Synthesis of the unusual lipid bis(monoacylglycero)phosphate in environmental bacteria

The bacterial membrane is constantly remodelled in response to environmental conditions and the external supply of precursor molecules. Some bacteria are able to acquire exogenous lyso-phospholipids and convert them to the corresponding phospholipids. Here, we report that some soil-dwelling bacteria have alternative options to metabolize lyso-phosphatidylglycerol (L-PG). We find that the plant-pathogen Agrobacterium tumefaciens takes up this mono-acylated phospholipid and converts it to two distinct isoforms of the non-canonical lipid bis(monoacylglycero)phosphate (BMP). Chromatographic separation and quadrupole-time-of-flight MS/MS analysis revealed the presence of two possible BMP stereo configurations acylated at either of the free hydroxyl groups of the glycerol head group. BMP accumulated in the inner membrane and did not visibly alter cell morphology and growth behaviour. The plant-associated bacterium Sinorhizobium meliloti was also able to convert externally provided L-PG to BMP. Other bacteria like Pseudomonas fluorescens and Escherichia coli metabolized L-PG after cell disruption, suggesting that BMP production in the natural habitat relies both on dedicated uptake systems and on head-group acylation enzymes. Overall, our study adds two previously overlooked phospholipids to the repertoire of bacterial membrane lipids and provides evidence for the remarkable condition-responsive adaptation of bacterial membranes.     

De novo biosynthesis of the late endosome lipid, bis(monoacylglycero)phosphate

Bis(monoacylglycero)phosphate (BMP) is a unique lipid enriched in the late endosomes participating in the trafficking of lipids and proteins through this organelle. The de novo biosynthesis of BMP has not been clearly demonstrated. We investigated whether phosphatidylglycerol (PG) and cardiolipin (CL) could serve as precursors of de novo BMP synthesis using two different cellular models: CHO cells deficient in phosphatidylglycerophosphate (PGP) synthase, the enzyme responsible for the first step of PG synthesis; and human lymphoblasts from patients with Barth syndrome (BTHS), characterized by mutations in tafazzin, an enzyme implicated in the deacylation-reacylation cycle of CL. The biosynthesis of both PG and BMP was reduced significantly in the PGP synthase-deficient CHO mutants. Furthermore, overexpression of PGP synthase in the deficient mutants induced an increase of BMP biosynthesis. In contrast to CHO mutants, BMP biosynthesis and its fatty acid composition were not altered in BTHS lymphoblasts. Our results thus suggest that in mammalian cells, PG, but not CL, is a precursor of the de novo biosynthesis of BMP. Despite the decrease of de novo synthesis, the cellular content of BMP remained unchanged in CHO mutants, suggesting that other pathway(s) than de novo biosynthesis are also used for BMP synthesis.     

Effect of lysosomal storage on bis(monoacylglycero)phosphate

BMP [bis(monoacylglycero)phosphate] is an acidic phospholipid and a structural isomer of PG (phosphatidylglycerol), consisting of lysophosphatidylglycerol with an additional fatty acid esterified to the glycerol head group. It is thought to be synthesized from PG in the endosomal/lysosomal compartment and is found primarily in multivesicular bodies within the same compartment. In the present study, we investigated the effect of lysosomal storage on BMP in cultured fibroblasts from patients with eight different LSDs (lysosomal storage disorders) and plasma samples from patients with one of 20 LSDs. Using ESI-MS/MS (electrospray ionization tandem MS), we were able to demonstrate either elevations or alterations in the individual species of BMP, but not of PG, in cultured fibroblasts. All affected cell lines, with the exception of Fabry disease, showed a loss of polyunsaturated BMP species relative to mono-unsaturated species, and this correlated with the literature reports of lysosomal dysfunction leading to elevations of glycosphingolipids and cholesterol in affected cells, processes thought to be critical to the pathogenesis of LSDs. Plasma samples from patients with LSDs involving storage in macrophages and/or with hepatomegaly showed an elevation in the plasma concentration of the C(18:1)/C(18:1) species of BMP when compared with control plasmas, whereas disorders involving primarily the central nervous system pathology did not. These results suggest that the release of BMP is cell/tissue-specific and that it may be useful as a biomarker for a subset of LSDs.     

