High myristic acid content in the cyanobacterium Cyanothece sp. PCC 8801 results from substrate specificity of lysophosphatidic acid acyltransferase

Analysis of fatty acids from the cyanobacterium Cyanothece sp. PCC 8801 revealed that this species contained high levels of myristic acid (14:0) and linoleic acid in its glycerolipids, with minor contributions from palmitic acid (16:0), stearic acid, and oleic acid. The level of 14:0 relative to total fatty acids reached nearly 50%. This 14:0 fatty acid was esterified primarily to the sn-2 position of the glycerol moiety of glycerolipids. This characteristic is unique because, in most of the cyanobacterial strains, the sn-2 position is esterified exclusively with C16 fatty acids, generally 16:0. Transformation of Synechocystis sp. PCC 6803 with the PCC8801_1274 gene for lysophosphatidic acid acyltransferase (1-acyl-sn-glycerol-3-phosphate acyltransferase) from Cyanothece sp. PCC 8801 increased the level of 14:0 from 2% to 17% in total lipids and the increase in the 14:0 content was observed in all lipid classes. These findings suggest that the high content of 14:0 in Cyanothece sp. PCC 8801 might be a result of the high specificity of this acyltransferase toward the 14:0-acyl-carrier protein.     

Glycerolipid synthesis by homogenate and oil bodies from developing mustard (Sinapis alba L.) seed

[1-¹⁴C]Oleic acid and [14-¹⁴C]erucic acid were converted to their acyl-CoA derivatives and incorporated into acyl lipids by a homogenate from developing mustard (Sinapis alba L.) seed and oil bodies, as well as supernatant isolated by centrifugation at 20000 g. In both homogenate and oil bodies, the oleoyl moieties from exogenous [1-¹⁴C]oleoyl-CoA were most extensively incorporated into phosphatidic acids, but very little into phosphatidylcholines. The pattern of labelling of acyl lipids by oleoyl versus erucoyl moieties from either of the corresponding fatty acids, added individually or as a mixed substrate, indicates that oleoyl-CoA directly acylates sn-glycerol-3-phosphate to yield lysophosphatidic acids and phosphatidic acids that are subsequently converted to mono- and diacylglycerols. In contrast, erucoyl-CoA predominantly acylates preformed mono-and diacylglycerols containing oleoyl moieties to yield triacylglycerols containing erucoyl moieties.     

Isolation of Novel Animal Cell Lines Defective in Glycerolipid Biosynthesis Reveals Mutations in Glucose-6-phosphate Isomerase

Glycerolipids are structural components for membranes and serve in energy storage. We describe here the use of a photodynamic selection technique to generate a population of Chinese hamster ovary cells that display a global deficiency in glycerolipid biosynthesis. One isolate from this population, GroD1, displayed a profound reduction in the synthesis of phosphatidylcholine, phosphatidylethanolamine, and triglycerides but presented high levels of phosphatidic acid and normal levels of phosphatidylinositol synthesis. This was accompanied by a reduction in phosphatidate phosphatase 1 (PAP1) activity. Expression cloning and sequencing of the cDNA obtained from GroD1 revealed a point mutation, Gly-189 → Glu, in glucose-6-phosphate isomerase (GPI), a glycolytic enzyme involved in an inherited disorder that results in anemia and neuromuscular symptoms in humans. GPI activity was reduced by 87% in GroD1. No significant differences were found in DNA synthesis, protein synthesis, and ATP levels, whereas glycerol 3-phosphate levels were increased in the mutant. Expression of wild-type hamster GPI restored GPI activity, glycerolipid biosynthesis, and PAP1 activity in GroD1. Two additional, independently isolated GPI-deficient mutants displayed similar phenotypes with respect to PAP1 activity and glycerolipid biosynthesis. These findings uncover a novel relationship between GPI, involved in carbohydrate metabolism, and PAP1, a lipogenic enzyme. These results may also help to explain neuromuscular symptoms associated with inherited GPI deficiency.     

Triacylglyceride composition and fatty acyl saturation profile of a psychrophilic and psychrotolerant fungal species grown at different temperatures

Pseudogymnoascus destructans is a psychrophilic fungus that infects cutaneous tissues in cave dwelling bats, and it is the causal agent for white nose syndrome (WNS) in North American (NA) bat populations. Geomyces pannorum is a related psychrotolerant keratinolytic species that is rarely a pathogen of mammals. In this study, we grew P. destructans and G. pannorum in static liquid cultures at favourable and suboptimal temperatures to: 1) determine if triacylglyceride profiles are species-specific, and 2) determine if there are differences in fatty acyl (FA) saturation levels with respect to temperature. Total lipids isolated from both fungal spp. were separated by thin-layer chromatography and determined to be primarily sterols (∼15 %), free fatty acids (FFAs) (∼45 %), and triacylglycerides (TAGs) (∼50 %), with minor amounts of mono-/diacylglycerides and sterol esters. TAG compositions were profiled by matrix-assisted laser desorption–ionization time-of-flight mass spectrometry (MALDI–TOF). Total fatty acid methyl esters (FAMEs) and acyl lipid unsaturation levels were determined by gas chromatography–mass spectrometry (GC–MS). Pseudogymnoascus destructans produced higher proportions of unsaturated 18C fatty acids and TAGs than G. pannorum. Pseudogymnoascus destructans and G. pannorum produced up to a two-fold increase in 18:3 fatty acids at 5 °C than at higher temperatures. TAG proportion for P. destructans at upper and lower temperature growth limits was greater than 50 % of total dried mycelia mass. These results indicate fungal spp. alter acyl lipid unsaturation as a strategy to adapt to cold temperatures. Differences between their glycerolipid profiles also provide evidence for a different metabolic strategy to support psychrophilic growth, which may influence P. destructans' pathogenicity to bats.     

