Synthesis and evaluation of lysophosphatidylserine analogues as inducers of mast cell degranulation. Potent activities of lysophosphatidylthreonine and its 2-deoxy derivative.

In response to various exogenous stimuli, mast cells (MCs) release a wide variety of inflammatory mediators stored in their cytoplasmic granules and this release initiates subsequent allergic reactions. Lysophosphatidylserine (lysoPS) has been known as an exogenous inducer to potentiate histamine release from MCs, though even at submicromolar concentrations. In this study, through SAR studies on lysoPS against MC degranulation, we identified lysoPT, a threonine-containing lysophospholipid and its 2-deoxy derivative as novel strong agonists. LysoPT and its 2-deoxy derivative induced histamine release from MCs both in vitro and in vivo at a concentration less than one-tenth that of lysoPS. Notably, lysoPT did not activate a recently proposed lysoPS receptor on MCs, GPR34, demonstrating the presence of another undefined receptor reactive to both lysoPS and lysoPT that is involved in MC degranulation. Thus, the present strong agonists, lysoPT and its 2-deoxy derivative, will be useful tools to understand the mechanisms of lysoPS-induced activation of degranulation of MCs.

Lysophosphatidylmethanol is a pan lysophosphatidic acid receptor agonist and is produced by autotaxin in blood.

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.

Focused lipidomics by tandem mass spectrometry.

In this paper we performed focused analyses of phospholipids by using the data of precursor ion scanning and neutral loss scanning of their polar head groups and fatty acyl moieties for the specific search of categorical phospholipids. By using precursor ion scanning or neutral loss scanning of polar head groups in the positive ion mode, more sensitive identification were obtained than that in the negative ion mode. Precursor ion scanning of carbonic anions in the negative ion mode was also effective to identify molecular species of phospholipids having specified fatty acyl moieties. By using these analytical methods, the detection limits of individual metabolites are going up to 5-20-fold of former conventional methods. The important factor is that by focusing in some limited categories of molecules, detection limit is greatly enhanced, thus minor but important molecules can be detected. Moreover, combination of LC-MS/MS and focused scanning for head group was revealed to be useful to identify very minor molecular species in the focused class of phospholipids.

High-resolution analysis by nano-electrospray ionization Fourier transform ion cyclotron resonance mass spectrometry for the identification of molecular species of phospholipids and their oxidized metabolites.

Nano-electrospray ionization (ESI) Fourier transform ion cyclotron resonance mass spectrometry (FTICRMS) was applied to identify the molecular species of phosphatidylethanolamine of Caenorhabditis elegans, which has a high concentration of phospholipids with a fatty acyl chain having an odd number of carbon atoms. The molecular species of diacyl phosphatidylethanolamine with one fatty acyl chain having an odd number of carbon atoms and one fatty acyl chain having an even number of carbon atoms was identified separately from alkyl-acyl phosphatidylethanolamine with an alkyl chain having an even number of carbon atoms and a fatty acyl chain having an even number of carbon atoms. Furthermore, nano-ESI-FTICRMS was applied to the direct identification of oxidized phosphatidylcholine from soybean. The mass peaks of individual molecular species of oxidative phosphatidylcholine, such as 34:3 diacyl phosphatidylcholine with peroxide (+2O) (m/z 788.544) or 34:2 diacyl phosphatidylcholine with peroxide (+2O) (m/z 790.560), can be separated from the peaks of the molecular species of the non-oxidative phospholipids. This suggests that the mass peaks with a difference of less than 0.1 mass units in their molecular weight can be separated and that their individual exact molecular compositions can be obtained by the FTICRMS analysis. The high resolution and high accuracy of FTICRMS are very effective in the analysis of molecular species of phospholipids and their derivatives.

Biochemical and molecular characterization of two phosphatidic acid-selective phospholipase A1s, mPA-PLA1alpha and mPA-PLA1beta.

We have identified a novel phospholipase A1, named mPA-PLA1beta, which is specifically expressed in human testis and characterized it biochemically together with previously identified mPA-PLA1alpha. The sequence of mPAPLA1beta encodes a 460-amino acid protein containing a lipase domain with significant homology to the previously identified phosphatidic acid (PA)-selective PLA1, mPA-PLA1alpha. mPA-PLA1beta contains a short lid and deleted beta9 loop, which are characteristics of PLA1 molecules in the lipase family, and is a member of a subfamily in the lipase family that includes mPA-PLA1alpha and phosphatidylserine-specific PLA1. Both mPA-PLA1beta and mPA-PLA1alpha recombinant proteins exhibited PA-specific PLA1 activity and were vanadate-sensitive. When mPAPLA1beta-expressing cells were treated with bacterial phospholipase D, the cells produced lysophosphatidic acid (LPA). In both mPA-PLA1alpha and beta-expressing cells, most of the PA generated by the phospholipase D (PLD) treatment was converted to LPA, whereas in control cells it was converted to diacylglycerol. When expressed in HeLa cells most mPA-PLA1alpha protein was recovered from the cell supernatant. By contrast, mPA-PLA1beta was recovered almost exclusively from cells. Consistent with this observation, we found that mPA-PLA1beta has higher affinity to heparin than mPA-PLA1alpha. We also found that the membrane-associated mPA-PLA1s were insoluble in solubilization by 1% Triton X-100 and were detected in Triton X-100-insoluble buoyant fractions of sucrose gradients. The present study raises the possibility that production of LPA by mPA-PLA1alpha and -beta occurs on detergent-resistant membrane domains of the cells where they compete with lipid phosphate phosphatase for PA.

A novel phosphatidic acid-selective phospholipase A1 that produces lysophosphatidic acid.

Lysophosphatidic acid (LPA) is a lipid mediator with diverse biological properties, although its synthetic pathways have not been completely solved. We report the cloning and characterization of a novel phosphatidic acid (PA)-selective phospholipase A(1) (PLA(1)) that produces 2-acyl-LPA. The PLA(1) was identified in the GenBank(TM) data base as a close homologue of phosphatidylserine (PS)-specific PLA(1) (PS-PLA(1)). When expressed in insect Sf9 cells, this enzyme was recovered from the Triton X-100-insoluble fraction and did not show any catalytic activity toward exogenously added phospholipid substrates. However, culture medium obtained from Sf9 cells expressing the enzyme was found to activate EDG7/LPA(3), a cellular receptor for 2-acyl-LPA. The activation of EDG7 was further enhanced when the cells were treated with phorbol ester or a bacterial phospholipase D, suggesting involvement of phospholipase D in the process. In the latter condition, an increased level of LPA, but not other lysophospholipids, was confirmed by mass spectrometry analyses. Expression of the enzyme is observed in several human tissues such as prostate, testis, ovary, pancreas, and especially platelets. These data show that the enzyme is a membrane-associated PA-selective PLA(1) and suggest that it has a role in LPA production.