Progesterone and a phospholipase inhibitor increase the endosomal bis(monoacylglycero)phosphate content and block HIV viral particle intercellular transmission

Progesterone, the cationic amphiphile U18666A and a phospholipase inhibitor (Methyl Arachidonyl Fluoro Phosphonate, MAFP) inhibited by 70%–90% HIV production in viral reservoir cells, i.e. human THP-1 monocytes and monocyte-derived macrophages (MDM). These compounds triggered an inhibition of fluid phase endocytosis (macropinocytosis) and modified cellular lipid homeostasis since endosomes accumulated filipin-stained sterols and Bis(Monoacylglycero)Phosphate (BMP). BMP was quantified using a new cytometry procedure and was increased by 1.25 times with MAFP, 1.7 times with U18666A and 2.5 times with progesterone. MAFP but not progesterone or U18666A inhibited the hydrolysis of BMP by the Pancreatic Lipase Related Protein 2 (PLRP2) as shown by in-vitro experiments. The possible role of sterol transporters in steroid-mediated BMP increase is discussed.     

Bis(monoacylglycero)phosphate: a secondary storage lipid in the gangliosidoses

Bis(monoacylglycero)phosphate (BMP) is a negatively charged glycerophospholipid with an unusual sn-1;sn-1′ structural configuration. BMP is primarily enriched in endosomal/lysosomal membranes. BMP is thought to play a role in glycosphingolipid degradation and cholesterol transport. Elevated BMP levels have been found in many lysosomal storage diseases (LSDs), suggesting an association with lysosomal storage material. The gangliosidoses are a group of neurodegenerative LSDs involving the accumulation of either GM1 or GM2 gangliosides resulting from inherited deficiencies in β-galactosidase or β-hexosaminidase, respectively. Little information is available on BMP levels in gangliosidosis brain tissue. Our results showed that the content of BMP in brain was significantly greater in humans and in animals (mice, cats, American black bears) with either GM1 or GM2 ganglioside storage diseases, than in brains of normal subjects. The storage of BMP and ganglioside GM2 in brain were reduced similarly following adeno-associated viral-mediated gene therapy in Sandhoff disease mice. We also found that C22:6, C18:0, and C18:1 were the predominant BMP fatty acid species in gangliosidosis brains. The results show that BMP accumulates as a secondary storage material in the brain of a broad range of mammals with gangliosidoses.     

Formation of acylphosphatidylglycerol by a lysosomal phosphatidylcholine:bis(monoacylglycero)phosphate acyl transferase

A delipidated soluble fraction prepared from a mitochondrial-lysosomal fraction of rabbit alveolar macrophages that catalyzes transacylation of lysophosphatidylglycerol to form bis(monoacylglycero)phosphate was also found to transfer oleic acid from [14C]dioleoyl phosphatidylcholine to form acylphosphatidylglycerol. The reaction was dependent on the presence of bis(monoacylglycero)phosphate and was maximal at a concentration of 44 microM when the ratio of fatty acid transferred to fatty acid released was 0.28. Addition of phosphatidylglycerol had only a small effect. Homogenates of rat liver also catalyzed the reaction and after subcellular fractionation the activity was localized to lysosomes. The lysosomal activity was solubilized by delipidation with butanol to give a preparation with a specific activity 2462 times that of the homogenate. Optimal activity of soluble preparations from both macrophages and liver was at pH 4.5, with little activity above 6.0. Release of free fatty acid was also stimulated under conditions of optimal acyl transfer. Both acyl transfer and release of fatty acid were inhibited by Ca2+, detergents, chlorpromazine, lysophosphatidylcholine, and oleic acid. When there was disproportional inhibition, acyl transfer was always more affected. These results suggest that sequential acylation of lysophosphatidylglycerol to form bis(monoacylglycero)phosphate and then acylphosphatidylglycerol constitute a mechanism in the lysosome for the transport and partition of fatty acids released by the lysosomal phospholipases.     

