Functions of plasmalogen lipids in health and disease

Plasmalogens are a unique class of membrane glycerophospholipids containing a fatty alcohol with a vinyl-ether bond at the sn-1 position, and enriched in polyunsaturated fatty acids at the sn-2 position of the glycerol backbone. These two features provide novel properties to these compounds. Although plasmalogens represent up to 20% of the total phospholipid mass in humans their physiological roles have been challenging to identify, and are likely to be particular to different tissues, metabolic processes and developmental stages. Their biosynthesis starts in peroxisomes, and defects at these steps cause the malformation syndrome, Rhizomelic Chondrodysplasia Punctata (RCDP). The RCDP phenotype predicts developmental roles for plasmalogens in bone, brain, lens, lung, kidney and heart. Recent studies have revealed secondary plasmalogen deficiencies associated with more common disorders and allow us to tease out additional pathways dependent on plasmalogen functions. In this review, we present current knowledge of plasmalogen biology in health and disease. This article is part of a Special Issue entitled: Metabolic Functions and Biogenesis of peroxisomes in Health and Disease.     

The YhhN protein of Legionella pneumophila is a Lysoplasmalogenase

Lysoplasmalogenase catalyzes hydrolytic cleavage of the vinyl-ether bond of lysoplasmalogen to yield fatty aldehyde and glycerophospho-ethanolamine or glycerophospho-choline. We recently purified lysoplasmalogenase from rat liver microsomes and identified the protein as TMEM86B, an integral membrane protein that is a member of the YhhN family found in numerous species of eukaryotes and bacteria. To test the hypothesis that bacterial YhhN proteins also function as lysoplasmalogenase enzymes, we cloned the Lpg1991 gene of Legionella pneumophila, which encodes a 216 amino acid YhhN protein (LpYhhN), and expressed it in Escherichia coli as a C-terminal-GFP-His8-fusion. Membranes were solubilized and the fusion protein was purified by nickel-affinity chromatography, cleaved with Tobacco Etch Virus protease, and subjected to a reverse nickel column to purify the un-tagged LpYhhN. Both the fusion protein and un-tagged LpYhhN exhibit robust lysoplasmalogenase activity, cleaving the vinyl-ether bond of lysoplasmalogen with a V_max of 12 µmol/min/mg protein and a K_m of 45 μM. LpYhhN has no activity on diradyl plasmalogen, 1-alkenyl-glycerol, and monoacylglycerophospho-ethanolamine or monoacylglycerophospho-choline; the pH optimum is 6.5–7.0. These properties are very similar to mammalian TMEM86B. Sequence analysis suggests that YhhN proteins contain eight transmembrane helices, an N-in/C-in topology, and about 5 highly conserved amino acid residues that may form an active site. This work is the first to demonstrate a function for a bacterial YhhN protein, as a vinyl ether bond hydrolase specific for lysoplasmalogen. Since L. pneumophila does not contain endogenous plasmalogens, we hypothesize that LpYhhN may serve to protect the bacterium from lysis by lysoplasmalogen derived from plasmalogens of the host.     

Effects of Growth Temperature on Fatty Acid and Alk-1-Enyl Group Compositions of Veillonella parvula and Megasphaera elsdenii Phospholipids

Veillonella parvula ATCC 10790, an anaerobic gram-negative coccus, contains diacyl and alk-1-enyl acyl (plasmalogen) forms of phosphatidylethanolamine and phosphatidylserine. We studied the effect of growth temperature on the lipid composition of this strain. There was a small increase in the phosphatidylethanolamine content but no change in the content of plasmalogens at the lower growth temperatures tested. The total acyl chains and the plasmalogen acyl chains contained between 73 and 80% mono-unsaturated fatty acids at all growth temperatures. The plasmalogen alk-1-enyl chains were significantly more unsaturated in cells grown at 30 and 25°C than in cells grown at 37°C. Differential scanning calorimetry of the hydrated phospholipids showed lower phase transition temperatures for the lipids from the cells grown at the lower temperatures. In Megasphaera elsdenii lipids, which are similar in composition to the lipids of V. parvula, the proportion of phosphatidylethanolamine also increased slightly at lower growth temperatures, with no significant change in the content of plasmalogens. M. elsdenii contained cyclopropane fatty acyl and alk-1-enyl chains in addition to the mono-unsaturated and saturated chains previously reported. As cells entered the stationary phase of growth at 30 and 42.5°C, there was a reciprocal increase in the proportion of cyclopropane acyl chains and decrease in the unsaturated moieties. The total proportion of cyclopropane and unsaturated acyl and alk-1-enyl chains was more than 65% at all growth temperatures studied, and there was no discernible increase in the sum of these moieties at the lower growth temperatures.     

