Dynamic modulation of the glycosphingolipid content in supported lipid bilayers by glycolipid transfer protein

Supported lipid bilayers (SLBs) are popular models of cell membranes. Owing to the importance of glycosphingolipids (GSLs) in modulating structure and function of membranes and membrane proteins, methods to tune the GSL content in SLBs would be desirable. Glycolipid transfer protein (GLTP) can selectively transfer GSLs between membrane compartments. Using the ganglioside GM1 as a model GSL, and two mass-sensitive and label-free characterization techniques—quartz crystal microbalance with dissipation monitoring and ellipsometry—we demonstrate that GLTP is an efficient and robust biochemical tool to dynamically modulate the GSL content of SLBs up to 10 mol % GM1, and to quantitatively control the GSL content in the bulk-facing SLB leaflet. By exploiting what we believe to be a novel tool, we provide evidence that GM1 distributes highly asymmetrically in silica-supported lipid bilayers, with ∼85% of the ganglioside being present in the bulk-facing membrane leaflet. We report also that the pentameric B-subunit of cholera toxin binds with close-to-maximal stoichiometry to GM1 in SLBs over a large range of GM1 concentrations. Furthermore, we quantify the liganding affinity of GLTP for GM1 in an SLB context to be 1.5 μM.     

A mechanism of membrane neutral lipid acquisition by the microsomal triglyceride transfer protein

The microsomal triglyceride transfer protein (MTP) and apolipoprotein B (apoB) belong to the vitellogenin (VTG) family of lipid transfer proteins. MTP is essential for the intracellular assembly and secretion of apoB-containing lipoproteins, the key intravascular lipid transport proteins in vertebrates. We report the predicted three-dimensional structure of the C-terminal lipid binding cavity of MTP, modeled on the crystal structure of the lamprey VTG gene product, lipovitellin. The cavity in MTP resembles those found in the intracellular lipid-binding proteins and bactericidal/permeability-increasing protein. Two conserved helices, designated A and B, at the entrance to the MTP cavity mediate lipid acquisition and binding. Helix A (amino acids 725-736) interacts with membranes in a manner similar to viral fusion peptides. Mutation of helix A blocks the interaction of MTP with phospholipid vesicles containing triglyceride and impairs triglyceride binding. Mutations of helix B (amino acids 781-786) and of N780Y, which causes abetalipoproteinemia, have no impact on the interaction of MTP with phospholipid vesicles but impair triglyceride binding. We propose that insertion of helix A into lipid membranes is necessary for the acquisition of neutral lipids and that helix B is required for their transfer to the lipid binding cavity of MTP.     

The synthesis and esterification of cholesterol by hepatocytes and H35 hepatoma cells are independent of the level of nonspecific lipid transfer protein

The level of the nonspecific lipid transfer protein (i.e., sterol carrier protein 2) is 16-fold lower in the Reuber H35 hepatoma cells as compared to the hepatocytes in culture (49 and 810 ng of protein per mg of 105000 X g supernatant protein, respectively). In order to establish whether there is a relationship between the level of nonspecific transfer protein and intracellular cholesterol metabolism, we have determined the biosynthesis and esterification of cholesterol in these hepatoma cells and hepatocytes. Both types of cells incorporated [3H]mevalonate into cholesterol and cholesterol ester. Incubation of both cell types with [3H]cholesterol in the medium resulted in a time-dependent uptake and subsequent conversion into cholesterol ester. In both instances, the amount of 3H label incorporated into cholesterol per mg of cellular protein was about 2-fold higher for the hepatoma cells. The kinetics of esterification of endogenously synthesized cholesterol were similar for both hepatoma cells and hepatocytes. Esterification of cholesterol derived from the medium proceeded 2-times faster in the hepatoma cells than in the hepatocytes. From the kinetics of cholesterol esterification we conclude that cells do not discriminate between cholesterol synthesized de novo and cholesterol derived from the medium. In addition, the proposition that the nonspecific lipid transfer protein is involved in cholesterol synthesis and esterification is not substantiated by this study.     

