J, Boroń B, Karcz D, Szczygieł M, Fiedor L (2015) Recent Progress in chemical modifications of chlorophylls and bacteriochlorophylls for the applications in photodynamic therapy. Curr Med Chem 22: 3054-3074. doi: 10.2174/0929867322666150818104034.
• Storch CH, Ehehalt R, Haefeli WE, 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. J Pharmacol Exp Ther 323: 257-264. doi: 10.1124/jpet.107.122994.
• Szafraniec MJ, Szczygieł M, Urbanska K, Fiedor L (2014) Determinants of the activity and substrate recognition of breast cancer resistance protein (ABCG2) Drug Metab Rev 46: 459-474. doi: 10.3109/03602532.2014.942037.
• 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. doi: 10.1021/jm7016368.
• Takano M, Yumoto R, Murakami T (2006) Expression and function of efflux drug transporters in the intestine. Pharmacol Ther 109: 137-161. doi: 10.1016/j.pharmthera.2005.06.005.
• Taylor SC, Posch A (2014) The design of a quantitative western blot experiment. Biomed Res Int 2014. doi: 10.1155/2014/361590.
• 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. doi: 10.1016/j.bbamem.2007.06.026.
• 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. doi: 10.1042/BJ20121485.
• Théou N, Gil S, Devocelle A, Julié C, Lavergne-Slove A, Beauchet A, Emile JF (2005) Multidrug resistance proteins in gastrointestinal stromal tumors: Site-dependent expression and initial response to imatinib. Clin Cancer Res 11: 7593-7598. doi: 10.1158/1078-0432.CCR-05-0710.
• Watson DH (2001) Food chemical safety. CRC Press.
• Xu J, Peng H, Chen Q, Liu Y, Dong Z, Zhang JT (2007) Oligomerization domain of the multidrug resistance-associated transporter ABCG2 and its dominant inhibitory activity. Cancer Res 67: 4373-4381. doi: 10.1158/0008-5472.CAN-06-3169.

Identifiers

DOI

10.18388/abp.2017_2299