Development of a selective fluorescence-based enzyme assay for glycerophosphodiesterase family members GDE4 and GDE7

• DOI: 10.1016/j.jlr.2021.100141
• Publication Date: 2021-10-19T15:00:55Z
• Authors: Keisuke Kitakaze; Kazuhito Tsuboi; Maho Tsuda; Yasuhiro Takenouchi; Hironobu Ishimaru; Yasuo Okamoto
• Keywords: 0303 health sciences; Phosphoric Diester Hydrolases; enzymology; Organophosphonates; Phosphatidic Acids; QD415-436; Naphthalenes; Biochemistry; Fluorescence; 3. Good health; phospholipids/metabolism; 03 medical and health sciences; HEK293 Cells; lysophospholipid; kinetics; Methods; MCF-7 Cells; Humans; Anilides; phospholipids/biosynthesis; phospholipases/D; Enzyme Assays

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.