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

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 int...

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Autores principales:	Keisuke Kitakaze, Kazuhito Tsuboi, Maho Tsuda, Yasuhiro Takenouchi, Hironobu Ishimaru, Yasuo Okamoto
Formato:	article
Lenguaje:	EN
Publicado:	Elsevier 2021
Materias:	enzymology kinetics lysophospholipid phospholipases/D phospholipids/biosynthesis phospholipids/metabolism Biochemistry QD415-436
Acceso en línea:	https://doaj.org/article/24778526397149548f656fa5d4264a44
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spelling	oai:doaj.org-article:24778526397149548f656fa5d4264a442021-11-10T04:17:18ZDevelopment of a selective fluorescence-based enzyme assay for glycerophosphodiesterase family members GDE4 and GDE70022-227510.1016/j.jlr.2021.100141https://doaj.org/article/24778526397149548f656fa5d4264a442021-01-01T00:00:00Zhttp://www.sciencedirect.com/science/article/pii/S0022227521001231https://doaj.org/toc/0022-2275Lysophosphatidic 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.Keisuke KitakazeKazuhito TsuboiMaho TsudaYasuhiro TakenouchiHironobu IshimaruYasuo OkamotoElsevierarticleenzymologykineticslysophospholipidphospholipases/Dphospholipids/biosynthesisphospholipids/metabolismBiochemistryQD415-436ENJournal of Lipid Research, Vol 62, Iss , Pp 100141- (2021)
institution	DOAJ
collection	DOAJ
language	EN
topic	enzymology kinetics lysophospholipid phospholipases/D phospholipids/biosynthesis phospholipids/metabolism Biochemistry QD415-436
spellingShingle	enzymology kinetics lysophospholipid phospholipases/D phospholipids/biosynthesis phospholipids/metabolism Biochemistry QD415-436 Keisuke Kitakaze Kazuhito Tsuboi Maho Tsuda Yasuhiro Takenouchi Hironobu Ishimaru Yasuo Okamoto Development of a selective fluorescence-based enzyme assay for glycerophosphodiesterase family members GDE4 and GDE7
description	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.
format	article
author	Keisuke Kitakaze Kazuhito Tsuboi Maho Tsuda Yasuhiro Takenouchi Hironobu Ishimaru Yasuo Okamoto
author_facet	Keisuke Kitakaze Kazuhito Tsuboi Maho Tsuda Yasuhiro Takenouchi Hironobu Ishimaru Yasuo Okamoto
author_sort	Keisuke Kitakaze
title	Development of a selective fluorescence-based enzyme assay for glycerophosphodiesterase family members GDE4 and GDE7
title_short	Development of a selective fluorescence-based enzyme assay for glycerophosphodiesterase family members GDE4 and GDE7
title_full	Development of a selective fluorescence-based enzyme assay for glycerophosphodiesterase family members GDE4 and GDE7
title_fullStr	Development of a selective fluorescence-based enzyme assay for glycerophosphodiesterase family members GDE4 and GDE7
title_full_unstemmed	Development of a selective fluorescence-based enzyme assay for glycerophosphodiesterase family members GDE4 and GDE7
title_sort	development of a selective fluorescence-based enzyme assay for glycerophosphodiesterase family members gde4 and gde7
publisher	Elsevier
publishDate	2021
url	https://doaj.org/article/24778526397149548f656fa5d4264a44
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Development of a selective fluorescence-based enzyme assay for glycerophosphodiesterase family members GDE4 and GDE7

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