Comparative study of serine-plasmalogens in human retina and optic nerve: identification of atypical species with odd carbon chains

The objective of this work was to detect and identify phosphatidylserine plasmalogen species in human ocular neurons represented by the retina and the optic nerve. Plasmalogens (vinyl-ether bearing phospholipids) are commonly found in the forms of phosphatidylcholine and phosphatidylethanolamine in numerous mammalian cell types, including the retina. Although their biological functions are unclear, the alteration of cellular plasmalogen content has been associated with several human disorders such as rhizomelic chondrodysplasia punctata Type 2 and primary open-angle glaucoma. By using liquid chromatography coupled to high-resolution and tandem mass spectrometry, we have identified for the first time several species of phosphatidylserine plasmalogens, including atypical forms having moieties with odd numbers of carbons and unsaturation in sn-2 position. Structural elucidation of the potential phosphatidylserine ether linked species was pursued by performing MS(3) experiments, and three fragments are proposed as marker ions to deduce which fatty acid is linked as ether or ester on the glycerol backbone. Interpretation of the fragmentation patterns based on this scheme enabled the assignment of structures to the m/z values, thereby identifying the phosphatidylserine plasmalogens.     

Interactions of plasmalogens and their diacyl analogs with singlet oxygen in selected model systems

Plasmalogens are phospholipids containing a vinyl-ether linkage at the sn-1 position of the glycerophospholipid backbone. Despite being quite abundant in humans, the biological role of plasmalogens remains speculative. It has been postulated that plasmalogens are physiological antioxidants with the vinyl-ether functionality serving as a sacrificial trap for free radicals and singlet oxygen. However, no quantitative data on the efficiency of plasmalogens at scavenging these reactive species are available. In this study, rate constants of quenching of singlet oxygen, generated by photosensitized energy transfer, by several plasmalogens and, for comparison, by their diacyl analogs were determined by time-resolved detection of phosphorescence at 1270nm. Relative rates of the interactions of singlet oxygen with plasmalogens and other lipids, in solution and in liposomal membranes, were measured by electron paramagnetic resonance oximetry and product analysis using HPLC-EC detection of cholesterol hydroperoxides and iodometric assay of lipid hydroperoxides. The results show that singlet oxygen interacts with plasmalogens significantly faster than with the other lipids, with the corresponding rate constants being 1 to 2 orders of magnitude greater. The quenching of singlet oxygen by plasmalogens is mostly reactive in nature and results from its preferential interaction with the vinyl-ether bond. The data suggest that plasmalogens could protect unsaturated membrane lipids against oxidation induced by singlet oxygen, providing that the oxidation products are not excessively cytotoxic.     

Posttranslational Regulation of Fatty Acyl-CoA Reductase 1, Far1, Controls Ether Glycerophospholipid Synthesis

Plasmalogens are a major subclass of ethanolamine and choline glycerophospholipids in which a long chain fatty alcohol is attached at the sn-1 position through a vinyl ether bond. This ether-linked alkyl bond is formed in peroxisomes by replacement of a fatty acyl chain in the intermediate 1-acyl-dihydroxyacetone phosphate with a fatty alcohol in a reaction catalyzed by alkyl dihydroxyacetone phosphate synthase. Here, we demonstrate that the enzyme fatty acyl-CoA reductase 1 (Far1) supplies the fatty alcohols used in the formation of ether-linked alkyl bonds. Far1 activity is elevated in plasmalogen-deficient cells, and conversely, the levels of this enzyme are restored to normal upon plasmalogen supplementation. Down-regulation of Far1 activity in response to plasmalogens is achieved by increasing the rate of degradation of peroxisomal Far1 protein. Supplementation of normal cells with ethanolamine and 1-O-hexadecylglycerol, which are intermediates in plasmalogen biosynthesis, accelerates degradation of Far1. Taken together, our results indicate that ether lipid biosynthesis in mammalian cells is regulated by a negative feedback mechanism that senses cellular plasmalogen levels and appropriately increases or decreases Far1.     

