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
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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