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Scintillation Proximity Assay to Detect the Changes in Cellular Dihydrosphingosine 1-Phosphate Levels

  • Methods
  • Published:
Lipids

Abstract

Compounds that modulate the activity of sphingosine 1-phosphate (S1P)-metabolizing enzymes are expected to be potential therapeutic agents for various diseases. Investigation of their potencies requires not only cell-free but also cell-based assays in which intracellular accumulation/depletion of S1P could be monitored. However, conventional methods have limitations to their simplicity, mainly due to the necessity of a separation process that separates S1P from its related substances. Here, we describe a method utilizing a scintillation proximity assay (SPA) for semi-quantifying intracellular [3H]-labeled dihydroS1P ([3H]dhS1P), which is also a substrate for S1P-metabolizing enzymes. We found that uncoated yttrium silicate SPA beads could selectively bind to and detect [3H]dhS1P rather than [3H]dihydrosphingosine (the non-phosphorylated form of [3H]dhS1P). Based on this, we developed a novel cell-based assay system which does not require any organic solvent extraction or chromatographic separation, and confirmed its practicality by using siRNA targeting S1P lyase (S1PL) and known S1PL inhibitors as models. Our results demonstrated that this assay is useful for rapid and easy evaluation of S1PL inhibitors, and could be potentially applicable for all compounds that modulate the activity of S1P-metabolizing enzymes.

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Abbreviations

A6770:

1-[4(5)-(Hydroxymethyl)-1H-imidazole-2-yl]ethanone

A6770-P:

[2-Acetyl-1H-imidazole-4(5)-yl]methoxyphosphate

dhCer:

Dihydroceramide

dhS1P:

Dihydrosphingosine 1-phosphate (Sphinganine 1-phosphate)

dhSph:

Dihydrosphingosine (Sphinganine)

DOP:

4-Deoxypyridoxine

DOP-P:

4-Deoxypyridoxine 5′-phosphate

FB1:

Fumonisin B1

SPA:

Scintillation proximity assay

SphK:

Sphingosine kinases

SPP:

Sphingosine 1-phosphate phosphatases

S1P:

Sphingosine 1-phosphate

S1PL:

Sphingosine 1-phosphate lyase

THI:

2-Acetyl-4-(tetrahydroxybutyl)imidazole

VB6:

Vitamin B6

VB6(−):

Vitamin B6-deficient

YSi:

Yttrium silicate

References

  1. Strub GM, Maceyka M, Hait NC, Milstien S, Spiegel S (2010) Extracellular and intracellular actions of sphingosine-1-phosphate. Adv Exp Med Biol 688:141–155

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  2. Marsolais D, Rosen H (2009) Chemical modulators of sphingosine-1-phosphate receptors as barrier-oriented therapeutic molecules. Nat Rev Drug Discov 8:297–307

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  3. Sanllehí P, Abad JL, Casas J, Delgado A (2016) Inhibitors of sphingosine-1-phosphate metabolism (sphingosine kinases and sphingosine-1-phosphate lyase). Chem Phys Lipids 197:69–81

    Article  PubMed  Google Scholar 

  4. Kumar A, Saba JD (2009) Lyase to live by: sphingosine phosphate lyase as a therapeutic target. Expert Opin Ther Targets 13:1013–1025

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  5. Pyne S, Bittman R, Pyne NJ (2011) Sphingosine kinase inhibitors and cancer: seeking the golden sword of Hercules. Cancer Res 71:6576–6582

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  6. Yatomi Y, Ruan F, Ohta J, Welch RJ, Hakomori S, Igarashi Y (1995) Quantitative measurement of sphingosine 1-phosphate in biological samples by acylation with radioactive acetic anhydride. Anal Biochem 230:315–320

    Article  CAS  PubMed  Google Scholar 

  7. Edsall LC, Spiegel S (1999) Enzymatic measurement of sphingosine 1-phosphate. Anal Biochem 272:80–86

    Article  CAS  PubMed  Google Scholar 

  8. Min JK, Yoo HS, Lee EY, Lee WJ, Lee YM (2002) Simultaneous quantitative analysis of sphingoid base 1-phosphates in biological samples by o-phthalaldehyde precolumn derivatization after dephosphorylation with alkaline phosphatase. Anal Biochem 303:167–175

