Exploring the metabolism of the signaling lipid lysophosphatidylserine in mammalian physiology and developing LC-MS-based lipidomics platforms

Abstract

Lysophosphatidylserine (lyso-PS) is an essential signaling lysophospholipid in mammals, and deregulation in its metabolism has been directly linked to an array of human autoimmune and neurological disorders such as PHARC (polyneuropathy, hearing loss, ataxia, retinitis pigmentosa, and cataract), an early onset autosomal recessive neurological disorder caused by deleterious mutations to the lyso-PS lipase ABHD12. Biochemically, ABHD12 catalyzes the hydrolysis of lyso-PS (lyso-PS lipase) and controls the concentrations and signaling pathways regulated by this potent signaling lysophospholipid in the mammalian brain. Since over 30 mutations in ABHD12 have been observed in human PHARC subjects and the biochemical activity of these pathogenic mutants remains unknown, we performed a bioinformatics study to identify functionally relevant conserved residues in the ABHD12 protein sequence that are likely important for its enzymatic activity. To validate these in silico findings, we generated numerous mutants of murine ABHD12, including those associated with human PHARC subjects, assayed them for their enzymatic activity, and performed the first thorough sequence-function relationship for mammalian ABHD12. Since lyso-PS has been extensively studied in the context of autoimmune and neurological disorders, our knowledge of lyso-PS metabolism is almost exclusively limited to the immune and central nervous systems (CNS) despite lyso-PSs being almost ubiquitously present in every tissue. Since lyso-PS levels are tightly governed by a lyso-PS lipase in the nervous system (ABHD12), we profiled the lyso-PS lipase activity associated with different mammalian tissues. We found that most tissues could degrade lyso-PS by virtue of membrane-associated metabolic serine hydrolase (mSH) enzymes; however, ABHD12 only influences lyso-PS metabolism exclusively in the brain. To bridge this gap, we used a repertoire of mSH inhibitors to screen the lipase activity reduction of tissue membrane fractions and identified the mSH lipase ABHD6 as a potential lyso-PS lipase in the liver and kidneys. Using specific pharmacological inhibitors in cell lines and mice, we functionally annotate ABHD6 as a lyso-PS lipase in primary hepatocytes and mouse liver and kidneys. We also validated the lyso-PS lipase activity of ABHD6 using an overexpression system in mammalian cells. These findings expand our understanding of lyso-PS metabolism in mammalian physiology and open up ABHD6 as a target for lyso-PS-mediated processes in the liver and kidneys. Lipids such as lyso-PS are challenging macromolecules to study because of a lack of techniques to quantify them in biological samples. While lipids are a diverse class with total species in the order of 10000s, their coverage in a typical LC-MS/MS experiment is around 2-5%, in contrast to 80-90% for other metabolites. In an effort to identify and quantify lipids in a variety of biological samples, we first used a modified folch method to extract lipids. Subsequently, we developed methods to perform untargeted and targeted lipid analysis on quadrupole-time of fight (Q-TOF) and triple quadrupole (Q-Q-Q) mass spectrometers, respectively. Our methods enabled relative and absolute quantification of lipid species such as fatty acyls, glycerophospholipids, glycerolipids, and saccharolipids in extracts from organisms ranging from mycobacteria, bacteria, zebrafish to human plasma. Our lipidomics analyses were instrumental in assigning novel functions to different proteins and for looking at the effects of various drug treatments.