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In situ glucosylceramide synthesis and its pharmacological inhibition analysed in cells by 13 C 5 ‐sphingosine precursor feeding and mass spectrometry
Author(s) -
Katzy Rebecca E.,
Ferraz Maria J.,
Hazeu Marc,
Overkleeft Hermen S.,
Aerts Johannes M. F. G.
Publication year - 2022
Publication title -
febs letters
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.593
H-Index - 257
eISSN - 1873-3468
pISSN - 0014-5793
DOI - 10.1002/1873-3468.14448
Subject(s) - sphingosine , ceramide , sphingolipid , enzyme , mass spectrometry , biochemistry , in situ , chemistry , metabolism , glycolipid , biosynthesis , chromatography , receptor , apoptosis , organic chemistry
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 IC 50 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.