Metabolism, function and mass spectrometric analysis of bis(monoacylglycero)phosphate and cardiolipin

Polyglycerophospholipids (PGPLs) such as bismonoacylglycerophosphate (BMP) and cardiolipin are important membrane phospholipid species for the maintenance of membrane integrity. While BMP serves as membrane curvature regulator in multivesicular bodies for efficient lysosomal enzyme function, cardiolipin stabilizes the electron transfer complex in the inner mitochondrial membrane, which is crucial for physiological ATP production. Beside their membrane modulatory functions PGPLs play an important role in various signaling events. Although a number of disease associations were found for PGPL species, detailed information about their molecular role still remains unknown. This article reviews the known biological functions of PGPLs and the existing mass spectrometric methods. We discuss the different analytical strategies and how ESI–MS/MS can expand our understanding of PGPL homeostasis.     

The stereoconfiguration of bis(monoacylglycero)phosphate synthesized in vitro in lysosomes of rat liver: comparison with the natural lipid.

A procedure for stereoanalysis of radiochemically labeled glycerophospholipids is described. It is based on the study of the labeled alpha-glycerophosphate which retains its original configuration when liberated upon alkaline hydrolysis of the lipids. The labeled alpha-glycerophosphate is oxidized enzymatically with sn-3-glycerophosphate dehydrogenase and the product, dihydroxyacetone phosphate, is degraded with alkali to inorganic phosphate. The nonoxidizable alpha-glycerophophate (sn-1-glycerophosphate), the beta-glycerophosphate, and the inorganic phosphate derived from sn-3-glycerophosphate are quantitated after separation by thin-layer chromatography. The procedure gave the expected results when applied to [3H]glycerol-and 32P-labeled phosphatidylcholine, bis( monoacylglycero)phosphate, and phosphatidylglycerol from natural resources. Bis(monoacylglycero)phosphate, known also as lysobisphosphatidic acid, was synthesized from ]32P]diphosphatidylglycerol and from phosphatidyl[1',3'-3H]glycerol in lysosomal preparations of rat liver according to Poorthuis and Hostetler (1978. J. Lipid Res. 19: 309-315). Stereoanalysis proved that the product was in both cases a derivate of sn-1-glycerophospho-sn-1'-glycerol.     

Promotion of vesicular stomatitis virus fusion by the endosome-specific phospholipid bis(monoacylglycero)phosphate (BMP)

Vesicular stomatitis virus (VSV) is a prototypic virus commonly used in studies of endocytosis and membrane trafficking. One proposed mechanism for VSV entry involves initial fusion with internal vesicles of multivesicular endosomes followed by back-fusion of these vesicles into the cytoplasm. One feature of endosomal internal vesicles is that they contain the lipid bis(monoacylglycero)phosphate (BMP). Here, we show that the presence of BMP significantly increases the rate of VSV G-mediated membrane fusion. The increased fusion was selective for VSV and was not evident for another enveloped virus, influenza virus. Our data provide a biological rationale for a two-step infection reaction during VSV entry, and suggest that BMP preferentially affects the ability of VSV G to mediate lipid mixing during membrane fusion.     