    Article  CAS  PubMed  Google Scholar 

  9. Lee YM, Venkataraman K, Hwang SI, Han DK, Hla T (2007) A novel method to quantify sphingosine 1-phosphate by immobilized metal affinity chromatography (IMAC). Prostaglandins Other Lipid Mediat 84:154–162

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  10. Mano N, Oda Y, Yamada K, Asakawa N, Katayama K (1997) Simultaneous quantitative determination method for sphingolipid metabolites by liquid chromatography/ionspray ionization tandem mass spectrometry. Anal Biochem 244:291–300

    Article  CAS  PubMed  Google Scholar 

  11. Berdyshev EV, Gorshkova IA, Garcia JG, Natarajan V, Hubbard WC (2005) Quantitative analysis of sphingoid base-1-phosphates as bisacetylated derivatives by liquid chromatography-tandem mass spectrometry. Anal Biochem 339:129–136

    Article  CAS  PubMed  Google Scholar 

  12. Billich A, Beerli C, Bergmann R, Bruns C, Loetscher E (2013) Cellular assay for the characterization of sphingosine-1-phosphate lyase inhibitors. Anal Biochem 434:247–253

    Article  CAS  PubMed  Google Scholar 

  13. Murata N, Sato K, Kon J, Tomura H, Okajima F (2000) Quantitative measurement of sphingosine 1-phosphate by radioreceptor-binding assay. Anal Biochem 282:115–120

    Article  CAS  PubMed  Google Scholar 

  14. Loetscher E, Schneider K, Beerli C, Billich A (2013) Assay to measure the secretion of sphingosine-1-phosphate from cells induced by S1P lyase inhibitors. Biochem Biophys Res Commun 433:345–348

    Article  CAS  PubMed  Google Scholar 

  15. Cook N, Harris A, Hopkins A, Hughes K (2002) Scintillation proximity assay (SPA) technology to study biomolecular interactions. Curr Protoc Protein Sci. doi:10.1002/0471140864.ps1908s27

    PubMed  Google Scholar 

  16. Long RF, Morrison AL (1954) The synthesis of 4-deoxypyridoxine phosphates. J Chem Soc 3854−3856. doi:10.1039/JR9540003854

  17. Ohtoyo M, Machinaga N, Inoue R, Hagihara K, Yuita H, Tamura M, Hashimoto R, Chiba J, Muro F, Watanabe J, Kobayashi Y, Abe K, Kita Y, Nagasaki M, Shimozato T (2016) Component of caramel food coloring, THI, causes lymphopenia indirectly via a key metabolic intermediate. Cell Chem Biol 23:555–560

    Article  CAS  PubMed  Google Scholar 

  18. Cliff MD, Pyne SG (1997) Asymmetric Synthesis of 2-Acetyl-4(5)-(1,2,3,4-tetrahydroxybutyl)imidazoles. J Org Chem 62:1023–1032

    Article  CAS  Google Scholar 

  19. Boumendjel A, Miller SPF (1994) Synthesis of an inhibitor of sphingosine-1-phosphate lyase. Tetrahedron Lett 35:819–822

    Article  CAS  Google Scholar 

  20. Itoh T, Doi H, Chin S, Nishimura T, Kasahara S (1988) Establishment of mouse thymic nurse cell clones from a spontaneous BALB/c thymic tumor. Eur J Immunol 18:821–824

    Article  CAS  PubMed  Google Scholar 

  21. Ikeda M, Kihara A, Kariya Y, Lee YM, Igarashi Y (2005) Sphingolipid-to-glycerophospholipid conversion in SPL-null cells implies the existence of an alternative isozyme. Biochem Biophys Res Commun 329:474–479

