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Search for the Endogenous Ligand of MrgB5 and MRGX4, Oxidized Phospholipid GPCRs Involved in Itch During Pruritic Skin Diseases
Yugo Takagi1, Jumpei Omi1, and Junken Aoki1
1Graduate School of Pharmaceutical Sciences, Tokyo University, 7-3-1, hongo, Bunkyo-ku, Tokyo, Japan
Background: Polyunsaturated fatty acids (PUFA) in membrane phospholipids (PLs) can be oxidized by reactive oxygen species. Recent advances in mass spectrometry (MS) have revealed that such oxidized phospholipids (oxPLs) can be detected in vivo in various pathological conditions. However, the precise roles as well as the active components and the targets of oxPLs have remained to be clarified. As part of ligand identification for orphan GPCRs, we recently found that some members of the MRG family, a GPCR family expressed in cutaneous sensory neuron (DRG neuron), respond to oxidized phospholipids. Such GPCRs include MrgB5 (mouse) and MRGX4 (human). Both MrgB5 and MRGX4 were activated by oxPL products prepared by air oxidation of di-PUFA-typed phosphatidylserine (PS) or phosphatidylglycerol (PG). Subcutaneous injection of one of these oxidized phospholipids (oxDLPG; oxidize products of di-linoleoyl PG) induced itch in mice in an MrgB5-dependent manner and MrgB5 deficient mice showed reduced itch response during contact hypersensitivity (CHS). These findings suggest that oxidized phospholipids play an important role in itch-sensing during CHS as a novel bioactive phospholipid, but the endogenous ligand of MrgB5 and the active component of oxidized phospholipids are not determined. In this study, we performed a lipidomic analysis of CHS skin and a detailed structural analysis of the active components of oxPLs activating MrgB5 and MRGX4.
Methods: In the CHS model, mice were sensitized and challenged with repeated treatment with DNFB. On day 9 and 12, mice skin was collected, and lipid fraction was extracted by using the B&D method and subjected to LC-MS analysis. Preparation of oxidized PG was performed by air-oxidation at room temperature for 1~21 days or by AAPH reaction for 1-72 hours. These oxPL products were subjected to both TGFα shedding assay to evaluate the GPCR activation and LC-MS analysis.
Results: We found that among various PS and PG acyl-chain species, di-linoleoyl PG (DLPG) increased dramatically (~10-fold) in the mouse CHS skin. DLPG was detected almost exclusively in the epidermis but bot in dermis, to which the peripheral terminals of DRG neurons extend in response to skin inflammation. Thus, DLPG potentially serves as a precursor for the endogenous ligand of MrgB5.
Oxidation produces an enormous number of oxPL products with distinct structures, making it difficult to analyze them. Thus, we prepared oxidized DLPGs using in vitro oxidation method and tried to identify the active component(s) that activated MrgB5 or MRGX4. During the time course of air-oxidation for 21 days, we detected 173 oxDLPG products out of 291 predicted oxDLPG products. GPCR activation potential of oxDLPG products over time and from LC-MS analysis, we found two stable oxDLPG products, which were found to have truncated to 9-carbons with aldehyde or carboxylic acid at the omega terminus. Next, we fractionated oxDLPG products by reverse-phase chromatography and found that both MrgB5 and MRGX4 responded to very broad fractions. Interestingly, MRGX4 and MrgB5 were activated differently by the fractions: MRGX4 was activated more strongly by the earlier fractions, while MrgB5 responded more strongly to the later fractions, suggesting that MRGX4 prefers more highly oxidized molecules than MrgB5. Finally, when DLPG was very highly oxidized by AAPH reaction, MRGX4 was strongly activated by the highly oxidized products, while MrgB5 was not. We identified three oxDLPG products as strong candidates for MRGX4 ligands, i.e., oxDLPG with two C9 aldehydes, two C9 carboxylic acid or a C9 aldehydes/a C9 carboxylic acid. We also identified two oxDLPG products as strong candidates for MrgB5 ligands, i.e., oxDLPG products with both a C9 aldehydes and an oxygenated linoleic acid and with a carboxylic acid and an oxygenated linoleic acid.
Conclusion: Present findings indicated that broad species of oxDLPGs which have C9 aldehydes/carboxylic acid plausibly act as ligands for MrgB5 and MRGX4, and that MRGX4 prefers more highly oxidized products. We also showed that DLPG significantly increased in the epidermis of CHS skin, where the highly oxidative environment forms due to inflammation. These results suggest that oxDLPG may act as the endogenous ligand of MrgB5.