Sterically stabilized liposomes of DPPC/DPPE-PEG:2000. A spin label ESR and spectrophotometric study

The chain dynamics and the thermotropic phase behavior of sterically stabilized liposomes obtained introducing in the host bilayer matrix of DPPC up to 7 mol% of the polymer-lipid DPPE-PEG:2000 were investigated by spin label electron spin resonance spectroscopy and spectrophotometry. The experimental data indicate that the dispersions have the dynamic and thermotropic characteristics of normal lamellar phase. Moreover, using spin labels that locate both in the interfacial and in the hydrocarbon regions, namely TEMPO-stearate, 5- and 16-PCSL, we find that relative to the unmodified DPPC bilayers, the polymer-grafted bilayers are loosely packed in the interfacial region and have reduced chain mobility in the gel phase. From the temperature dependence of the partition coefficient (P(c)), of the spin probe DTBN between the aqueous and the fluid hydrophobic regions of the bilayers and from the melting curves of the absorbance at 400 nm, we observe a slight influence on the endothermic phase transitions when increasing the concentration of the polymer-lipid in the DPPC bilayers, the influence being more evident in the pre-transition.     

Accumulation of bis(monoacylglycero)phosphate and gangliosides in mouse models of neuronal ceroid lipofuscinosis

The neuronal ceroid lipofuscinoses comprise a group of inherited severe neurodegenerative lysosomal disorders characterized by lysosomal dysfunction and massive accumulation of fluorescent lipopigments and aggregated proteins. To examine the role of lipids in neurodegenerative processes of these diseases, we analysed phospho- and glycolipids in the brains of ctsd−/− and nclf mice, disease models of cathepsin D and CLN6 deficiency, respectively. Both ctsd−/− and nclf mice exhibited increased levels of GM2 and GM3 gangliosides. Immunohistochemically GM2 and GM3 staining was found preferentially in neurons and glial cells, respectively, of ctsd−/− mice. Of particular note, a 20-fold elevation of the unusual lysophospholipid bis(monoacylglycero)phosphate was specifically detected in the brain of ctsd−/− mice accompanied with sporadic accumulation of unesterified cholesterol in distinct cells. The impaired processing of the sphingolipid activator protein precursor, an in vitro cathepsin D substrate, in the brain of ctsd−/− mice may provide the mechanistic link to the storage of lipids. These studies show for the first time that cathepsin D regulates the lysosomal phospho- and glycosphingolipid metabolism suggesting that defects in the composition, trafficking and/or recycling of membrane components along the late endocytic pathway may be critical for the pathogenesis of early onset neuronal ceroid lipofuscinoses.     

Effect of polar carotenoids on the oxygen diffusion-concentration product in lipid bilayers. An EPR spin label study.

The oxygen diffusion-concentration product was determined in phosphatidylcholine (PC) bilayers from oxygen broadening of the spin label EPR spectra. The use of fatty acid spin labels makes it possible to do structural and oximetric measurements with the same sample. We find that polar carotenoids, zeaxanthin and violaxanthin, increase ordering of hydrocarbon chains in saturated (dimyristoyl-PC) and unsaturated (egg yolk PC) membranes and also significantly decrease the oxygen diffusion-concentration product in the hydrocarbon region of these membranes. At 25 degrees C in the presence of 10 mol% of carotenoids, the product is about 30% smaller than in pure PC membranes. Intercalation of carotenoids decreases the oxygen diffusion-concentration product in the central part of the bilayer and has little effect on the product in the polar head group region. In contrast, cholesterol molecules significantly reduce the product on and near the membrane surface, and do not change it (saturated PC) or increase it (unsaturated PC) in the middle of the bilayer (Subczynski, W.K., Hyde, J.S. and Kusumi, A. (1989) Proc. Natl. Acad. Sci. USA 86, 4474-4478). The decrease of oxygen diffusion-concentration product may be a mechanism of carotenoid protective activity, which should be effective in plant and animal cells in the light as well as in the dark.     