Kinetics of fluorescent-labeled phosphatidylcholine transfer between nonspecific lipid transfer protein and phospholipid vesicles

Recently, rat liver nonspecific lipid transfer protein (nsLTP) was shown to form a fluorescent complex when allowed to equilibrate with self-quenching vesicles prepared from the fluorescent phospholipid 1-palmitoyl-2-[12-[(7-nitro-2,1,3-benzoxadiazol-4- yl)amino]dodecanoyl]phosphatidylcholine (P-C12-NBD-PC) [Nichols, J. W. (1987) J. Biol. Chem. 262, 14172-14177]. Investigation of the mechanism of complex formation was continued by studying the kinetics of transfer of P-C12-NBD-PC between nsLTP and phospholipid vesicles using a transfer assay based on resonance energy transfer between P-C12-NBD-PC and N-(lissamine rhodamine B sulfonyl)dioleoylphosphatidylethanolamine. The principles of mass action kinetics (which predict initial lipid transfer rates as a function of protein and vesicle concentration) were used to derive equations for two distinct mechanisms: lipid transfer by the diffusion of monomers through the aqueous phase and lipid transfer during nsLTP-membrane collisions. The results of these kinetics studies indicated that the model for neither mechanism alone adequately predicted the initial rates of formation and dissolution of the P-C12-NBD-PC-nsLTP complex. The initial rate kinetics for both processes were predicted best by a model in which monomer diffusion and collision-dependent transfer occur simultaneously. These data support the hypothesis that the phospholipid-nsLTP complex functions as an intermediate in the transfer of phospholipids between membranes.     

Paraquat potentiates iron-induced microsomal lipid peroxidation: modulation by the diet lipid composition

SUMMARY

The study concerns the role of two combined factors—lipid composition of the microsomal membranes and the iron concentration in the incubation medium—in lipid peroxidation catalysed by paraquat (P⁺⁺). Rats were subjected to diets containing 5% lipids composed of either tripalmitin (T), peanut oil/rapeseed oil (v/v) (C) or fish oil (F). The level of polyunsaturated fatty acids in the microsomal membranes was higher in C and F than in T. The level of vitamin E was lowest in F. The activity of the system ‘Cyt P450-NADPH cyt c reductase’ increased in the order T<C<F. The iron concentrations initiating a basal NADPH-dependent lipid peroxidation have been established. p⁺⁺ potentiates this peroxidation due to additional reduction of Fe³⁺ by p^+., rather than by O₂^.- as is usually thought to occur. The sensitivity of the membranes to the potentiating effect of P^{+ +} is mainly determined by a high level of polyunsaturated fatty acids, but also by a low level of the antioxidant vitamin E.     

Control of lipid membrane stability by cholesterol content

Cholesterol has a concentration-dependent effect on membrane organization. It is able to control the membrane permeability by inducing conformational ordering of the lipid chains. A systematic investigation of lipid bilayer permeability is described in the present work. It takes advantage of the transmembrane potential difference modulation induced in vesicles when an external electric field is applied. The magnitude of this modulation is under the control of the membrane electrical permeability. When brought to a critical value by the external field, the membrane potential difference induces a new membrane organization. The membrane is then permeable and prone to solubilized membrane protein back-insertion. This is obtained for an external field strength, which depends on membrane native permeability. This approach was used to study the cholesterol effect on phosphatidylcholine bilayers. Studies have been performed with lipids in gel and in fluid states. When cholesterol is present, it does not affect electropermeabilization and electroinsertion in lipids in the fluid state. When lipids are in the gel state, cholesterol has a dose-dependent effect. When present at 6% (mol/mol), cholesterol prevents electropermeabilization and electroinsertion. When cholesterol is present at more than 12%, electropermeabilization and electroinsertion are obtained under milder field conditions. This is tentatively explained by a cholesterol-induced alteration of the hydrophobic barrier of the bilayer core. Our results indicate that lipid membrane permeability is affected by the cholesterol content.     

Characterization and structural analyses of nonspecific lipid transfer protein 1 from mung bean

Plant nonspecific lipid transfer proteins (nsLTPs) are thermal stable proteins that are capable of transferring lipid molecules between bilayers in vitro. This family of proteins, abundant in plants, is proposed to be involved in defense, pollination, and germination; the in vivo biological function remains, however, elusive. Here we report the purification and sequencing of an nsLTP1 from mung bean sprouts. We have also determined the solution structure of this nsLTP1, which represents the first 3D structure of the dicotyledonous nsLTP1 family. The global fold of mung bean nsLTP1 is similar to those of the monocotyledonous nsLTP1 structures and consists of four alpha-helices stabilized by four disulfide bonds. There are, however, some notable differences in the C-terminal tails and internal hydrophobic cavities. Circular dichroism and fluorescence spectroscopy were used to compare the thermodynamics and lipid transfer properties of mung bean nsLTP1 with those of rice nsLTP1. Docking of a lipid molecule into the solution structure of mung bean nsLTP1 reveals similar binding cavities and hydrophobic interactions as in rice nsLTP1, consistent with their comparable lipid transfer properties measured experimentally.     

