A Bioassay Using a Pentadecanal Derivative to Measure S1P Lyase Activity

• Published in: International journal of molecular sciences Vol. 22; no. 3; p. 1438
• Main Authors: Shin, Kyong-Oh, Shamshiddinova, Maftuna, Lee, Jung-No, Lee, Kwang-Sik, Lee, Yong-Moon
• Format: Journal Article
• Language: English
• Published: Switzerland MDPI AG 01-02-2021; MDPI
• Subjects: 5,5-dimethyl cyclohexanedione; Acetic acid; Ammonium; Ammonium acetate; Bioassays; By products; Carbon; Chromatography; Enzyme kinetics; Fluorescence; fluorescence detection; FTY720; hexadecenal; High-performance liquid chromatography; HPLC; Kinases; Lipids; pentadecanal; Proteins; Quantitative analysis; Reaction time; Reagents; Sensitivity analysis; Sphinganine; Sphingosine 1-phosphate; sphingosine 1-phosphate lyase; Tumor cell lines; sphingosine 1-phosphate lyase; hexadecenal; pentadecanal; HPLC; 5,5-dimethyl cyclohexanedione; fluorescence detection
• ISSN: 1422-0067; 1661-6596; 1422-0067
• DOI: 10.3390/ijms22031438

Sphingosine-1-phosphate (S1P) is a unique lipid ligand binding to S1P receptors to transduce various cell survival or proliferation signals via small G proteins. S1P lyase (S1PL) is the specific enzyme that degrades S1P to phosphoethanolamine and (2E)-hexadecenal and therefore regulates S1P levels. S1PL also degrades dihydrosphingosine-1-phosphate (Sa1P), with a higher affinity to produce hexadecanal. Here, we developed a newly designed assay using a C17-Sa1P substrate that degrades into pentadecanal and phosphoethanolamine. For higher sensitivity in pentadecanal analysis, we developed a quantitative protocol as well as a 5,5-dimethyl cyclohexanedione (5,5-dimethyl CHD) derivatization method. The derivatization conditions were optimized for the reaction time, temperature, and concentrations of the 5,5-dimethyl CHD reagent, acetic acid, and ammonium acetate. The S1PL reaction in the cell lysate after spiking 20 µM of C17-Sa1P for 20 min was linear to the total protein concentrations of 50 µg. The S1PL levels (4 pmol/mg/min) were readily detected in this HPLC with fluorescence detection (λex = 366 nm, λem = 455 nm). The S1PL-catalyzed reaction was linear over 30 min and yielded a K
value of 2.68 μM for C17-Sa1P. This new method was validated to measure the S1PL activity of mouse embryonal carcinoma cell lines of the standard cell (F9-0), S1PL knockdown cells (F9-2), and S1PL-overexpressed cells (F9-4). Furthermore, we treated F9-4 cells with different S1PL inhibitors such as FTY720, 4-deoxypyridoxine (DOP), and the deletion of pyridoxal-5-phosphate (P5P), an essential cofactor for S1PL activity, and observed a significant decrease in pentadecanal relative to the untreated cells. In conclusion, we developed a highly sensitive S1PL assay using a C17-Sa1P substrate for pentadecanal quantification for application in the characterization of S1PL activity in vitro.