A Bioassay Using a Pentadecanal Derivative to Measure S1P Lyase Activity

Autor: Kwang Sik Lee, Jung-No Lee, Kyong-Oh Shin, Yong-Moon Lee, Maftuna Shamshiddinova
Rok vydání: 2021
Předmět:
0301 basic medicine
Lysis
Ligands
High-performance liquid chromatography
lcsh:Chemistry
Mice
chemistry.chemical_compound
5
5-dimethyl cyclohexanedione
Limit of Detection
sphingosine 1-phosphate lyase
Bioassay
lcsh:QH301-705.5
Chromatography
High Pressure Liquid
Spectroscopy
Chromatography
General Medicine
hexadecenal
Computer Science Applications
Ethanolamines
Biological Assay
Ammonium acetate
Protein Binding
pentadecanal
Article
Catalysis
Inorganic Chemistry
03 medical and health sciences
Acetic acid
Cell Line
Tumor
Animals
Physical and Theoretical Chemistry
Derivatization
Molecular Biology
Aldehyde-Lyases
Fluorescent Dyes
Aldehydes
030102 biochemistry & molecular biology
Cyclohexanones
Organic Chemistry
Substrate (chemistry)
030104 developmental biology
lcsh:Biology (General)
lcsh:QD1-999
chemistry
Cell culture
fluorescence detection
Mutation
Linear Models
HPLC
Zdroj: International Journal of Molecular Sciences
Volume 22
Issue 3
International Journal of Molecular Sciences, Vol 22, Iss 1438, p 1438 (2021)
ISSN: 1422-0067
DOI: 10.3390/ijms22031438
Popis: 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.
Databáze: OpenAIRE
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