Molecular cloning of the anther-preferential nonspecific lipid transfer protein cDNA in hot pepper by mRNA differential display

To better understand the molecular control of anther development, an anther-preferential mRNA was isolated from hot pepper (Capsicum annuum) using mRNA differentially display. Using the displayed fragment as a probe, a full-length cDNA named CaLTP was isolated. A nucleotide sequence analysis of CaLTP revealed that the clone contains an open reading frame of 123 amino acids, which exhibits a 60-23% identity with nonspecific lipid transfer proteins (nsLTP). Northern and RT-PCR analysis of the clone confirmed that CaLTP mRNA was predominant to anther tissues. The basal expression level in the leaves was slightly induced only by abscisic acid (ABA) treatment. Southern analysis reveals that CaLTP is present as a single-copy gene in hot pepper genome. We hypothesize that CaLTP might have an important role in protecting the reproductive tissues from environmental stresses.     

Simplified spectrophotometric assay for microsomal 3-hydroxy-3-methylglutaryl CoA reductase by measurement of coenzyme A

A new assay for 3-hydroxy-3-methylglutaryl CoA reductase (mevalonate:NADP oxidoreductase [acylating CoA], EC 1.1.1.34) is based upon the measurement of released coenzyme A (SH) during the reduction of 3-hydroxy-3-methylglutaryl CoA to mevalonate. Coenzyme A was measured in the presence of dithiothreitol, required for activity, by reaction with 5,5'-dithiobis(2-nitrobenzoic acid). Sodium arsenite forms a complex with the dithiol, but not with monothiols. Thus, reduced coenzyme A reacts instantaneously with the reagent and dithiothreitol reacts slowly. The absorbance due to the coenzyme A-5,5'-dithiobis(2-nitrobenzoic acid) reaction is determined by extrapolating the linear (dithiol) absorbance-time curve to the time of addition of the reagent. After subtraction of control absorbance (deletion of NADPH), the concentration of CoA-SH is calculated from epsilon(max) = 1.36 x 10(4) at 412 nm. The method of protein removal and reduction of sulfhydryl groups on the enzyme are critical. This method provides an immediate assay. Recovery of reduced coenzyme A was 98.7%. The assay is applicable for microsomes or purified enzyme and has an effective range of 0.5-50 nmoles of coenzyme A. It was applied to kinetic measurement of the pigeon liver microsomal enzyme reaction. The apparent K(m) value for 3-hydroxy-3-methylglutaryl CoA was 1.75 x 10(-5) M, and for NADPH the value was 6.81 x 10(-4) M. This method was compared with the dual-label method at high and low levels of activity. The data were not statistically different.     

Cabbage cryoprotectin is a member of the nonspecific plant lipid transfer protein gene family

We have recently purified a protein (cryoprotectin) from the leaves of cold-acclimated cabbage (Brassica oleracea) to electrophoretic homogeneity, which protects thylakoids isolated from the leaves of nonacclimated spinach (Spinacia oleracea) from freeze-thaw damage. Sequencing of cryoprotectin showed the presence of at least three isoforms of WAX9 proteins, which belong to the class of nonspecific lipid transfer proteins. Antibodies raised against two synthetic peptides derived from the WAX9 proteins recognized a band of approximately 10 kD in western blots of crude cryoprotectin preparations. This protein and the cryoprotective activity could be precipitated from solution by the antiserum. We show further that cryoprotectin is structurally and functionally different from WAX9 isolated from the surface wax of cabbage leaves. WAX9 has lipid transfer activity for phosphatidylcholine, but no cryoprotective activity. Cryoprotectin, on the other hand, has cryoprotective, but no lipid transfer activity. The cryoprotective activity of cryoprotectin was strictly dependent on Ca(2+) and Mn(2+) and could be inhibited by chelating agents, whereas the lipid transfer activity of WAX9 was higher in the presence of ethylenediaminetetraacetate than in the presence of Ca(2+) and Mn(2+).     

