ABSTRACT
Choline is an essential nutrient for all living cells and is produced extracellularly by sequential degradation of phosphatidylcholine (PC). However, little is known about how choline is produced extracellularly. Here, we report that ENPP6, a choline-specific phosphodiesterase, hydrolyzes glycerophosphocholine (GPC), a degradation product of PC, as a physiological substrate and participates in choline metabolism. ENPP6 is highly expressed in liver sinusoidal endothelial cells and developing oligodendrocytes, which actively incorporate choline and synthesize PC. ENPP6-deficient mice exhibited fatty liver and hypomyelination, well known choline-deficient phenotypes. The choline moiety of GPC was incorporated into PC in an ENPP6-dependent manner both in vivo and in vitro. The crystal structure of ENPP6 in complex with phosphocholine revealed that the choline moiety of the phosphocholine is recognized by a choline-binding pocket formed by conserved aromatic and acidic residues. The present study provides the molecular basis for ENPP6-mediated choline metabolism at atomic, cellular and tissue levels.
Subject(s)
Phosphatidylcholines/chemistry , Phosphatidylcholines/metabolism , Phosphoric Diester Hydrolases/chemistry , Phosphoric Diester Hydrolases/metabolism , Animals , Crystallography, X-Ray , Endothelial Cells/enzymology , Fatty Liver/enzymology , Fatty Liver/genetics , Liver/enzymology , Mice , Mice, Knockout , Oligodendroglia/enzymology , Organ Specificity , Phosphatidylcholines/genetics , Structure-Activity Relationship , Substrate SpecificityABSTRACT
Lysophosphatidic acid (LPA) and sphingosine-1-phosphate (S1P) are second-generation lysophospholipid mediators that exert multiple biological functions through their own cognate receptors. They are both present in the blood stream, activate receptors with similar structures (endothelial differentiation gene receptors), have similar roles in the vasculature and are vasoactive. However, it is unclear whether these lysophospholipid mediators cross-talk downstream of each receptor. Here, we provide in vivo evidence that LPA signaling counteracted S1P signaling. When autotaxin (Atx), an LPA-producing enzyme, was overexpressed in zebrafish embryos by injecting atx mRNA, the embryos showed cardia bifida, a phenotype induced by down-regulation of S1P signaling. A similar cardiac phenotype was not induced when catalytically inactive Atx was introduced. The cardiac phenotype was synergistically enhanced when antisense morpholino oligonucleotides (MO) against S1P receptor (s1pr2/mil) or S1P transporter (spns2) was introduced together with atx mRNA. The Atx-induced cardia bifida was prominently suppressed when embryos were treated with an lpar1 receptor antagonist, Ki16425, or with MO against lpar1. These results provide the first in vivo evidence of cross-talk between LPA and S1P signaling.
Subject(s)
Embryo, Nonmammalian/abnormalities , Embryo, Nonmammalian/enzymology , Heart Defects, Congenital/embryology , Lysophospholipids/biosynthesis , Lysophospholipids/metabolism , Phosphoric Diester Hydrolases/metabolism , Sphingosine/analogs & derivatives , Zebrafish/embryology , Animals , Down-Regulation/drug effects , Embryo, Nonmammalian/drug effects , Embryo, Nonmammalian/pathology , HEK293 Cells , Heart Defects, Congenital/enzymology , Heart Defects, Congenital/pathology , Humans , Isoxazoles/pharmacology , Phenotype , Phosphoric Diester Hydrolases/genetics , Propionates/pharmacology , RNA, Messenger/genetics , RNA, Messenger/metabolism , Receptors, Lysophosphatidic Acid/agonists , Receptors, Lysophosphatidic Acid/metabolism , Signal Transduction/drug effects , Sphingosine/metabolismABSTRACT
An enantionselective synthesis of both enantiomers of Ki16425, which possesses selective LPA antagonistic activity, was achieved. The isoxazole core was constructed by a 1,3-dipolar cycloaddition of nitrile oxide with alkyne and condensation with the optically active α-phenethyl alcohol segment, which was prepared by an enantioselective reduction of arylmethylketone. Biological evaluation of both enantiomers of Ki16425 revealed that the (R)-isomer showed much higher antagonistic activity for LPA(1) and LPA(3) receptors.
