The origin of long-chain fatty acids required for de novo ether lipid/plasmalogen synthesis.

Chornyi S, Ofman R, Koster J, and Waterham HR. (2023) The origin of long-chain fatty acids required for de novo ether lipid/plasmalogen synthesis. Journal of Lipid Research

Plasmalogens are a class of ether phospholipid that contain a vinyl-ether bond at the sn-1 position which cause these lipids to have a more compact structure. This class has roles in vesicular fusion, lipid raft formation, cholesterol transport, and membrane structure and fluidity. Ether phospholipid synthesis begins in the peroxisome by enzymes such as glyceronephosphate O-acyltransferase (GNPAT), alkylglycerone phosphate synthase (AGPS), and fatty acyl-CoA reductase 1 and 2 (FAR1 and FAR2). GNPAT is the catalyst to convert dihydroxyacetone phosphate (DHAP) and acyl-CoA into acyl-DHAP. Following this, AGPS catalyzes the substitution of the acyl chain of acyl-DHAP with a long-chain fatty alcohol which results in alkyl-DHAP. FAR1 provides the long-chain fatty alcohol for this substitution, then alkyl-DHAP is transported to the endoplasmic reticulum for the rest of the synthesis steps. GNPAT has specificity to react with certain long-chain acyl-CoAs such as hexadecanoyl- (C16:0), tetradecanoyl- (C14:0), cis- or trans-9- hexadecenoyl- (C16:1) CoA. It is unknown whether these long-chain acyl-CoAs are produced in peroxisomes or if they needed to be transported into the peroxisome from the cytosol by transporters. Chornyi et al were interested in determining the origin of long-chain acyl-CoAs needed in the biosynthetic step performed by GNPAT.

To start, the authors had to develop an assay to quantify ether phospholipid synthesis within HeLa cells. To specifically detect the production of new ether lipids without labeling, cells were cultured in the presence of 40 µM of the odd-chain long-chain fatty alcohol 1-heptadecanol (C17:0 alcohol) since ether lipids typically contain a long-chain fatty alcohol with C16:0, C18:0, or C18:1 and odd-chain are far less common. Therefore, they could measure new ether lipids that had incorporated 1-heptadecanol. Including 1-heptadecanol caused the synthesis of many ether phospholipid species that contained odd numbered side-chains and decreased the species with even numbered side chains. Interestingly, when cells that were deficient in PEX1 activity, peroxisomal biogenesis factor 1 which is essential for the form and function of peroxisomes, were supplemented with 1-heptadecanol there was a decrease in all ether phospholipids, even those with 1-heptadecanol incorporated, compared to wild-type cells. In addition, they also looked at de novo synthesis in cells that could not synthesize ether phospholipids because of gene deletions in other enzymes involved in the synthetic steps including AGPS, PEX7, and FAR1. The loss of any of these enzymes resulted in an inability to produce ether phospholipids de novo. As the role of FAR1 is to provide the long-chain alcohol during synthesis, when FAR1 deficient cells were supplemented with 1-heptadecanol the production of ether phospholipids was recovered.

Chornyi et al also wanted to determine which transporters are needed to import acyl-CoAs into human peroxisomes. ATP-binding cassette transporters subfamily D (ABCD) transporters are thought to be likely contenders for the transporters responsible for importing acyl CoA esters for the synthesis step performed by GNPAT. As ABCD1 and ABCD3 are present in HeLa cells, these were studied to determine the involvement of this class of transporter. ABCD1 or ABCD2 genes were disrupted then peroxisomal beta-oxidation activity was measured. Compared to wild-type HeLa cells and cells lacking ABCD3 activity, those that lacked ABCD1 activity had a moderate decrease in hexacosanoic acid beta-oxidation but when both ABCD1 and ABCD3 were lacking, there was a much larger reduction. These findings suggest that it is mainly the responsibility of ABCD1 to transport the long-chain acyl-CoA esters into the peroxisome. When de novo ether phospholipid synthesis was measured there was no difference in the cells without ABCD1 activity, a significant decrease in synthesis in the ABCD3 deficient cells, and completely stopped in the cells deficient in both ABCD1 and ABCD3 activity. This indicates that ABCD3 is the main transporter for long-chain acyl-CoA esters that are used in ether lipid synthesis.

In this study the authors were interested in determining whether the long-chain acyl-CoAs needed for ether phospholipid, including plasmalogens, synthesis are transported into the peroxisome or if they are produced in that organelle and, if a transporter is required, which is responsible for this. They developed a novel method to measure the amount of de novo synthesis by using cells deficient in enzymatic activity from specific enzymes in the ether phospholipid synthetic route and supplemented with an odd-numbered long-chain fatty alcohol since those are less common in ether lipids. They found that when wild-type cells were supplemented with 1-heptadecanol there was an increase in ether phospholipids with odd-numbered long-chain fatty alcohols and a decrease in even-numbered long-chain fatty alcohols. However, when cells that were deficient in enzymes in the ether phospholipid synthesis pathway were supplemented they were still unable to produce ether phospholipids de novo. Interestingly, when FAR1, the gene that encodes the enzyme that provides the long-chain acyl CoA to ether phospholipid synthesis, was knocked out, supplementation with 1-heptadecanol recovered the production of ether phospholipids by providing that necessary acyl CoA. In addition they also determined that it is ABCD3 transporter that is responsible for the transportation of acyl CoAs into the peroxisome for ether phospholipid synthesis. The authors suggest that this work could indicate that a deficiency in ABCD3 activity could also result in a plasmalogen deficient phenotype similar to Rhizomelic chondrodysplasia punctata, although only one case of a partial ABCD3 deficiency has been reported. Further work will help elucidate the plasmalogen and ether phospholipid synthesis pathways and could provide new ideas for treating an ether lipid deficiency.