Peroxisomes and ether lipid biosynthesis in rat testis and epididymis

Plasmalogens are a main component of the spermatozoon membrane, playing a crucial role in their maturation. The initial steps in plasmalogen biosynthesis are catalyzed by two peroxisomal enzymes, dihydroxyacetonephosphate acyltransferase and alkyl-dihydroxyacetonephosphate synthase. The localization of both enzymes in the membrane of peroxisomes implies that plasmalogen-producing cells should contain this organelle. To unravel the putative source of spermatozoan plasmalogens we investigated which cell types in the testis and epididymis are endowed with peroxisomes. To this extent, testicular and epididymal tissue was analyzed at the protein and RNA levels by means of light and electron microscopical immunocytochemistry as well as by Western and Northern blotting. Proteins and mRNAs of peroxisomal enzymes, especially those of dihydroxyacetonephosphate acyltransferase and alkyl-dihydroxyacetonephosphate synthase, were detected in the testis and epididymis. In the testis, peroxisomes were localized exclusively in Leydig cells and not in cells of the seminiferous tubules, implying that the latter do not contribute to the biosynthesis of plasmalogens of the sperm membrane. In contrast, peroxisomes could be clearly visualized in the epithelial cells of the epididymis. The results suggest that peroxisomes in epithelial cells of the rat epididymis play a pivotal role in the biosynthesis of plasmalogens destined for delivery to the sperm plasma membrane.     

Functions and biosynthesis of plasmalogens in health and disease

Plasmalogens (1-O-alk-1'-enyl-2-acyl glycerophospholipids) constitute a special class of phospholipids characterized by the presence of a vinyl-ether bond at the sn-1 position. Although long considered as biological peculiarities, interest in this group of phospholipids has grown in recent years, thanks to the realization that plasmalogens are involved in different human diseases. In this review, we summarize the current state of knowledge with respect to the enzymatic synthesis of plasmalogens, the characteristic topology of the enzymes involved and the biological roles that have been assigned to plasmalogens.     

Differential turnover of polyunsaturated fatty acids in plasmalogen and diacyl glycerophospholipids of isolated cardiac myocytes

To investigate the relative turnover of esterified polyunsaturated fatty acids in diacylglycerophospholipids and plasmalogens in isolated cardiac myocytes, we characterized the phospholipid composition and distribution of radiolabel in different phospholipid classes and in individual molecular species of diradyl choline (CGP) and ethanolamine (EGP) glycerophospholipids after incubation of isolated cardiac myocytes with [3H]arachidonate or [14C]linoleate. Plasmalogens in CGP (55%) and EGP (42%) quantitatively accounted for the total plasmalogen content (39%) of cardiac myocyte phospholipids. Plasmalogens comprised 86% and 51% of total arachidonylated CGP and EGP mass, respectively, and [3H]arachidonate was primarily incorporated into plasmalogens in both CGP (65%) and EGP (61%) classes. The specificity activity of [3H]arachidonylated diacyl-CGP was approximately 2- to 5-fold greater than that of [3H]arachidonylated choline plasmalogen, whereas comparable specific activities were found in the [3H]arachidonate-labeled ethanolamine plasmalogen and diacyl-EGP pools. Of the total linoleate-containing CGP and EGP mass, 54% and 57%, respectively, was esterified to plasmalogen molecular species. However, [14C]linoleate was almost exclusively incorporated into diacyl-CGP (96%) and diacyl-EGP (86%). The specific activities of [14C]linoleate-labeled diacyl-CGP and diacyl-EGP were 5- to 20-fold greater than that of the [14C]linoleate-labeled plasmalogen pools. The differential incorporation of polyunsaturated fatty acids in plasmalogens and diacylglycerophospholipids demonstrates that the metabolism of the sn-2 fatty acyl moiety in these phospholipid subclasses is differentially regulated, possibly fulfilling separate and distinct physiologic roles.     

