A Bioassay Using a Pentadecanal Derivative to Measure S1P Lyase Activity.

Kyong-Oh Shin,Jung-No Lee,Yong-Moon Lee,Maftuna Shamshiddinova,Kwang-Sik Lee

doi:10.3390/ijms22031438

Abstract

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 Km 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.

Highlights

sphingosine 1-phosphate (S1P) levels in our body seem to be controlled by three specific enzymes: S1P synthesis by sphingosine kinases and S1P degradations by S1P lyase (S1PL) and S1P phosphatase [7,8]
The Hantzsch reaction is well characterized by the cyclization of two ß-diketones and aldehyde in the presence of ammonium acetate [19]
By a precise comparison of previously reported reaction conditions, pentadecenal successfully derivatized in the customary reaction conditions with 1% 5,5-dimethyl CHD and yielded a high fluorescent peak area (Figure 2A)

Summary

Introduction

Radioactive substrate usage requires multiple cumbersome separation steps, such as phase separation and scraping the tritium-labeled reaction product from a thin-layer chromatographic plate. The fluorescent substrates, such as 7-nitrobenz-2-oxa-1,3-diazol S1P (NBD-S1P), were employed to enhance the detectability by fluorescently monitoring S1PL products [16]. Fluorescent substrates, the appearance of multiple fluorescent byproducts, or a higher Km value are the main disadvantages of each method To overcome these disadvantages, a specific chemical method for targeting aldehyde products was introduced, producing intense mass fragmentation ions such as semicarbazone [17], 2-diphenylacetyl-1,3-indandione-1hydrazone (DAIH) derivatives, and biotinylated aminosphingosine-1-[(33)P]phosphate (S1(33)P-biotin) [15,18]. The ideal designed reagent is nonfluorescent and reacts with an aldehyde to selectively form a fluorogenic structure

Methods

Results

Discussion

Conclusion