SignificanceCardiolipins are a unique class of phospholipids in mitochondrial membranes that are crucial for cellular bioenergetics as they stabilize respiratory chain complexes. In contrast to most other phospholipids, cardiolipins are substituted with four, rather than only two fatty acyl side chains. Consequently, this opens up a vast number of different theoretically possible molecular lipid species. Experimentally assessing the molecular diversity of cardiolipin species is analytically challenging. In this study we successfully combine tandem mass spectrometry with a mathematical structural modeling approach, to achieve the comprehensive characterization of complex biological cardiolipin compositions.
A significant fraction of the glycerophospholipids in the human body is composed of plasmalogens, particularly in the brain, cardiac, and immune cell membranes. A decline in these lipids has been observed in such diseases as Alzheimer’s and chronic obstructive pulmonary disease. Plasmalogens contain a characteristic 1-O-alk-1′-enyl ether (vinyl ether) double bond that confers special biophysical, biochemical, and chemical properties to these lipids. However, the genetics of their biosynthesis is not fully understood, since no gene has been identified that encodes plasmanylethanolamine desaturase (E.C. 1.14.99.19), the enzyme introducing the crucial alk-1′-enyl ether double bond. The present work identifies this gene as transmembrane protein 189 (TMEM189). Inactivation of theTMEM189gene in human HAP1 cells led to a total loss of plasmanylethanolamine desaturase activity, strongly decreased plasmalogen levels, and accumulation of plasmanylethanolamine substrates and resulted in an inability of these cells to form labeled plasmalogens from labeled alkylglycerols. Transient expression of TMEM189 protein, but not of other selected desaturases, recovered this deficit. TMEM189 proteins contain a conserved protein motif (pfam10520) with eight conserved histidines that is shared by an alternative type of plant desaturase but not by other mammalian proteins. Each of these histidines is essential for plasmanylethanolamine desaturase activity. Mice homozygous for an inactivatedTmem189gene lacked plasmanylethanolamine desaturase activity and had dramatically lowered plasmalogen levels in their tissues. These results assign theTMEM189gene to plasmanylethanolamine desaturase and suggest that the previously characterized phenotype ofTmem189-deficient mice may be caused by a lack of plasmalogens.
Tetrahydrobiopterin is a cofactor synthesized from GTP with well-known roles in enzymatic nitric oxide synthesis and aromatic amino acid hydroxylation. It is used to treat mild forms of phenylketonuria. Less is known about the role of tetrahydrobiopterin in lipid metabolism, although it is essential for irreversible ether lipid cleavage by alkylglycerol monooxygenase. Here we found intracellular alkylglycerol monooxygenase activity to be an important regulator of alkylglycerol metabolism in intact murine RAW264.7 macrophage-like cells. Alkylglycerol monooxygenase was expressed and active also in primary mouse bone marrowderived monocytes and "alternatively activated" M2 macrophages obtained by interleukin 4 treatment, but almost missing in M1 macrophages obtained by IFN-γ and lipopolysaccharide treatment. The cellular lipidome of RAW264.7 was markedly changed in a parallel way by modulation of alkylglycerol monooxygenase expression and of tetrahydrobiopterin biosynthesis affecting not only various ether lipid species upstream of alkylglycerol monooxygenase but also other more complex lipids including glycosylated ceramides and cardiolipins, which have no direct connection to ether lipid pathways. Alkylglycerol monooxygenase activity manipulation modulated the IFN-γ/lipopolysaccharide-induced expression of inducible nitric oxide synthase, interleukin-1β, and interleukin 1 receptor antagonist but not transforming growth factor β1, suggesting that alkylglycerol monooxygenase activity affects IFN-γ/lipopolysaccharide signaling. Our results demonstrate a central role of tetrahydrobiopterin and alkylglycerol monooxygenase in ether lipid metabolism of murine macrophages and reveal that alteration of alkylglycerol monooxygenase activity has a profound impact on the lipidome also beyond the class of ether lipids. alkylglycerols | lipidomics | macrophages | RAW264.7 | tetrahydrobiopterin
Deficient ether lipid biosynthesis in rhizomelic chondrodysplasia punctata and other disorders is associated with a wide range of severe symptoms including small stature with proximal shortening of the limbs, contractures, facial dysmorphism, congenital cataracts, ichthyosis, spasticity, microcephaly, and mental disability. Mouse models are available but show less severe symptoms. In both humans and mice, it has remained elusive which of the symptoms can be attributed to lack of plasmanyl or plasmenyl ether lipids. The latter compounds, better known as plasmalogens, harbor a vinyl ether double bond conferring special chemical and physical properties. Discrimination between plasmanyl and plasmenyl ether lipids is a major analytical challenge, especially in complex lipid extracts with many isobaric species. Consequently, these lipids are often neglected also in recent lipidomic studies. Here, we present a comprehensive LC–MS/MS based approach that allows unequivocal distinction of these two lipid subclasses based on their chromatographic properties. The method was validated using a novel plasmalogen-deficient mouse model, which lacks plasmanylethanolamine desaturase and therefore cannot form plasmenyl ether lipids. We demonstrate that plasmanylethanolamine desaturase deficiency causes an accumulation of plasmanyl species, a too little studied but biologically important substance class.
