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Autotaxin (ATX) is a secreted enzyme that produces a bioactive lysophospholipid, lysophosphatidic acid (LPA). ATX plays a role in vascular and neural development in embryos but its mechanisms remain unclear. At the beginning of this study, only one zebrafish atx gene (atxa) was known and had been investigated. In this study, we generated ATX knockout (KO) fish by TALEN targeting atxa. Unexpectedly, atxa KO fish showed neither vascular defects nor reduction of ATX activity, implying the existence of one or more other ATXs in the genome. By a BLAST search using ATXa protein fragments as a query, we found a genomic sequence that closely resembled atxa exons 13, 14 and 15. Consequently, we cloned a cDNA encoding a second zebrafish autotaxin (ATXb), and found that it was transcribed in various tissues. The atxb gene encoded a protein of 832 amino acids (compared to 850 amino acids in ATXa) with 60% amino acid identity to ATXa and clustered with ATXs from other species. A recombinant ATXb protein showed lysophospholipase D (lysoPLD) activities with substrate specificities similar to those of ATXa and mammalian ATXs. These results indicate that ATXb is a second zebrafish ATX, which possibly shares redundant roles with ATXa in embryonic development., (© The Author(s) 2019. Published by Oxford University Press on behalf of the Japanese Biochemical Society. All rights reserved.)

Lysophosphatidylserine (1-oleoyl-2 R-lysophosphatidylserine, LysoPS) has been shown to have lipid mediator-like actions such as stimulation of mast cell degranulation and suppression of T lymphocyte proliferation, although the mechanisms of LysoPS actions have been elusive. Recently, three G protein-coupled receptors (LPS1/GPR34, LPS2/P2Y10 and LPS3/GPR174) were found to react specifically with LysoPS, raising the possibility that LysoPS serves as a lipid mediator that exerts its role through these receptors. Previously, we chemically synthesized a number of LysoPS analogues and evaluated them as agonists for mast-cell degranulation. Here, we used a transforming growth factor-α (TGFα) shedding assay to see if these LysoPS analogues activated the three LysoPS receptors. Modification of the serine moiety significantly reduced the ability of the analogues to activate the three LysoPS receptors, whereas modification of other parts resulted in loss of activity in receptor-specific manner. We found that introduction of methyl group to serine moiety (1-oleoyl-lysophosphatidylallothreonine) and removal of sn-2 hydroxyl group (1-oleoyl-2-deoxy-LysoPS) resulted in reduction of reactivity with LPS1 and LPS3, respectively. Accordingly, we synthesized a LysoPS analogue with the two modifications (1-oleoyl-2-deoxy-lysophosphatidylallothreonine) and found it to be an LPS2-selective agonist. These pharmacological tools will definitely help to identify the biological roles of these LysoPS receptors., (© The Authors 2014. Published by Oxford University Press on behalf of the Japanese Biochemical Society. All rights reserved.)

Lysophosphatidic acid (LPA) is one of the simplest glycerophospholipids with one fatty acid chain and a phosphate group as a polar head. Although LPA had been viewed just as a metabolic intermediate in de novo lipid synthetic pathways, it has recently been paid much attention as a lipid mediator. LPA exerts many kinds of cellular processes, such as cell proliferation and smooth muscle contraction, through cognate G protein-coupled receptors. Because lipids are not coded by the genome directly, it is difficult to know their patho- and physiological roles. However, recent studies have identified several key factors mediating the biological roles of LPA, such as receptors and producing enzymes. In addition, studies of transgenic and gene knockout animals for these LPA-related genes, have revealed the biological significance of LPA. In this review we will summarize recent advances in the studies of LPA production and its roles in both physiological and pathological conditions., (© The Authors 2014. Published by Oxford University Press on behalf of the Japanese Biochemical Society. All rights reserved.)

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.