Effect of high electrolyte concentration on the phase transition behaviour of DPPC vesicles: a spin label study

We have investigated by Electron Spin Resonance spectroscopy, the effects of high electrolyte concentration on the phase transitions of unilamellar vesicles of dipalmitoylphosphatidylcholine at the pH values of 5.0 and 9.0. Using the 5-nitroxide stearic acid as spin probe we have found that, at both pH values, the lipid main phase transition is not quite affected by variations of the electrolyte concentration up to the value of 3 M. Instead, the pretransition at pH 5.0 disappears in the presence of 1 M electrolyte, and at pH 9.0, the pretransition temperature shifts upward from 25.5 to 31.0 degrees C when the electrolyte concentration reaches the value of 3 M. The observed results on the pre- and main phase transition widths, transition temperatures and their cooperativity indicate that the presence of salt in the bulk solution leads to structural changes of the lipid bilayer which essentially concern either the polar zone or the hydrogen belt region of the DPPC vesicles. The extent of observed perturbation depends on salt concentration.     

A spin label study of the lipid boundary layer of mitochondrial NADH-ubiquinone oxidoreductase

Mitochondrial NADH-ubiquinone oxidoreductase (Complex I) is a lipoprotein enzyme containing phosphatidylcholine (PC), phosphatidylethanolamine (PE) and cardiolipin. Enzyme preparations containing endogenous cardiolipin and a range of either soyabean PC or dimyristoylphosphatidylcholine (DMPC) concentrations have been made. Using a spin-labelled fatty acid, two probe environments differing in mobility have been shown to be present. The fatty acid probe has a relative binding constant (or partition coefficient between lipid and protein) of unity. The boundary layer or lipid annulus reported by the probe has a value of approx. 300 lipid molecules per molecule of enzyme FMN in preparations containing soyabean PC, or DMPC above the phase transition temperature of the latter. In soyabean PC-replaced enzyme the apparent size of the boundary layer is independent of temperature between 30 degrees C and 14 degrees C but shows a modest increase to about 400 lipid molecules per molecule of FMN between 14 degrees C and 2 degrees C. Complex I replaced with high concentrations of DMPC gives non-linear Arrhenius plots of NADH-ubiquinone oxidoreductase activity. The results of the ESR experiments show that both boundary layer and bulk lipid must be motionally restricted for this to occur. Thus, the change in activity is probably not caused by an effect exerted directly on the catalytic activity of the enzyme but is more likely due to restriction of free diffusion of ubiquinone to its site of reduction.     

Integrated analysis of the conformation of a protein-linked spin label by crystallography,EPR and NMR spectroscopy

Long-range structural information derived from paramagnetic relaxation enhancement observed in the presence of a paramagnetic nitroxide radical is highly useful for structural characterization of globular, modular and intrinsically disordered proteins, as well as protein–protein and protein-DNA complexes. Here we characterized the conformation of a spin-label attached to the homodimeric protein CylR2 using a combination of X-ray crystallography, electron paramagnetic resonance (EPR) and NMR spectroscopy. Close agreement was found between the conformation of the spin label observed in the crystal structure with interspin distances measured by EPR and signal broadening in NMR spectra, suggesting that the conformation seen in the crystal structure is also preferred in solution. In contrast, conformations of the spin label observed in crystal structures of T4 lysozyme are not in agreement with the paramagnetic relaxation enhancement observed for spin-labeled CylR2 in solution. Our data demonstrate that accurate positioning of the paramagnetic center is essential for high-resolution structure determination.     

Investigations of thermotropic phase changes in peripheral nerve of frog and rat. A spin label study

The temperature-dependent fluidity of myelin of frog and rat peripheral nerve (Nervus ischiadicus) was studied using the spin label technique. In frog nerve a phase change was detected at 38 °C. In rat nerve no sharp phase change could be established, and the lipid-depleted frog and rat nerve also showed no transition. From the spectral data, it was concluded that in frog and rat nerve the lipid-protein interactions are different, i.e. species dependent. Ca²⁺-depletion of frog nerve caused a loss of transition, while rat nerve remained unaffected. Thus it was indicated that, in frog nerve, Ca²⁺ is involved in the phase change. In the total lipid extract of frog nerve a phase change centered at 32 °C occurred, while the total lipid extract of rat nerve again showed no transition. It is suggested that a connexion exists between our results and investigations on the temperature dependence of an axonal conduction block of nerve.     

