In situ glucosylceramide synthesis and its pharmacological inhibition analysed in cells by 13C5‐sphingosine precursor feeding and mass spectrometry. Issue 18 (13th July 2022)

• Record Type: Journal Article
• Authors: Katzy, Rebecca E.; Ferraz, Maria J.; Hazeu, Marc; Overkleeft, Hermen S.; Aerts, Johannes M. F. G.
• Is Part Of: FEBS letters. Volume 596:Issue 18(2022)
• Page Start: 2400
• Page End: 2408
• Publication Date: 2022-07-13
• Subjects: 13C‐labelled lipids -- glucosylceramide synthase -- glycosphingolipid metabolism -- mass spectrometry; Biochemistry -- Periodicals; Biophysics -- Periodicals; Molecular biology -- Periodicals; Biochimie -- Périodiques; Biochemistry; Biophysics; Molecular biology; Periodicals; 572.05
• DOI: 10.1002/1873-3468.14448
• Languages: English
• ISSNs: 0014-5793

Abstract

Abstract : Glycosphingolipids (GSLs) fulfil diverse functions in cells. Abnormalities in their metabolism are associated with specific pathologies and, consequently, the pharmacological modulation of GSLs is considered a therapeutic avenue. The accurate measurement of in situ metabolism of GSLs and the modulatory impact of drugs is warranted. Employing synthesised sphingosine and sphinganine containing 13 C atoms, we developed a method to monitor the de novo synthesis of glucosylceramide, the precursor of complex GSLs, by the enzyme glucosylceramide synthase (GCS). We show that feeding cells with isotope‐labelled precursor combined with liquid chromatography–mass spectrometry (MS)/MS analysis allows accurate determination of the IC50 values of therapeutically considered inhibitors (iminosugars and ceramide mimics) of GCS in cultured cells. Acquired data were comparable to those obtained with an earlier method using artificial fluorescently labelled ceramide to feed cells. Abstract : Abnormalities in the metabolism of glycosphingolipids (GSLs) are associated with a variety of pathologies. Thus, their pharmacological modulation is of therapeutic interest. We show that employing isotope‐labelled precursor lipids in combination with liquid chromatography–mass spectrometry (MS)/MS allows for monitoring the synthesis of glucosylceramide, the precursor of complex GSLs, by the enzyme glucosylceramide synthase (GCS), and for accurate determination of the IC50 values for inhibitors of GCS in cultured cells.