Reversibility of Propranolol-Induced Changes in the Biosynthesis of Monoacylglycerol, Diacylglycerol, Triacylglycerol, and Phospholipids in the Retina

Abstract: The biosynthesis and metabolism of phospholipids and neutral glycerides were studied in the bovine retina. Radioactive glycerol was used as a precursor. Phentolamine and d - and dl -propranolol were found to produce similar effects on lipid metabolism in the retina. Marked stimulation of phosphatidylinositol (PhI) synthesis and maximal inhibition of phosphatidylcholine (PhC), diacylglycerol (DG), and triacylglycerol (TG) formation were observed within 5 min after exposure to 0.5 m M dl -propranolol. Pulse-chase experiments showed a high turnover rate in DG and a reversibility of the propranolol-induced changes produced during the synthesis of PhC, TG, DG, monoacylglycerol (MG), and phosphatidylserine. All reversals of the drug-induced biosynthetic profiles approached control values 60 min after incubation in drug-free medium. However, complete reversal was not achieved in any of the cases under these conditions. Propranolol appeared to inhibit both the formation of DG from phosphatidic acid and the further metabolism of DG, probably to MG. Phosphatidylethanolamine biosynthesis showed some recovery from this inhibition. Synthesis of Phi was greatly stimulated by preincubation with propranolol and was further enhanced by reincubation in the presence of propranolol. However, this effect was not reversed by reincubation without the drug. The active de novo biosynthesis of retinal phospholipids and glycerides is a very dynamic pathway that may be redirected by amphiphilic drugs. In addition, the partial reversal of modifications induced in the flux of [2-³H]glycerol through the lipids can occur during short-term reincubations of retinas in drug-free medium.     

Visualization of lipid droplet composition by direct organelle mass spectrometry

An expanding appreciation for the varied functions of neutral lipids in cellular organisms relies on a more detailed understanding of the mechanisms of lipid production and packaging into cytosolic lipid droplets (LDs). Conventional lipid profiling procedures involve the analysis of tissue extracts and consequently lack cellular or subcellular resolution. Here, we report an approach that combines the visualization of individual LDs, microphase extraction of lipid components from droplets, and the direct identification of lipid composition by nanospray mass spectrometry, even to the level of a single LD. The triacylglycerol (TAG) composition of LDs from several plant sources (mature cotton (Gossypium hirsutum) embryos, roots of cotton seedlings, and Arabidopsis thaliana seeds and leaves) were examined by direct organelle mass spectrometry and revealed the heterogeneity of LDs derived from different plant tissue sources. The analysis of individual LDs makes possible organellar resolution of molecular compositions and will facilitate new studies of LD biogenesis and functions, especially in combination with analysis of morphological and metabolic mutants. Furthermore, direct organelle mass spectrometry could be applied to the molecular analysis of other subcellular compartments and macromolecules.     

Identification of plant-like galactolipids in Chromera velia, a photosynthetic relative of malaria parasites

Apicomplexa are protist parasites that include Plasmodium spp., the causative agents of malaria, and Toxoplasma gondii, responsible for toxoplasmosis. Most Apicomplexa possess a relict plastid, the apicoplast, which was acquired by secondary endosymbiosis of a red alga. Despite being nonphotosynthetic, the apicoplast is otherwise metabolically similar to algal and plant plastids and is essential for parasite survival. Previous studies of Toxoplasma gondii identified membrane lipids with some structural features of plastid galactolipids, the major plastid lipid class. However, direct evidence for the plant-like enzymes responsible for galactolipid synthesis in Apicomplexan parasites has not been obtained. Chromera velia is an Apicomplexan relative recently discovered in Australian corals. C. velia retains a photosynthetic plastid, providing a unique model to study the evolution of the apicoplast. Here, we report the unambiguous presence of plant-like monogalactosyldiacylglycerol and digalactosyldiacylglycerol in C. velia and localize digalactosyldiacylglycerol to the plastid. We also provide evidence for a plant-like biosynthesis pathway and identify candidate galactosyltranferases responsible for galactolipid synthesis. Our study provides new insights in the evolution of these important enzymes in plastid-containing eukaryotes and will help reconstruct the evolution of glycerolipid metabolism in important parasites such as Plasmodium and Toxoplasma.