Polyunsaturated fatty acid incorporation into plasmalogens in plasma membrane of glioma cells is preceded temporally by acylation in microsomes.

Plasmalogens (1-O-alk-1'-enyl-2-acyl-sn-glycero-3-phosphoethanolamine) are major phospholipids in many tissues and cells, particularly of neural origin. Using cultured C6 glioma cells and subcellular fractions isolated on Percoll gradients we investigated selectivity for esterification of several polyunsaturated fatty acids (PUFA) in the sn-2 position of plasmalogens compared to [1-14C]hexadecanol, representative of de novo synthesis of the ether-linked sn-1 position. In whole cells at a final concentration of 105 microM PUFA, 2-4 nmol plasmalogen/mg protein was labeled in 4 h and 10-14 nmol in 24 h, representing 8-15% and 35-50%, respectively, of initial plasmalogen mass. Incorporation of label from hexadecanol was lower than PUFA incorporation (20:5(n-3) greater than 20:4(n-6) greater than 18:3(n-3) much greater than 18:2(n-6)) suggesting deacylation-reacylation at the sn-2 position. Plasmalogens accounted for 50% of total cell ethanolamine phospholipids and 75% in plasma membrane. Using a novel, improved method for extraction of subcellular fractions containing Percoll, plasma membrane also was enriched in plasmalogen relative to microsomes (107.4 +/- 5.2 vs. 40.0 +/- 2.9 nmol/mg protein). Selectivity for esterification at the sn-2 position of plasmalogens with respect to chain length and unsaturation of the fatty acyl chain was similar in both subcellular fractions and reflected that of whole cells. Labeling of plasma membrane with PUFA and fatty alcohol lagged behind that of microsomes. Chase experiments in cells prelabeled with [1-14C]18:3(n-3) for 2 h showed no significant reduction of label in plasmalogen of any subcellular fraction although accumulation of label in the microsomal fraction was slowed initially. Reduction of plasmalogen label (40-50%) did occur in microsomes and plasma membrane when cells prelabeled for 24 h were switched to chase medium with or without chase fatty acid. Our data suggest that esterification of PUFA to plasmalogen may occur at the endoplasmic reticulum with subsequent translocation to plasma membrane resulting in accumulation of relatively stable pools of plasmalogen that are not readily accessible for deacylation-reacylation exchange with newly appearing PUFA. Alternatively, deacylation-reacylation may occur in a more stable phospholipid pool within the plasma membrane but would involve a slower process than at the endoplasmic reticulum.     

Characterization of 5-aminolevulinate synthase from Agrobacterium radiobacter,screening new inhibitors for 5-aminolevulinate dehydratase from Escherichia coli and their potential use for high 5-aminolevulinate production

The hemA gene encoding 5-aminolevulinate synthase (ALAS) from Agrobacterium radiobacter zju-0121 showed 92.6% homology with that from A. radiobacter ATCC4718 and contained several rare codons. To enhance the expression of this gene, Escherichia coli Rosetta(DE3), which is a rare codon optimizer strain, was used as the host to construct an efficient recombinant strain. And the encoded protein was over-expressed as fusion protein and was purified by affinity purification on Ni-NTA agarose and by gel filtration chromatography on Sephadex G-25 Medium resin. The recombinant protein was partly characterized, and d-glucose, d-fructose, d-xylose, d-mannose, l-arabinose, d-galactose, lactose, sucrose and maltose were detected to have no distinct inhibition on this recombinant ALAS. Meanwhile, 20 mM d-glucose or d-xylose inhibited about 20% activity of ALA dehydratase (ALAD) from Escherichia coli Rosetta(DE3). Combining d-xylose as a new inhibitor for ALAD with d-glucose in fed-batch culture and based on the optimal culture system using Rosetta(DE3)/pET28a-hemA, the yield of ALA achieved was 7.3 g/l (56 mM) under the appropriate conditions in the fermenter.     

