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Bioorthogonal azido-S1P works as substrate for S1PR1

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-functionalized 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 synthesized 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. (
      • Lang J.
      • Bohn P.
      • Bhat H.
      • Jastrow H.
      • Walkenfort B.
      • Cansiz F.
      • et al.
      Acid ceramidase of macrophages traps herpes simplex virus in multivesicular bodies and protects from severe disease.
      ) yielded N-Boc-protected azido-sphingosine, followed by phosphorylation of the primary alcohol to the target molecule. To investigate if S1P-N3 works as a substrate for S1PR1, living human embryonic kidney 293T (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 localized 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 internalization 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 adequate endogenous levels of S1PR3 and S1PR5, with S1P-N3. Staining by click reaction with a DBCO-BODIPY dye and its visualization within the cells by confocal microscopy showed that the S1P-dye conjugate and its metabolites were mainly localized intracellularly at the nuclear membrane and in the endoplasmic reticulum (C). Until now, colabeling 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 visualized and localized 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 h, cells were imaged in the presence or absence of 1 μM S1P-N3. To visualize subcellular localization of S1P-N3, U2Os cells were incubated for 40 min 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 63×/1.4 Oil M27 objective and 488 nm laser light. Schematic illustrations were created with Biorender.com.

      Conflict of interest

      The authors declare that they have no conflicts of interest with the contents of this article.

      Author Contributions

      M. S. and Jürgen Seibel conceptualization; Jan Schlegel methodology; C. S. and Jan Schlegel investigation; C. S. writing–original draft; Jan Schlegel, M. S., and Jürgen Seibel writing–review & editing; M. S. and Jürgen Seibel supervision.
      Funding and additional information
      This work was funded by the Deutsche Forschungsgemeinschaft - RTG 2581, project number 417857878.

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