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Optimization and Application of a Biotinylation Method for Quantification of Plasma Membrane Expression of Transporters in Cells

  • Protocols in Pharmaceutical Sciences
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Abstract

Quantitative proteomics, using LC-MS/MS, is increasingly used to quantify drug transporters present in tissues and cells. Most of these investigations quantify total transporter expression in the cells by utilizing a total membrane fraction, not only the plasma membrane. Here, we report development and optimization of a biotinylation method to quantify protein expression of transporters in the plasma membrane of cells. The Pierce cell surface isolation protocol was optimized for plasma membrane isolation. Incubation of OATP1B1-expressing CHO cells with 0.78 mg/mL of membrane impermeable biotinylation reagent (sulfo-NHS-SS-biotin) at 37°C for 1 h resulted in optimum isolation of the plasma membrane. Subsequently, the expression of transporters in the plasma membrane as a percent of the total was determined by quantitative proteomics using LC-MS/MS. Mean (±SD) plasma membrane expression of OATP1B1 in plated OATP1B1-expressing CHO, MDCKII, and HEK293 cells was found to be 79.7% (±4.7%), 67.7% (±12.2%), and 65.3% (±6.8%) of total cell OATP1B1 expression. Mean (±SD) plasma membrane expression of OATP1B3 in plated OATP1B3-expressing HEK293 cells, OATP2B1 in plated OATP2B1-expressing MDCKII cells, and sodium/taurocholate co-transporting polypeptide (NTCP) in plated NTCP-expressing CHO cells was 63.2% (±1.6%), 37.1% (±15.7%), and 71.7% (±1.2%), respectively. This method of quantifying transporter protein expression in the plasma membrane will be useful in the future to predict transporter-mediated drug disposition.

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REFERENCES

  1. Feng B, Varma MV, Costales C, Zhang H, Tremaine L. In vitro and in vivo approaches to characterize transporter-mediated disposition in drug discovery. Expert Opin Drug Discovery. 2014;9(8):873–90.

    Article  CAS  Google Scholar 

  2. Varma MV, Bi YA, Kimoto E, Lin J. Quantitative prediction of transporter- and enzyme-mediated clinical drug-drug interactions of organic anion-transporting polypeptide 1B1 substrates using a mechanistic net-effect model. J Pharmacol Exp Ther. 2014;351(1):214–23.

    Article  PubMed  Google Scholar 

  3. Bosgra S, van de Steeg E, Vlaming ML, Verhoeckx KC, Huisman MT, Verwei M, et al. Predicting carrier-mediated hepatic disposition of rosuvastatin in man by scaling from individual transfected cell-lines in vitro using absolute transporter protein quantification and PBPK modeling. Eur J Pharm Sci. 2014;65:156–66.

    Article  CAS  PubMed  Google Scholar 

  4. Vildhede A, Mateus A, Khan EK, Lai Y, Karlgren M, Artursson P, et al. Mechanistic modeling of pitavastatin disposition in sandwich-cultured human hepatocytes: a proteomics-informed bottom-up approach. Drug Metab Dispos. 2016;44(4):505–16.

    Article  CAS  PubMed  Google Scholar 

  5. Jones HM, Barton HA, Lai Y, Bi YA, Kimoto E, Kempshall S, et al. Mechanistic pharmacokinetic modeling for the prediction of transporter-mediated disposition in humans from sandwich culture human hepatocyte data. Drug Metab Dispos. 2012;40(5):1007–17.

    Article  CAS  PubMed  Google Scholar 

  6. Menochet K, Kenworthy KE, Houston JB, Galetin A. Use of mechanistic modeling to assess interindividual variability and interspecies differences in active uptake in human and rat hepatocytes. Drug Metab Dispos. 2012;40(9):1744–56.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  7. Vildhede A, Wisniewski JR, Noren A, Karlgren M, Artursson P. Comparative proteomic analysis of human liver tissue and isolated hepatocytes with a focus on proteins determining drug exposure. J Proteome Res. 2015;14(8):3305–14.