Glycolipid transfer protein interaction with bilayer vesicles: modulation by changing lipid composition

Glycosphingolipids (GSLs) are important constituents of lipid rafts and caveolae, are essential for the normal development of cells, and are adhesion sites for various infectious agents. One strategy for modulating GSL composition in lipid rafts is to selectively transfer GSL to or from these putative membrane microdomains. Glycolipid transfer protein (GLTP) catalyzes selective intermembrane transfer of GSLs. To enable effective use of GLTP as a tool to modify the glycolipid content of membranes, it is imperative to understand how the membrane regulates GLTP action. In this study, GLTP partitioning to membranes was analyzed by monitoring the fluorescence resonance energy transfer from tryptophans and tyrosines of GLTP to N-(5-dimethyl-aminonaphthalene-1-sulfonyl)-1,2-dihexadecanoyl-sn-glycero-3-phospho-ethanolamine present in bilayer vesicles. GLTP partitioned to POPC vesicles even when no GSL was present. GLTP interaction with model membranes was nonpenetrating, as assessed by protein-induced changes in lipid monolayer surface pressure, and nonperturbing in that neither membrane fluidity nor order were affected, as monitored by anisotropy of 1,6-diphenyl-1,3,5-hexatriene and 6-dodecanoyl-N,N-dimethyl-2-naphthylamine, even though the tryptophan anisotropy of GLTP increased in the presence of vesicles. Ionic strength, vesicle packing, and vesicle lipid composition affected GLTP partitioning to the membrane and led to the following conclusion: Conditions that increase the ratio of bound/unbound GLTP do not guarantee increased transfer activity, but conditions that decrease the ratio of bound/unbound GLTP always diminish transfer. A model of GLTP interaction with the membrane, based on the partitioning equilibrium data and consistent with the kinetics of GSL transfer, is presented and solved mathematically.     

Dietary oxidized cholesterol decreases expression of hepatic microsomal triglyceride transfer protein in rats

The aim of this study was to compare the effects of dietary oxidized cholesterol and pure cholesterol on plasma and very low density lipoprotein (VLDL) lipids and on some parameters of VLDL assembly and secretion in rats fed two different dietary fats. Four groups of male growing Sprague-Dawley rats were fed diets containing pure or oxidized cholesterol (5 g/kg diet) with either coconut oil or salmon oil as dietary fat (100 g/kg diet) for 35 days. Rats fed oxidized cholesterol supplemented diets had significantly lower concentrations of triglycerides and cholesterol in plasma and VLDL than rats fed pure cholesterol supplemented diets irrespective of the type of fat. In addition, rats fed oxidized cholesterol supplemented diets had significantly lower relative concentrations of microsomal triglyceride transfer protein messenger ribonucleic acid (mRNA) than rats fed pure cholesterol supplemented diets. In contrast, hepatic lipid concentrations and the relative concentration of apolipoprotein B mRNA were not influenced by the dietary factors investigated. Parameters of hepatic lipogenesis (relative mRNA concentration of sterol regulatory element binding protein-1c and activity of glucose-6-phosphat dehydrogenase) were significantly reduced by feeding fish oil compared to coconut oil, but were not affected by the type of cholesterol. In conclusion, the data of this study suggest, that dietary oxidized cholesterol affects VLDL assembly and/or secretion by reducing the synthesis of MTP but not by impairing hepatic lipogenesis or synthesis of apolipoprotein B.     

Sphingolipids and cellular cholesterol homeostasis. Effect of ceramide on cholesterol trafficking and HMG CoA reductase activity

We previously showed that degradation of cellular sphingomyelin (SM) by SMase C results in a greater stimulation of cholesterol translocation to endoplasmic reticulum, compared to its degradation by SMase D. Here we investigated the hypothesis that the effect of SMase C is partly due to the generation of ceramide, rather than due to depletion of SM alone. Inhibition of hydroxymethylglutaryl CoA reductase (HMGCR) activity was used as a measure of cholesterol translocation. Treatment of fibroblasts with SMase C resulted in a 90% inhibition of HMGCR, whereas SMase D treatment inhibited it by 29%. Treatment with exogenous ceramides, or increasing the endogenous ceramide levels also inhibited HMGCR by 60-80%. Phosphorylation of HMGCR was stimulated by SMase C or exogenous ceramide. The effects of ceramide and SMase D were additive, indicating the independent effects of SM depletion and ceramide generation. These results show that ceramide regulates sterol trafficking independent of cellular SM levels.     