GPR34 is a G protein-coupled receptor belonging to the P2Y family. Here, we attempted to resolve conflicting reports about whether it is a functional lysophosphatidylserine (LysoPS) receptor. In HEK293 cells expressing human, mouse or rat GPR34 and Gα chimera between Gαq and Gαi1(Gq/i1), LysoPS quickly elevated intracellular Ca(2+) ion levels ([Ca(2+)](i)). LysoPS also stimulated alkaline phosphatase (AP)-tagged TGFα (AP-TGFα) release in GPR34-expressing HEK293 cells and induced the migration of CHO-K1 cells expressing GPR34. Other lysophospholipids did not induce these actions. Replacement of the serine residue of LysoPS abolished the reactivity of LysoPS with GPR34, indicating that GPR34 strictly recognizes the serine head group of LysoPS. Recombinant phosphatidylserine-specific phospholipase A(1) (PS-PLA(1)) that deacylates fatty acid at the sn-1 position of PS and produces 2-acyl-LysoPS, but not catalytically inactive mutant PS-PLA(1), stimulated the release of AP-TGFα from GPR34-expressing cells. Consistent with the result, LysoPS was detected in the cells treated with wild-type PS-PLA(1) but not with the mutant PS-PLA(1). PS treated with PLA(1) was much more effective at stimulating AP-TGFα release than PS treated with PLA(2). In addition, migration-resistant 2-acyl-1-deoxy-LysoPS, a 2-acyl-LysoPS analogue, was much more potent than 1-acyl-2-deoxy-LysoPS. The present studies confirm that GPR34 is a cellular receptor for LysoPS, especially with a fatty acid at the sn-2 position.

Autotaxin (ATX) is lysophospholipase D, which converts lysophospholipids such as lysophosphatidylcholine (LPC) to lysophosphatidic acid (LPA), a bioactive lipid mediator with multiple biological roles. ATX is present in high concentrations in various biological fluids and is responsible for LPA production in these fluids. The plasma ATX level is altered in some patho-physiological conditions. Three splicing isoforms of ATX have been reported so far (ATXα, β and γ). In this study, we identified and characterized ATXδ, a novel alternative splice variant of ATX, which has a four-amino acid deletion in the L2 linker region of ATXβ. ATXδ was found to be the second major isoform following ATXβ and fully active. ATXβ and ATXδ showed similar divalent cation sensitivity and cell motility-stimulating activity. ATXβ and ATXδ are present in wide range of organism from fish to mammals. Among them, only ATXδ was found in Gallus gallus and Xenopus laevis, suggesting the indispensable role of the isoform. ATXδ was expressed in various human tissues with different expression patterns from that of ATXβ. These results show that ATXδ is a second major ATX isoform sharing similar biochemical characters with the major isoform, ATXβ, and is a potential biomarker.

Lysophosphatidic acid (LPA) is a simple phospholipid but has numerous biological effects through a series of G-protein-coupled receptors specific to LPA. In general, LPA is short-lived when applied in vivo, which hinders most pharmacological experiments. In our continuing study to identify stable LPA analogues capable of in vivo applications, we identified here lysophosphatidylmethanol (LPM) as a stable and pan-LPA receptor agonist. A synthetic LPM activated all five LPA receptors (LPA(1-5)), and stimulates both cell proliferation and LPA-receptor-dependent cell motility. In addition, LPM showed a hypertensive effect in rodent when applied in vivo. We found that, when fetal calf serum was incubated in the presence of methanol, formation of LPM occurred rapidly, whereas it was completely blocked by depletion of autotaxin (ATX), a plasma enzyme that converts lysophosphatidylcholine (LPC) to LPA. When recombinant ATX was incubated with LPC in the presence of methanol, both LPM and LPA were produced with a ratio of 1:10, showing that ATX has transphosphatidylation activity in addition to its lysophospholipase D activity. Administration of methanol in mice resulted in the formation of several micromoles of LPM in plasma, which is much higher than that of LPA. The present study identified LPM as a novel and stable lysophospholipid mediator with LPA-like activities and ATX as a potential synthetic enzyme for LPM.

Ro09-0198 is a cyclic peptide isolated from Streptoverticillium griseoverticillatum which recognizes strictly the structure of phosphatidylethanolamine (PE) and forms an equimolar complex with the phospholipid on biological membranes. To use the peptide as a probe for analyzing the transbilayer movement of PE, we labeled the amino-terminal amino acid of the peptide with biotin without changing either the reactivity or specificity of the peptide. The amount of the peptide bound to the membrane was measured by enzyme linked immunosorbent assay (ELISA) after extraction of the peptide from the membrane. The peptide showed a strict temperature-dependent binding to human erythrocytes and the binding increased with increasing temperature. Since the peptide bound to PE in a temperature-independent manner and the binding to membrane PE is not affected by membrane proteins, the present temperature-dependent binding of the peptide to the cell membranes was likely to reflect temperature-dependent translocation of PE. The binding of the peptide to erythrocytes differed greatly among animal species. The peptide also showed temperature-dependent binding to a human histocytic lymphoma cell line, U937, suggesting that the peptide will provide a novel and convenient probe for analyzing the transbilayer movement of PE in eukaryotic cells.