Plasmanylethanolamine desaturase (PEDS) (EC 1.14.99.19) introduces the 1-prime double bond into plasmalogens, one of the most abundant phospholipids in the human body. This labile membrane enzyme has not been purified and its coding sequence is unknown. Previous assays for this enzyme used radiolabeled substrates followed by multistep processing. We describe here a straight-forward method for the quantification of PEDS in enzyme incubation mixtures using pyrene-labeled substrates and reversed-phase HPLC with fluorescence detection. After stopping the reaction with hydrochloric acid in acetonitrile, the mixture was directly injected into the HPLC system without the need of lipid extraction. The substrate, 1-O-pyrenedecyl-2-acyl-sn-glycero-3-phosphoethanolamine, and the lyso-substrate, 1-O-pyrenedecyl-sn-glycero-3-phosphoethanolamine, were prepared from RAW-12 cells deficient in PEDS activity and were compared for their performance in the assay. Plasmalogen levels in mouse tissues and in cultured cells did not correlate with PEDS levels, indicating that the desaturase might not be the rate limiting step for plasmalogen biosynthesis. Among selected mouse organs, the highest activities were found in kidney and in spleen. Incubation of intact cultivated mammalian cells with 1-O-pyrenedecyl-sn-glycerol, extraction of lipids, and treatment with hydrochloric or acetic acid in acetonitrile allowed sensitive monitoring of PEDS activity in intact cells.
The polypeptide distribution of lipoprotein density fractions isolated from normal human serum by rate zonal ultracentrifugation was investigated by polyacrylamide gel electrophoresis and qualitative and quantitative immunochemical methods. I n very-low-density lipoproteins all known apoA and apoC peptides were present in amounts similar to these preparations from fixed-angle rotors. Low-density apolipoproteins consisted primarily of apoB (98 Oi0) but some small amounts of apoAIII and apoCIII were also present. No region of 100 apoB, uncontaminated by A and C peptides could be isolated by subfractionation of the low-density peak. There were marked differences in thc protein part of the high-density lipoprotein subfractions 2 and 3, as compared to these subfractions separated in the angle-head rotor. The weight ratio apoAI : apoAII was found to be approximately three times as high for subfraction 2 as for subfraction 3. I n addition, apoAIII was detectable only in subfraction 3. Of the known apoC peptides, apoCII was present in both fractions only in trace amounts, while the concentration of apoCI was in the order of I o/o and that of apoCIII, and of apoCIII, in the order of 1.5 O i 0 . Apolipoprotein B was not detectable in subfraction 2 . Two density regions were found with small amounts of lipoproteins consisting only of apoAI polypeptides. A peak corresponding to subfraction 1 of the highdensity lipoprotein from fixed-angle rotor preparations could not be detected in the eluate of zonal rotors after 24-h runs; the low-density lipoprotein peak was symmetrical in all experiments. Lipoproteins present in the bottom fraction amounted to about 2.5-4O/, of the total lipoprotein content. The only apolipoprotein found in this fraction was apoA1.It now seems to be widely accepted that individual lipoprotein density classes are composed of several lipoprotein families, i.e. separate entities with identical prot,ein moities [l -41. Although from analytical isoelectric focusing experiments a minimum of 8 different lipoprotein subpopulations can be expected [5], only three main lipoprotein families could be isolated from human plasma on a preparative scale and characterized by chemical and physicochemical means. Lipoprotein A represents the major family in high-density lipoprotein subfractions 2 and 3 and consists of two major nonidentical polypeptides, ApoAl and ApoII [2,6]. In addition, small amounts of a third apolipoprotein which probably also belongs to the lipoprotein A family could be found in subfraction 3 and was therefore designated as ApoAIII [7]. The protein part of lipoprotein B, the major family of low-density lipoprotein subfraction 3 has not been characterized entirely, and there seems to be some disagreement concerning the amount, type and molecular weight of the polypeptides found in this lipoprotein [8,9]. Apolipoprotein C, first demonstrated in very-low-density lipoprotein as a third class of apolipoproteins [lo], was found to be composed of four different polypeptides [ll]. These polypeptides, which w...
In a prospective controlled trial, we studied the effect of tight metabolic control on the outcomes of 102 gestational diabetes mellitus (GDM) pregnancies compared with outcomes of 102 matched nondiabetic control pregnancies. Women with GDM were treated to achieve and maintain a blood glucose concentration of less than 130 mg/dl at 1 h after breakfast. Treatment consisted of a diet low in oligosaccharides and fat and, if necessary, once daily insulin. By the end of gestation, 88 of the 102 women with GDM received insulin at a mean dose of 18 U/day. Duration of insulin therapy ranged from 3 to 32 wk with a median of 11 wk. Perinatal outcome of GDM pregnancies under this management equaled that of control pregnancies. The full spectrum of excess morbidity from GDM was prevented, and normal distribution of birth weight and normal rates of macrosomia, dystrophy, hypoglycemia, hypocalcemia, hyperbilirubinemia, fetal acidosis, and low Apgar scores were achieved. No mortality was observed. In addition to the two main study groups, we also studied a third group of 24 women with GDM whose treatment lasted less than or equal to 5 wk due to late diagnosis. This suboptimally treated group demonstrated a significant (P less than .05) increase of macrosomia and umbilical artery acidosis compared with the well-treated GDM group. The study reported herein demonstrates that excess mortality and morbidity typically observed in GDM can be prevented by early institution of tight metabolic control, which required insulin in 86% of our patients.
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