Development of a selective fluorescence-based enzyme assay for glycerophosphodiesterase family members GDE4 and GDE7
Autor: | Yasuhiro Takenouchi, Hironobu Ishimaru, Keisuke Kitakaze, Kazuhito Tsuboi, Maho Tsuda, Yasuo Okamoto |
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Jazyk: | angličtina |
Rok vydání: | 2021 |
Předmět: |
LPE
lysophosphatidylethanolamine enzymology phospholipids/phosphatidic acid Organophosphonates Phosphatidic Acids QD415-436 Naphthalenes Biochemistry GDE glycerophosphodiesterase Fluorescence phospholipids/metabolism chemistry.chemical_compound cDNA complementary DNA Endocrinology 3-ccPA 3-carbacyclic phosphatidic acid Lysophosphatidic acid LNCaP Methods Humans Anilides fluorescent substrate NP-40 Nonidet P-40 Enzyme Assays LPC lysophosphatidylcholine Chemistry Phosphoric Diester Hydrolases HEK 293 cells Cell migration Cell Biology Phosphatidic acid HEK293T human embryonic kidney 293T O/E overexpressing BrP-LPA α-bromomethylene phosphonate analog of LPA HEK293 Cells Lysophospholipase lysophospholipid kinetics glycerophosphodiesterase MCF-7 Cells ATX autotoxin Autotaxin phospholipids/biosynthesis Intracellular phospholipases/D lysophosphatidic acid LPA lysophosphatidic acid lyso-PLD lysophospholipase D |
Zdroj: | Journal of Lipid Research Journal of Lipid Research, Vol 62, Iss, Pp 100141-(2021) |
ISSN: | 1539-7262 0022-2275 |
Popis: | Lysophosphatidic acid (LPA) is a lipid mediator that regulates various processes, including cell migration and cancer progression. Autotaxin (ATX) is a lysophospholipase D-type exoenzyme that produces extracellular LPA. In contrast, glycerophosphodiesterase (GDE) family members GDE4 and GDE7 are intracellular lysophospholipases D that form LPA, depending on Mg2+ and Ca2+, respectively. Since no fluorescent substrate for these GDEs has been reported, in the present study, we examined whether a fluorescent ATX substrate, FS-3, could be applied to study GDE activity. We found that the membrane fractions of human GDE4- and GDE7-overexpressing human embryonic kidney 293T cells hydrolyzed FS-3 in a manner almost exclusively dependent on Mg2+ and Ca2+, respectively. Using these assay systems, we found that several ATX inhibitors, including α-bromomethylene phosphonate analog of LPA and 3-carbacyclic phosphatidic acid, also potently inhibited GDE4 and GDE7 activities. In contrast, the ATX inhibitor S32826 hardly inhibited these activities. Furthermore, FS-3 was hydrolyzed in a Mg2+-dependent manner by the membrane fraction of human prostate cancer LNCaP cells that express GDE4 endogenously but not by those of GDE4-deficient LNCaP cells. Similar Ca2+-dependent GDE7 activity was observed in human breast cancer MCF-7 cells but not in GDE7-deficient MCF-7 cells. Finally, our assay system could selectively measure GDE4 and GDE7 activities in a mixture of the membrane fractions of GDE4- and GDE7-overexpressing human embryonic kidney 293T cells in the presence of S32826. These findings allow high-throughput assays of GDE4 and GDE7 activities, which could lead to the development of selective inhibitors and stimulators as well as a better understanding of the biological roles of these enzymes. Graphical abstract |
Databáze: | OpenAIRE |
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