Synthesis of MAG and lyso-PS lipids with varying lipid tails

Abstract

My doctoral research described in this thesis involves the synthesis, and characterization of mono-acyl glycerol (MAG) and lysophosphatidylserine (lyso-PS) lipids with varying lipid tails. PHARC syndrome is a neurodegenerative disease abbreviated based on its symptoms i.e. polyneuropathy, hearing loss, ataxia, retinitis pigmentosa, and cataract. It has been attributed to a mutation in the abhd12 gene which encodes for lysophosphatidylserine (lyso-PS) lipase ABHD12. ABHD12 is an integral membrane enzyme which a member of the serine hydrolase family. Mice models for PHARC i.e. ABHD12 knock-out (KO) mice exhibits locomotor defects, microglial activation, and accumulation of lyso-PS in the cerebellum. Biochemical characterization and pathways downstream of lyso-PS is unclear in PHARC syndrome. The diastereomeric complexity in the lyso-PS structure has caused its commercial paucity to study its biological role in detail. Hence, in this study, I have chemically synthesized a library of lyso-PSs chain lengths such as medium, long, and very-long-chain fatty acyl chains to investigate their role in (neuro) immunological processes. We used these lipids to understand the enzyme kinetics of ABHD12. We found that ABHD12 is highly stereospecific and strongly prefers the (R) configuration of Me-lyso-PSs over (S) analogs. Next, we used our synthetic Me-lyso-PS library to investigate the pathways that can be triggered through lyso-PS signaling. We measured release of calcium, cytokines, and histamine that were involved in the immune cell activation and phosphorylation pathways in immune cells as a function of lyso-PS treatment. VLC lyso-PS have been found to elicit immune responses in the form of heightened cytokine release via the toll-like receptor 2 (TLR2) signalling pathway. We have also observed that upon LC lyso-PS stimulation, there is an increase in the cytosolic Ca2+, cAMP, and phospho-ERK levels which we hypothesize might be due to the activation of a yet unknown GPCR. This study suggests the intricate balance between LC and VLC lysoPS which influence significant biological processes via specific receptors. Currently, I have synthesized a library of (R) (Me-lyso-PS) with unsaturated fatty acid chains. However, using this synthetic strategy we are synthesizing bifunctional Me-lyso-PS lipid probes in an attempt to map the interacting partners of lyso-PS using chemical proteomics.