Degradation of membrane-bound ganglioside GM1. Stimulation by bis(monoacylglycero)phosphate and the activator proteins SAP-B and GM2-AP

According to our hypothesis (Fürst, W., and Sandhoff, K. (1992) Biochim. Biophys. Acta 1126, 1-16) glycosphingolipids of the plasma membrane are digested after endocytosis as components of intraendosomal and intralysosomal vesicles and membrane structures. The lysosomal degradation of glycosphingolipids with short oligosaccharide chains by acid exohydrolases requires small, non-enzymatic cofactors, called sphingolipid activator proteins (SAPs). A total of five activator proteins have been identified as follows: namely the saposins SAP-A, -B, -C, and -D, which are derived from the single chain SAP-precursor protein (prosaposin), and the GM2 activator protein. A deficiency of prosaposin results in the storage of ceramide and sphingolipids with short oligosaccharide head groups. The loss of the GM2 activator protein blocks the degradation of the ganglioside GM2. The enzymatic hydrolysis of the ganglioside GM1 is catalyzed by beta-galactosidase, a water-soluble acid exohydrolase. The lack of ganglioside GM1 accumulation in patients suffering from either prosaposin or GM2 activator protein deficiency has led to the hypothesis that SAPs are not needed for the hydrolysis of the ganglioside GM1 in vivo. In this study we demonstrate that an activator protein is required for the enzymatic degradation of membrane-bound ganglioside GM1 and that both SAP-B and the GM2 activator protein significantly enhance the degradation of the ganglioside GM1 by acid beta-galactosidase in a liposomal, detergent-free assay system. These findings offer a possible explanation for the observation that no storage of the ganglioside GM1 has been observed in patients with either isolated prosaposin or isolated GM2 activator deficiency. We also demonstrate that anionic phospholipids such as bis(monoacylglycero)phosphate and phosphatidylinositol, which specifically occur in inner membranes of endosomes and in lysosomes, are essential for the activator-stimulated hydrolysis of the ganglioside GM1. Assays utilizing surface plasmon resonance spectroscopy showed that bis(monoacylglycero)phosphate increases the binding of both beta-galactosidase and activator proteins to substrate-carrying membranes.     

Effect of the nature of the 3beta-substitution in manoyl oxides on the thermotropic behavior of DPPC lipid bilayer and on DPPC liposomes

Functionalized manoyl oxide derivatives have been proved over the years to evoke several biological responses. Among them, 3beta-hydroxy-manoyl oxide (1) and 3beta-acetoxy-manoyl oxide (2) have been shown to exhibit in vitro antimicrobial and cytotoxic activity, while N-imidazole-3 beta-thiocarbonyl ester of manoyl oxide (3) was found to exhibit potent cytotoxic effect. Their partitioning into phospholipid bilayers may lead to membrane structure modifications that are crucial in liposome development as they may influence their maintenance and integrity. DSC was used to study the modifications induced in DPPC bilayers by incorporating increasing concentrations of the three manoyl oxide derivatives. All derivatives were found to strongly affect the bilayer structural organization in terms of a decrease of the cooperativity, the fluidization and partially destabilization of the gel phase and the induction of a lateral phase separation in clustering domains. Derivatives 1 and 3 were incorporated into DPPC liposomes and their physicochemical stability was monitored at 4 degrees C. The stability of liposomes was strongly influenced by the presence of 1 and 3 at any molar ratio studied. DPPC/1 liposomes were found to retain its stability for 48 h at low concentration of 10% mol, while at higher concentrations up to 30% mol they collapsed into aggregated material. In all cases DPPC/3 liposomes were found unstable and sticky aggregated structures precipitated from the bulk suspension.