    Article  CAS  PubMed  Google Scholar 

  22. Brandish PE, Hill LA, Zheng W, Scolnick EM (2003) Scintillation proximity assay of inositol phosphates in cell extracts: high-throughput measurement of G-protein-coupled receptor activation. Anal Biochem 313:311–318

    Article  CAS  PubMed  Google Scholar 

  23. Bembenek ME, Roy R, Li P, Chee L, Jain S, Parsons T (2004) A homogeneous scintillation proximity format for monitoring the activity of recombinant human long-chain-fatty-acyl-CoA synthetase 5. Assay Drug Dev Technol 2:300–307

    Article  CAS  PubMed  Google Scholar 

  24. Ohtoyo M, Tamura M, Machinaga N, Muro F, Hashimoto R (2015) Sphingosine 1-phosphate lyase inhibition by 2-acetyl-4-(tetrahydroxybutyl)imidazole (THI) under conditions of vitamin B6 deficiency. Mol Cell Biochem 400:125–133

    Article  CAS  PubMed  Google Scholar 

  25. Stockmann-Juvala H, Savolainen K (2008) A review of the toxic effects and mechanisms of action of fumonisin B1. Hum Exp Toxicol 27:799–809

    Article  CAS  PubMed  Google Scholar 

  26. Kharel Y, Mathews TP, Kennedy AJ, Houck JD, Macdonald TL, Lynch KR (2011) A rapid assay for assessment of sphingosine kinase inhibitors and substrates. Anal Biochem 411:230–235

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  27. Kashem MA, Wa C, Wolak JP, Grafos NS, Ryan KR, Sanville-Ross ML, Fogarty KE, Rybina IV, Shoultz A, Molinaro T, Desai SN, Rajan A, Huber JD, Nelson RM (2014) A high-throughput scintillation proximity assay for sphingosine-1-phosphate lyase. Assay Drug Dev Technol 12:293–302

    Article  CAS  PubMed  Google Scholar 

  28. Fyrst H, Saba JD (2008) Sphingosine-1-phosphate lyase in development and disease: sphingolipid metabolism takes flight. Biochim Biophys Acta 1781:448–458

    Article  CAS  PubMed  PubMed Central  Google Scholar 

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Acknowledgments

The authors are grateful to Dr. Mika Ikeda for practical advice, and the project team members for their useful discussions. The authors would also like to thank Drs. Ryuta Koishi, Gen Kudo and Hiroshi Yokota for their suggestions, and Drs. Tohru Takahashi and Hidehiko Furukawa for their support.

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Authors and Affiliations

  1. Modality Research Laboratories, R&D Division, Daiichi Sankyo Co., Ltd., 1-2-58, Hiromachi, Shinagawa-ku, Tokyo, 140-8710, Japan

    Mamoru Ohtoyo, Masakazu Tamura & Ryuji Hashimoto

  2. Pain & Neuroscience Laboratories, R&D Division, Daiichi Sankyo Co., Ltd., 1-2-58, Hiromachi, Shinagawa-ku, Tokyo, 140-8710, Japan

    Nobuo Machinaga

  3. Manufacturing Department I, Production Division, Kitasato Daiichi Sankyo Vaccine Co., Ltd., 6-111, Arai, Kitamoto, Saitama, 364-0026, Japan

    Fumihito Muro

Authors
  1. Mamoru Ohtoyo
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  2. Masakazu Tamura
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  3. Nobuo Machinaga
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  4. Fumihito Muro
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  5. Ryuji Hashimoto
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Corresponding author

Correspondence to Mamoru Ohtoyo.

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Conflict of interest

All authors are employees of Daiichi Sankyo Co., Ltd. M.O and M.T are the inventors of the patent application relating to the cell-based assay method described in this paper, and all rights of the patent have been assigned to the company.

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Ohtoyo, M., Tamura, M., Machinaga, N. et al. Scintillation Proximity Assay to Detect the Changes in Cellular Dihydrosphingosine 1-Phosphate Levels. Lipids 51, 1207–1216 (2016). https://doi.org/10.1007/s11745-016-4187-0

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  • DOI: https://doi.org/10.1007/s11745-016-4187-0

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