Advertisement

Bioorthogonal azido-S1P works as substrate for S1PR1

  • Christine Sternstein
    Affiliations
    Institute of Organic Chemistry, Julius-Maximilians-Universität Würzburg, Am Hubland, 97074 Würzburg, Germany
    Search for articles by this author
  • Author Footnotes
    3 Current Address: Department of Women’s and Children’s Health, Karolinska Institutet, SciLifeLab, Tomtebodavägen 23B, Solna, 171 65 Stockholm, Sweden
    Jan Schlegel
    Footnotes
    3 Current Address: Department of Women’s and Children’s Health, Karolinska Institutet, SciLifeLab, Tomtebodavägen 23B, Solna, 171 65 Stockholm, Sweden
    Affiliations
    Department of Biotechnology and Biophysics, Biocenter, Julius-Maximilians-Universität Würzburg, Am Hubland, 97074 Würzburg, Germany
    Search for articles by this author
  • Markus Sauer
    Affiliations
    Department of Biotechnology and Biophysics, Biocenter, Julius-Maximilians-Universität Würzburg, Am Hubland, 97074 Würzburg, Germany
    Search for articles by this author
  • Jürgen Seibel
    Correspondence
    For correspondence: Jürgen Seibel. jü[email protected]
    Affiliations
    Institute of Organic Chemistry, Julius-Maximilians-Universität Würzburg, Am Hubland, 97074 Würzburg, Germany
    Search for articles by this author
  • Author Footnotes
    3 Current Address: Department of Women’s and Children’s Health, Karolinska Institutet, SciLifeLab, Tomtebodavägen 23B, Solna, 171 65 Stockholm, Sweden
Open AccessPublished:November 09, 2022DOI:https://doi.org/10.1016/j.jlr.2022.100311
      Sphingosine-1-phosphate (S1P) and its G protein coupled receptors S1PR1-5 play an important role in cellular processes and are associated with many diseases.
      • O'Sullivan C.
      • Dev K.K.
      The structure and function of the S1P1 receptor.
      ,
      • McGinley M.P.
      • Cohen J.A.
      Sphingosine 1-phosphate receptor modulators in multiple sclerosis and other conditions.
      For that reason, the investigation of the S1P metabolism and its intracellular tracking is of high scientific interest. Azido-functionalised sphingolipids are tolerated by biological systems and can be modified via click chemistry with alkyne-substituted molecules.[
      • Fink J.
      • Seibel J.
      Click reactions with functional sphingolipids.
      ] We synthesised a clickable S1P derivative with a terminal azido function (S1P-N3) in 11 steps combining two synthetic approaches. Following the protocol by Lang et al. yielded N-Boc-protected azido-sphingosine,[
      • Lang J.
      • Bohn P.
      • Bhat H.
      • Jastrow H.
      • Walkenfort B.
      • Cansiz F.
      • Fink J.
      • Bauer M.
      • Olszewski D.
      • Ramos-Nascimento A.
      • Duhan V.
      • Friedrich S.-K.
      • Becker K.A.
      • Krawczyk A.
      • Edwards M.J.
      • Burchert A.
      • Huber M.
      • Friebus-Kardash J.
      • Göthert J.R.
      • Hardt C.
      • Probst H.C.
      • Schumacher F.
      • Köhrer K.
      • Kleuser B.
      • Babiychuk E.B.
      • Sodeik B.
      • Seibel J.
      • Greber U.F.
      • Lang P.A.
      • Gulbins E.
      • Lang K.S.
      Acid ceramidase of macrophages traps herpes simplex virus in multivesicular bodies and protects from severe disease.
      ] followed by phosphorylation of the primary alcohol to the target molecule. To investigate if S1P-N3 works as a substrate for S1PR1, living HEK293T cells, showing very low transcription of S1PRs, were transfected with S1PR1-GFP and incubated with S1P-N3. Prior to the addition of the lipid, the receptor was localised within the plasma membrane (A). After feeding with S1P-N3, a considerable amount of the receptor was detected within the cell (B). This suggests the activation and consecutive internalisation of S1PR1 by our modified substrate. These findings strongly indicate that S1P-N3 is still functional, making it a powerful tool for studying the S1P metabolism.[
      • Fink J.
      • Seibel J.
      Click reactions with functional sphingolipids.
      ] The applicability for this was confirmed by incubating U2Os cells, showing high endogenous levels of S1PR3 and S1PR5, with S1P-N3. Staining by click reaction with a DBCO-BODIPY dye and its visualisation within the cells by confocal microscopy showed that the S1P-dye conjugate and its metabolites were mainly localised intracellularly at the nuclear membrane and in the endoplasmic reticulum (C). Until now, co-labelling experiments of the clicked S1P-N3 within the S1PR1-GFP construct were not successful. As the alkyl chain of S1P is known to interact with the intracellular region of the S1PR1-binding pocket, the terminal azido-function is probably buried and not accessible for the click reaction.[
      • O'Sullivan C.
      • Dev K.K.
      The structure and function of the S1P1 receptor.
      ] We demonstrated that S1PR1-GFP of HEK293T cells accepts S1P-N3 as a substrate and that S1P-N3 and its metabolites can be visualised and localised within U2Os cells.

      Equipment and Methods

      HEK293T cells were seeded into 8 well chambered cover glass (Cellvis) and transfected with S1PR1-GFP (kindly provided by Prof. Meyer zu Heringdorf) using PEI 25K (Polysciences). After 24 hours cells were imaged in the presence or absence of 1 μM S1P-N3. To visualise subcellular localization of S1P-N3, U2Os cells were incubated 40 minutes at 37°C and 5% CO2 with 1 μM S1P-N3 and clicked with 1 μM BODIPY- FL-PEG4-DBCO (Jena Bioscience) for 20 min. Both fluorophores, GFP and BODIPY, were imaged using a Zeiss LSM 700 AxioObserver microscope equipped with Plan-Apochromat 63x/1.4 Oil M27 objective and 488nm laser light. Schematic illustrations were created with Biorender.com.

      References

        • O'Sullivan C.
        • Dev K.K.
        The structure and function of the S1P1 receptor.
        Trends Pharmacol. Sci. 2013; 34: 401-412
        • McGinley M.P.
        • Cohen J.A.
        Sphingosine 1-phosphate receptor modulators in multiple sclerosis and other conditions.
        Lancet. 2021; 398: 1184-1194
        • Fink J.
        • Seibel J.
        Click reactions with functional sphingolipids.
        Biol. Chem. 2018; 399: 1157-1168
        • Lang J.
        • Bohn P.
        • Bhat H.
        • Jastrow H.
        • Walkenfort B.
        • Cansiz F.
        • Fink J.
        • Bauer M.
        • Olszewski D.
        • Ramos-Nascimento A.
        • Duhan V.
        • Friedrich S.-K.
        • Becker K.A.
        • Krawczyk A.
        • Edwards M.J.
        • Burchert A.
        • Huber M.
        • Friebus-Kardash J.
        • Göthert J.R.
        • Hardt C.
        • Probst H.C.
        • Schumacher F.
        • Köhrer K.
        • Kleuser B.
        • Babiychuk E.B.
        • Sodeik B.
        • Seibel J.
        • Greber U.F.
        • Lang P.A.
        • Gulbins E.
        • Lang K.S.
        Acid ceramidase of macrophages traps herpes simplex virus in multivesicular bodies and protects from severe disease.
        Nat. commun. 2020; 11: 1338