Plasmalogen synthesis is regulated via alkyl-dihydroxyacetonephosphate-synthase by amyloid precursor protein processing and is affected in Alzheimer's disease

Lipids play an important role as risk or protective factors in Alzheimer's disease, which is characterized by amyloid plaques composed of aggregated amyloid-beta. Plasmalogens are major brain lipids and controversially discussed to be altered in Alzheimer's disease (AD) and whether changes in plasmalogens are cause or consequence of AD pathology. Here, we reveal a new physiological function of the amyloid precursor protein (APP) in plasmalogen metabolism. The APP intracellular domain was found in vivo and in vitro to increase the expression of the alkyl-dihydroxyacetonephosphate-synthase (AGPS), a rate limiting enzyme in plasmalogen synthesis. Alterations in APP dependent changes of AGPS expression result in reduced protein and plasmalogen levels. Under the pathological situation of AD, increased amyloid-beta level lead to increased reactive oxidative species production, reduced AGPS protein and plasmalogen level. Accordingly, phosphatidylethanol plasmalogen was decreased in the frontal cortex of AD compared to age matched controls. Our findings elucidate that plasmalogens are decreased as a consequence of AD and regulated by APP processing under physiological conditions.     

Reactive brominating species produced by myeloperoxidase target the vinyl ether bond of plasmalogens: disparate utilization of sodium halides in the production of alpha-halo fatty aldehydes

Plasmalogens are a phospholipid molecular subclass that are enriched in the plasma membrane of many mammalian cells. The present study demonstrates that reactive brominating species produced by myeloperoxidase, as well as activated neutrophils, attack the vinyl ether bond of plasmalogens. Reactive brominating species produced by myeloperoxidase target the vinyl ether bond of plasmalogens, resulting in the production of a neutral lipid and lysophosphatidylcholine. Gas chromatography-mass spectrometry and proton NMR analyses of this neutral lipid demonstrated that it was 2-bromohexadecanal (2-BrHDA). In comparison to myeloperoxidase-generated reactive chlorinating species, reactive brominating species attacked the plasmalogen vinyl ether bond at neutral pH. In the presence of a 20-fold molar excess of NaCl compared with NaBr, myeloperoxidase-derived reactive halogenating species favored the production of 2-BrHDA over that of 2-chlorohexadecanal. Additionally, 2-BrHDA was preferentially produced from plasmalogen treated with hypochlorous acid in the presence of NaBr. The potential physiological significance of this pathway was suggested by the demonstration that both 2-BrHDA and 2-bromooctadecanal were produced by PMA-stimulated neutrophils. Taken together, the present studies demonstrate the targeting of the vinyl ether bond of plasmalogens by the reactive brominating species produced by myeloperoxidase and by activated neutrophils, resulting in the production of novel brominated fatty aldehydes.     

Cloning and expression in Escherichia coli of the glutamate dehydrogenase gene,gdh, from Corynebacterium melassecola

A hybrid plasmid containing a fragment of the Corynebacterium melassecola chromosome cloned into pBR325 restored growth of glutamate auxotrophs of Escherichia coli strains that have mutations in the genes for glutamate dehydrogenase and glutamate synthase. A 3.1-kilobase pair region was shown by complementation analysis and enzyme measurements to carry the glutamate dehydrogenase gene, gdh. Glutamate dehydrogenase encoded by gdh carried on recombinant plasmids was elevated over 100-fold in E. coli cells. The gdh promoter was located by in vitro fusion to a promoter-deficient galK gene.     

Plasmalogens in the yeast Pullularia pullulans

Choline and ethanolamine phosphoglycerides have been found in plasmalogen form in P. pullilans. Plasmalogens had not been described in yeasts up to now.     

Overproduction of carotenoids in Thermus thermophilus

Phytoene synthase encoded by the crtB gene is one of the rate-limiting enzymes for carotenoid production in Thermus thermophilus. We introduced a multicopy recombinant plasmid, pCOP1, in which the Thermus crtB gene was cloned, into carotenoid overproducing mutants of T. thermophilus. The overproducing mutants carrying a pCOP1 produced about twenty times as much carotenoids as the parental strain did.     