Refined solution structure of a liganded type 2 wheat nonspecific lipid transfer protein

The refined structure of a wheat type 2 nonspecific lipid transfer protein (ns-LTP2) liganded with l-alpha-palmitoylphosphatidylglycerol has been determined by NMR. The (15)N-labeled protein was produced in Pichia pastoris. Physicochemical conditions and ligandation were intensively screened to obtain the best NMR spectra quality. This ns-LTP2 is a 67-residue globular protein with a diameter of about 30 A. The structure is composed of five helices forming a right superhelix. The protein presents an inner cavity, which has been measured at 341 A(3). All of the helices display hydrophobic side chains oriented toward the cavity. The phospholipid is found in this cavity. Its fatty acid chain is completely inserted in the protein, the l-alpha-palmitoylphosphatidylglycerol glycerol moiety being located on a positively charged pocket on the surface of the protein. The superhelix structure of the protein is coiled around the fatty acid chain. The overall structure shows similarities with ns-LTP1. Nevertheless, large three-dimensional structural discrepancies are observed for the H3 and H4 alpha-helices, the C-terminal region, and the last turn of the H2 helix. The lipid is orthogonal to the orientation observed in ns-LTP1. The volume of the hydrophobic cavity appears to be in the same range as the one of ns-LTP1, despite the fact that ns-LTP2 is shorter by 24 residues.     

Free cholesterol is a potent regulator of lipid transfer protein function

This study investigates the effect of altered lipoprotein free cholesterol (FC) content on the transfer of cholesteryl ester (CE) and triglyceride (TG) from very low- (VLDL), low- (LDL), and high-(HDL) density lipoproteins by the plasma-derived lipid transfer protein (LTP). The FC content of VLDL and HDL was selectively altered by incubating these lipoproteins with FC/phospholipid dispersions of varying composition. FC-modified lipoproteins were then equilibrated with [3H] TG, [14C]CE-labeled lipoproteins of another class to facilitate the subsequent modification of the radiolabeled donor lipoproteins. LTP was added and the extent of radiolabeled TG and CE transfer determined after 1 h. With either LDL or VLDL as lipid donor, an increase in the FC content of these lipoproteins caused a concentration-dependent inhibition (up to 50%) of CE transfer from these particles, without any significant effect on TG transfer. In contrast, with HDL as donor, increasing the HDL FC content had little effect on CE transfer from HDL, but markedly stimulated (up to 2.5-fold) the transfer of TG. This differential effect of FC on the unidirectional transfer of radiolabeled lipids from VLDL and HDL led to marked effects on LTP-facilitated net mass transfer of lipids. During long-term incubation of a constant amount of LTP with FC-modified VLDL and HDL, the extent of net mass transfer was linearly related to lipoprotein FC content; a 4-fold increase in FC content resulted in a 3-fold stimulation of the CE mass transferred to VLDL, which was coupled to an equimolar, reciprocal transfer of TG mass to HDL. Since lipid transfer between lipoproteins is integral to the process of reverse cholesterol transport, we conclude that lipoprotein FC levels are a potent, positive regulator of the pathways involved in sterol clearance. FC may modulate lipid transfer by altering the availability of CE and TG to LTP at the lipoprotein surface.     

Hepatic cholesterol synthesis and hydroxymethylglutaryl CoA reductase activity after injection of methylazoxymethanol acetate

Cholesterol synthesis and 3-hydroxy-3-methylglutaryl CoA reductase (HMG-CoA reductase) in the liver of rats at various times (7, 22, 45 and 314 days) after injection with the carcinogen, methylazoxymethanol acetate (MAMA) is reported. Seven days after treatment, an increase in both cholesterol synthesis and HMG-CoA reductase activity was observed. Elevated HMG-CoA reductase activity and reduced dietary feedback was present 22 days after carcinogen. Cholesterol synthesis was normal at this time but dietary cholesterol failed to significantly reduce synthesis. Forty-five days after carcinogen both cholesterol synthesis and HMG-CoA reductase activity had returned to normal. Both parameters were normal 314 days after carcinogen. The enzyme gamma-glutamyl transferase was also elevated at 7, 22 and 314 days. These results indicate that HMG-CoA reductase activity and cholesterol synthesis exhibit different regulatory characteristics during the early stages of hepatocarcinogenesis initiated by MAMA injection.