    Article  CAS  PubMed  Google Scholar 

  8. Prasad B, Unadkat JD. Optimized approaches for quantification of drug transporters in tissues and cells by MRM proteomics. AAPS J. 2014;16(4):634–48.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  9. Lundquist P, Loof J, Sohlenius-Sternbeck AK, Floby E, Johansson J, Bylund J, et al. The impact of solute carrier (SLC) drug uptake transporter loss in human and rat cryopreserved hepatocytes on clearance predictions. Drug Metab Dispos. 2014;42(3):469–80.

    Article  PubMed  Google Scholar 

  10. Roma MG, Crocenzi FA, Mottino AD. Dynamic localization of hepatocellular transporters in health and disease. World J Gastroenterol. 2008;14(44):6786–801.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  11. Bow DA, Perry JL, Miller DS, Pritchard JB, Brouwer KL. Localization of P-gp (Abcb1) and Mrp2 (Abcc2) in freshly isolated rat hepatocytes. Drug Metab Dispos. 2008;36(1):198–202.

    Article  CAS  PubMed  Google Scholar 

  12. Cole SR, Ashman LK, Ey PL. Biotinylation: an alternative to radioiodination for the identification of cell surface antigens in immunoprecipitates. Mol Immunol. 1987;24(7):699–705.

    Article  CAS  PubMed  Google Scholar 

  13. Kumar V, Prasad B, Patilea G, Gupta A, Salphati L, Evers R, et al. Quantitative transporter proteomics by liquid chromatography with tandem mass spectrometry: addressing methodologic issues of plasma membrane isolation and expression-activity relationship. Drug Metab Dispos. 2015;43(2):284–8.

    Article  PubMed  Google Scholar 

  14. Steck TL, Weinstein RS, Straus JH, Wallach DF. Inside-out red cell membrane vesicles: preparation and purification. Science. 1970;168(3928):255–7.

    Article  CAS  PubMed  Google Scholar 

  15. Eggleton P, Michalak M. Calreticulin. 2nd ed. New York: Landes Bioscience/Eurekah.com; Kluwer Academic/Plenum; 2003. p. 282.

    Book  Google Scholar 

  16. Wang L, Prasad B, Salphati L, Chu X, Gupta A, Hop CE, et al. Interspecies variability in expression of hepatobiliary transporters across human, dog, monkey, and rat as determined by quantitative proteomics. Drug Metab Dispos. 2015;43(3):367–74.

    Article  PubMed  Google Scholar 

  17. Prasad B, Lai Y, Lin Y, Unadkat JD. Interindividual variability in the hepatic expression of the human breast cancer resistance protein (BCRP/ABCG2): effect of age, sex, and genotype. J Pharm Sci. 2013;102(3):787–93.

    Article  CAS  PubMed  Google Scholar 

  18. User Guide: Pierce cell surface protein isolation kit: Thermo Fisher: [Pierce cell surface protein isolation kit] 2017. Available from: https://tools.thermofisher.com/content/sfs/manuals/MAN0011518_Pierce_Cell_Surface_Protein_Isolat_UG.pdf.

  19. Loder MK, Melikian HE. The dopamine transporter constitutively internalizes and recycles in a protein kinase C-regulated manner in stably transfected PC12 cell lines. J Biol Chem. 2003;278(24):22168–74.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  20. Powell J, Farasyn T, Kock K, Meng X, Pahwa S, Brouwer KL, et al. Novel mechanism of impaired function of organic anion-transporting polypeptide 1B3 in human hepatocytes: post-translational regulation of OATP1B3 by protein kinase C activation. Drug Metab Dispos. 2014;42(11):1964–70.