Roles of AtpI and Two YidC-Type Proteins from Alkaliphilic Bacillus pseudofirmus OF4 in ATP Synthase Assembly and Nonfermentative Growth

AtpI, a membrane protein encoded by many bacterial atp operons, is reported to be necessary for c-ring oligomer formation during assembly of some ATP synthase complexes. We investigated chaperone functions of AtpI and compared them to those of AtpZ, a protein encoded by a gene upstream of atpI that has a role in magnesium acquisition at near-neutral pH, and of SpoIIIJ and YqjG, two YidC/OxaI/Alb3 family proteins, in alkaliphilic Bacillus pseudofirmus OF4. A strain with a chromosomal deletion of atpI grew nonfermentatively, and its purified ATP synthase had a c-ring of normal size, indicating that AtpI is not absolutely required for ATP synthase function. However, deletion of atpI, but not atpZ, led to reduced stability of the ATP synthase rotor, reduced membrane association of the F₁ domain, reduced ATPase activity, and modestly reduced nonfermentative growth on malate at both pH 7.5 and 10.5. Both spoIIIJ and yqjG, but not atpI or atpZ, complemented a YidC-depleted Escherichia coli strain. Consistent with such overlapping functions, single deletions of spoIIIJ or yqjG in the alkaliphile did not affect membrane ATP synthase levels or activities, but functional specialization was indicated by YqjG and SpoIIIJ showing respectively greater roles in malate growth at pH 7.5 and 10.5. Expression of yqjG was elevated at pH 7.5 relative to that at pH 10.5 and in ΔspoIIIJ strains, but it was lower than constitutive spoIIIJ expression. Deletion of atpZ caused the largest increase among the mutants in magnesium concentrations needed for pH 7.5 growth. The basis for this phenotype is not yet resolved.     

Cloning and Heterologous Expression of an Enantioselective Amidase from Rhodococcus erythropolis Strain MP50

The gene for an enantioselective amidase was cloned from Rhodococcus erythropolis MP50, which utilizes various aromatic nitriles via a nitrile hydratase/amidase system as nitrogen sources. The gene encoded a protein of 525 amino acids which corresponded to a protein with a molecular mass of 55.5 kDa. The deduced complete amino acid sequence showed homology to other enantioselective amidases from different bacterial genera. The nucleotide sequence approximately 2.5 kb upstream and downstream of the amidase gene was determined, but no indications for a structural coupling of the amidase gene with the genes for a nitrile hydratase were found. The amidase gene was carried by an approximately 40-kb circular plasmid in R. erythropolis MP50. The amidase was heterologously expressed in Escherichia coli and shown to hydrolyze 2-phenylpropionamide, α-chlorophenylacetamide, and α-methoxyphenylacetamide with high enantioselectivity; mandeloamide and 2-methyl-3-phenylpropionamide were also converted, but only with reduced enantioselectivity. The recombinant E. coli strain which synthesized the amidase gene was shown to grow with organic amides as nitrogen sources. A comparison of the amidase activities observed with whole cells or cell extracts of the recombinant E. coli strain suggested that the transport of the amides into the cells becomes the rate-limiting step for amide hydrolysis in recombinant E. coli strains.     

Mechanisms of lipid peroxidation in erythrocytes of vitamin E-deficient rats and in phospholipid model systems

Erythrocytes from vitamin E-deficient and control rats were peroxidized by glucose oxidase-glucose or dialuric acid. Losses of polyunsaturated fatty acids from membrane phospholipids, and of dimethylacetals from plasmalogens, were quantitated by gas-liquid chromatography. Similar treatment of solubilized or micellar phospholipids or plasmalogens in vitro showed that in both erythrocytes and micellar systems, arachidonic acid and the 16-carbon plasmalogen are most susceptible to peroxidation by either reagent. The same narrow concentration range of dialuric acid found effective in peroxidizing erythrocytes from tocopherol-deficient rats was also found effective in peroxidizing micellar phospholipids in vitro.Partially peroxidized erythrocytes from tocopherol-deficient rats were subjected to treatment with phospholipase A or phospholipase C. Hemolysis by either phospholipase was accelerated in partially peroxidized cells as compared to controls, suggesting that peroxidation exposes both polar and nonpolar lipid sites in the erythrocyte membrane.