PLAAT1 Exhibits Phosphatidylcholine:Monolysocardiolipin Transacylase Activity.

Tissue-specific cardiolipin fatty acyl profiles are achieved by remodeling of de novo synthesized cardiolipin, and four remodeling enzymes have thus far been identified. We studied the enzyme phospholipase A and acyltransferase 1 (PLAAT1), and we report the discovery that it has phosphatidylcholine (PC):monolysocardiolipin (MLCL) transacylase activity. Subcellular localization was analyzed by differential centrifugation and immunoblotting. Total levels of major phospholipids, and the fatty acyl profile of cardiolipin, were analyzed in HEK293 cells expressing murine PLAAT1 using gas chromatography. Apparent enzyme kinetics of affinity-purified PLAAT1 were calculated using radiochemical enzyme assays. This enzyme was found to localize predominantly to the endoplasmic reticulum (ER) but was detected at low levels in the mitochondria-associated ER matrix. Cells expressing PLAAT1 had higher levels of total cardiolipin, but not other phospholipids, and it was primarily enriched in the saturated fatty acids myristate, palmitate, and stearate, with quantitatively smaller increases in the n-3 polyunsaturated fatty acids linolenate, eicosatrienoate, and eicosapentanoate and the monounsaturated fatty acid erucate. Affinity-purified PLAAT1 did not catalyze the transacylation of MLCL using 1-palmitoyl-2-[14C]-linoleoyl-PC as an acyl donor. However, PLAAT1 had an apparent Vmax of 1.61 µmol/min/mg protein and Km of 126 µM using [9,10-3H]-distearoyl-PC as an acyl donor, and 0.61 µmol/min/mg protein and Km of 16 µM using [9,10-3H]-dioleoyl-PC. PLAAT1 is therefore a novel PC:MLCL transacylase.

Microglial cell activation increases saturated and decreases monounsaturated fatty acid content, but both lipid species are proinflammatory.

Neuroinflammation is a component of age-related neurodegenerative diseases and cognitive decline. Saturated (SFA) and monounsaturated (MUFA) fatty acids are bioactive molecules that may play different extrinsic and intrinsic roles in neuroinflammation, serving as exogenous ligands for cellular receptors, or endogenous components of cell structural, energetic and signaling pathways. We determined the fatty acyl profile of BV2 microglial cells before and after acute activation with lipopolysaccharide (LPS). We also investigated the effect of SFA and MUFA pretreatment on the production of an invasive, neurotoxic phenotype in BV2 cells. Acute activation of BV2 microglia resulted in an increase in the relative content of SFA (12:0, 16:0, 18:0, 20:0, 22:0, and 24:0 increased significantly), and a relative decrease in the content of MUFA (16:1n7, 18:1n7, 18:1n9, 20:1n9, 24:1n9 decreased significantly). In agreement, the major stearoyl-CoA desaturase (SCD) isoform in BV2 cells, SCD2, was significantly down-regulated by LPS. We next treated cells with SFA (16:0 or 18:0) or MUFA (16:1n7 or 18:1n9), and found that levels of secreted IL6 were increased, as was secreted MMP9-mediated proteolytic activity. To test the functional significance, we treated SH-SY5Y neuronal cells with conditioned medium from BV2 cells pretreated with fatty acids, and found a small but significant induction of cell death. Our findings suggest differential intrinsic roles for SFA and MUFA in activated microglial cells, but similar extrinsic roles for these fatty acid species in inducing activation. Expansion of SFA is important during microglial cell activation, but either supplemental SFA or MUFA may contribute to chronic low-grade neuroinflammation.