    Article  PubMed  PubMed Central  Google Scholar 

  21. Sun T, Yu SH, Zhao P, Meng L, Moremen KW, Wells L, et al. One-step selective exoenzymatic labeling (SEEL) strategy for the biotinylation and identification of glycoproteins of living cells. J Am Chem Soc. 2016;138(36):11575–82.

    Article  CAS  PubMed  Google Scholar 

  22. Ho RH, Leake BF, Roberts RL, Lee W, Kim RB. Ethnicity-dependent polymorphism in Na+-taurocholate cotransporting polypeptide (SLC10A1) reveals a domain critical for bile acid substrate recognition. J Biol Chem. 2004;279(8):7213–22.

    Article  CAS  PubMed  Google Scholar 

  23. Lee W, Glaeser H, Smith LH, Roberts RL, Moeckel GW, Gervasini G, et al. Polymorphisms in human organic anion-transporting polypeptide 1A2 (OATP1A2): implications for altered drug disposition and central nervous system drug entry. J Biol Chem. 2005;280(10):9610–7.

    Article  CAS  PubMed  Google Scholar 

  24. Urquhart BL, Ware JA, Tirona RG, Ho RH, Leake BF, Schwarz UI, et al. Breast cancer resistance protein (ABCG2) and drug disposition: intestinal expression, polymorphisms and sulfasalazine as an in vivo probe. Pharmacogenet Genomics. 2008;18(5):439–48.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  25. Kopplow K, Letschert K, Konig J, Walter B, Keppler D. Human hepatobiliary transport of organic anions analyzed by quadruple-transfected cells. Mol Pharmacol. 2005;68(4):1031–8.

    Article  CAS  PubMed  Google Scholar 

  26. Kotani N, Maeda K, Watanabe T, Hiramatsu M, Gong LK, Bi YA, et al. Culture period-dependent changes in the uptake of transporter substrates in sandwich-cultured rat and human hepatocytes. Drug Metab Dispos. 2011;39(9):1503–10.

    Article  CAS  PubMed  Google Scholar 

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ACKNOWLEDGEMENTS

The authors thank Dr. Bruno Stieger, University Hospital Zurich, for OATP1B1-expressing CHO cells, Dr. Markus Keiser, University of Greifswald for OATP1B1-expressing MDCKII cells, Solvo Biotechnology for OATP1B1-expressing HEK293, OATP1B3-expressing HEK293, OATP2B1-expressing MDCKII, and NTCP-expressing CHO cells. The authors thank Dr. Sarah Billington for her help in reviewing this manuscript. Vineet Kumar was supported by the Simcyp Grant and Partnership Scheme (Certera).

AUTHORSHIP CONTRIBUTIONS

Participated in research design: Kumar, Nguyen, Unadkat.

Conducted experiments: Kumar, Nguyen.

Contributed new reagents or analytic tools: Tóth, Juhasz.

Performed data analysis: Kumar, Nguyen, Unadkat.

Wrote or contributed to the writing of the manuscript: Kumar, Unadkat.

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Authors and Affiliations

  1. Department of Pharmaceutics, University of Washington, P. O. Box 357610, Seattle, Washington, 98195, USA

    Vineet Kumar, Tot Bui Nguyen & Jashvant D. Unadkat

  2. SOLVO Biotechnology, Gyár utca 2, Budaörs, 2040, Hungary

    Beáta Tóth & Viktoria Juhasz

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  1. Vineet Kumar
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  2. Tot Bui Nguyen
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  3. Beáta Tóth
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  4. Viktoria Juhasz
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  5. Jashvant D. Unadkat
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Correspondence to Jashvant D. Unadkat.

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Kumar, V., Nguyen, T.B., Tóth, B. et al. Optimization and Application of a Biotinylation Method for Quantification of Plasma Membrane Expression of Transporters in Cells. AAPS J 19, 1377–1386 (2017). https://doi.org/10.1208/s12248-017-0121-5

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  • DOI: https://doi.org/10.1208/s12248-017-0121-5

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