Activity of neutral and alkaline ceramidases on fluorogenic N-acylated coumarin-containing aminodiols.
Ceramidases catalyze the cleavage of ceramides into sphingosine and fatty acids. Previously, we reported on the use of the RBM14 fluorogenic ceramide analogs to determine acidic ceramidase activity. In this work, we investigated the activity of other amidohydrolases on RBM14 compounds. Both bacterial and human purified neutral ceramidases (NCs), as well as ectopically expressed mouse neutral ceramidase hydrolyzed RBM14 with different selectivity, depending on the N-acyl chain length. On the other hand, microsomes from alkaline ceramidase (ACER)3 knockdown cells were less competent at hydrolyzing RBM14C12, RBM12C14, and RBM14C16 than controls, while microsomes from ACER2 and ACER3 overexpressing cells showed no activity toward the RBM14 substrates. Conversely, N-acylethanolamine-hydrolyzing acid amidase (NAAA) overexpressing cells hydrolyzed RBM14C14 and RBM14C16 at acidic pH. Overall, NC, ACER3, and, to a lesser extent, NAAA hydrolyze fluorogenic RBM14 compounds. Although the selectivity of the substrates toward ceramidases can be modulated by the length of the N-acyl chain, none of them was specific for a particular enzyme. Despite the lack of specificity, these substrates should prove useful in library screening programs aimed at identifying potent and selective inhibitors for NC and ACER3.
Consejo Superior de Investigaciones Científicas (CSIC), Institut de Química Avançada de Catalunya (IQAC-CSIC), Research Unit on Bioactive Molecules (RUBAM), 08034 Barcelona, Spain.
Mireia
M
Casasampere
Consejo Superior de Investigaciones Científicas (CSIC), Institut de Química Avançada de Catalunya (IQAC-CSIC), Research Unit on Bioactive Molecules (RUBAM), 08034 Barcelona, Spain.
Luz
L
Camacho
Consejo Superior de Investigaciones Científicas (CSIC), Institut de Química Avançada de Catalunya (IQAC-CSIC), Research Unit on Bioactive Molecules (RUBAM), 08034 Barcelona, Spain.
Francesca
F
Cingolani
Consejo Superior de Investigaciones Científicas (CSIC), Institut de Química Avançada de Catalunya (IQAC-CSIC), Research Unit on Bioactive Molecules (RUBAM), 08034 Barcelona, Spain.
Josefina
J
Casas
Consejo Superior de Investigaciones Científicas (CSIC), Institut de Química Avançada de Catalunya (IQAC-CSIC), Research Unit on Bioactive Molecules (RUBAM), 08034 Barcelona, Spain.
Meritxell
M
Egido-Gabás
Consejo Superior de Investigaciones Científicas (CSIC), Institut de Química Avançada de Catalunya (IQAC-CSIC), Research Unit on Bioactive Molecules (RUBAM), 08034 Barcelona, Spain.
José Luís
JL
Abad
Consejo Superior de Investigaciones Científicas (CSIC), Institut de Química Avançada de Catalunya (IQAC-CSIC), Research Unit on Bioactive Molecules (RUBAM), 08034 Barcelona, Spain.
Carmen
C
Bedia
Department of Medicine, State University of New York at Stony Brook, Stony Brook, NY 11794-8155.
Ruijuan
R
Xu
Department of Medicine, State University of New York at Stony Brook, Stony Brook, NY 11794-8155.
Kai
K
Wang
Department of Medicine, State University of New York at Stony Brook, Stony Brook, NY 11794-8155.
Daniel
D
Canals
Department of Medicine, State University of New York at Stony Brook, Stony Brook, NY 11794-8155.
Yusuf A
YA
Hannun
Department of Medicine, State University of New York at Stony Brook, Stony Brook, NY 11794-8155.
Cungui
C
Mao
Consejo Superior de Investigaciones Científicas (CSIC), Institut de Química Avançada de Catalunya (IQAC-CSIC), Research Unit on Bioactive Molecules (RUBAM), 08034 Barcelona, Spain.
Gemma
G
Fabrias
D000215:Acylation; D055574:Alkaline Ceramidase / Q000172:deficiency / Q000235:genetics / Q000378:metabolism*; D000818:Animals; D002518:Ceramides / Q000378:metabolism* / Q000493:pharmacokinetics; D003374:Coumarins / Q000493:pharmacokinetics; D005456:Fluorescent Dyes / Q000493:pharmacokinetics; D055785:Gene Knockdown Techniques; D045325:HCT116 Cells; D057809:HEK293 Cells; D006367:HeLa Cells; D006801:Humans; D006868:Hydrolysis; D013058:Mass Spectrometry; D051379:Mice; D055576:Neutral Ceramidase / Q000172:deficiency / Q000235:genetics / Q000378:metabolism*; D013107:Sphingolipids / Q000378:metabolism; D013329:Structure-Activity Relationship; D013379:Substrate Specificity
D002518:Ceramides; D003374:Coumarins; D005456:Fluorescent Dyes; D013107:Sphingolipids; D055574:Alkaline Ceramidase; D055576:Neutral Ceramidase
ceramides; enzymology; high throughput screening; lipids; mass spectrometry; sphingolipids; umbelliferone