The mevalonate pathway in the bloodstream form of Trypanosoma brucei. Identification of dolichols containing 11 and 12 isoprene residues

The major surface antigen of the bloodstream form of Trypanosoma brucei, the variant surface glycoprotein, is attached to the plasma membrane via a glycosylphosphatidylinositol anchor. The biosynthesis of the glycosylphosphatidylinositol anchor, as well as the assembly of the asparagine-linked oligosaccharide chains found on the variant surface glycoproteins, involves polyisoprenoid lipids that act as sugar carriers. Preliminary observations (Menon, A.K., Schwarz, R.T., Mayor, and Cross, G.A.M. (1990) J. Biol. Chem. 265, 9033-9042) suggested that the sugar carriers in T. brucei were short-chain polyisoprenoids containing substantially fewer isoprene residues than polyisoprenols in mammalian cells. In this paper we describe metabolic labeling experiments with [3H]mevalonate, as well as chromatographic and mass spectrometric analyses of products of the mevalonate pathway in T. brucei. We report that cells of the bloodstream form of T. brucei contain a limited spectrum of short chain dolichols and dolichol phosphates (11 and 12 isoprene residues). The total dolichol content was estimated to be 0.28 nmol/10(9) cells; the dolichyl phosphate content was 0.07 nmol/10(9) cells. The same spectrum of dolichol chain lengths was also found in a polar lipid that could be labeled with [3H]mevalonate, [3H]glucosamine, and [3H]mannose, and which was characterized as Man5GlcNAc2-PP-dolichol. The most abundant product of the mevalonate pathway identified in T. brucei was cholesterol (140 nmol/10(9) cells). Ubiquinone (0.09 nmol/10(9) cells) with a solanesol side chain was also identified.     

Involvement of sterol carrier protein-2 in dolichol biosynthesis.

The effect of sterol carrier protein-2 (SCP-2) on dolichol biosynthesis by rat liver microsomes was investigated. cis-Prenyltransferase activity was stimulated 7-fold in the presence of 5 micrograms of purified SCP-2/mg of microsomal protein, which was similar to the increase obtained by adding detergent. The polyisoprenoid pattern obtained in the presence of SCP-2 was the same as that present in rat liver, in contrast to the pattern appearing upon incubation of microsomes with detergent, which gave shorter polyisoprenoids. Like SCP-2, the cytosolic fraction from rat liver also stimulated cis-prenyltransferase. Incubation with cytosol pretreated with anti-SCP-2 showed no stimulatory effect and led to the accumulation of shorter polyisoprenoids. SCP-2 had no appreciable effect on polyprenol alpha-saturase, dolichol kinase, dolichyl phosphate phosphatase, or acyl-CoA:dolichol acyltransferase. The results demonstrate that SCP-2 greatly stimulates and may regulate the condensation reactions mediated by cis-prenyltransferase in the process of dolichol biosynthesis and permits polymerization of the polyisoprenoid to its natural chain length.     

Dolichol induces membrane leakage of liposomes composed of phosphatidylethanolamine and phosphatidylcholine

Dolichol promotes the leakage of membranes in liposomes composed of phosphatidylethanolamine and phosphatidylcholine but not liposomes composed only of phosphatidylcholine. The membrane leakage was assayed by measuring the entrapment of TEMPOcholine, a cationic spin probe, in liposomes using ESR methods. The percent of membrane leakage induced by dolichol was found to be linearly proportional to the concentrations of dolichol. It is proposed that dolichol enhances the formation of non-bilayer configurations in liposomes containing phosphatidylethanolamine, thereby membrane leakage.     

Cationic polypeptide-induced fusion of acidic liposomes

Fusion of acidic liposomes was induced by Mg²⁺, Ca²⁺, polylysine and polymyxin B. The extent of fusion and the concomitant change in liposome permeability induced by divalent cations depended on the concentration of liposomes in the suspension as well as on the cation concentration. In contradistinction, the extent of fusion and the change in permeability induced by the polypeptides depended only on the polycation concentration. The difference in the pattern of interaction, between the liposomes and the various cations, is a result of different binding affinities. The binding of the polypeptides to the liposomes, in contrast to divalent cations, is practically irreversible. The potential of polylysine to induce fusion of acidic phosphatidylethanolamine-devoid liposomes was used to demonstrate that in order to obtain fusion, both membranes involved must be susceptible, at least to a certain degree, to fusion by the proper inducer. When lysophosphatidylcholine substituted for phosphatidylcholine in phosphatidylethanolamine-rich acidic liposomes, extensive polylysine-induced fusion was obtained without concomitant spillage of the liposome contents.     

Lateral diffusion in model membranes is independent of the size of the hydrophobic region of molecules.

We have systematically investigated the probe size and shape dependence of lateral diffusion in model dimyristoyl phosphatidylcholine membranes. Linear hydrophobic polymers, which differ in length by an order of magnitude, were used to explore the effect on the lateral diffusion coefficient of hydrodynamic restrictions in the bilayer interior. The polymers employed are isoprenoid alcohols--citronellol, solanesol, and dolichol. Tracer lateral diffusion coefficients were measured by fluorescence photobleaching recovery. Despite the large difference in lengths, the nitrobenzoxadiazole labelled alcohols all diffuse at the rate of lipid self-diffusion (5.0 x 10(-12) m2 s-1, 29 degrees C) in the liquid crystal phase. Companion measurements in isotropic polymer solution, in gel phase lipid membranes and with nonpolar fluorescent polyaromatic hydrocarbons, show a marked dependence of the lateral diffusion coefficient on the probe molecule size. Our results in the liquid crystal phase are in accord with free area theory which asserts that lateral diffusion in the membrane is restricted by the surface-free area. Probe molecules which are significantly longer than the host phospholipid, seven times longer in the case of dolichol, are still restricted in their lateral motion by the surface properties of the bilayer in the liquid crystal phase. Fluorescence quenching experiments indicate that the nitrobenzoxadiazole label does not reside at the aqueous interface, although it must reside in close proximity according to the diffusion measurements.     

The glucose transport activity of human erythrocyte membranes. Reconstitution in phospholipid liposomes and fractionation by molecular sieve and ion exchange chromatography

Human erythrocyte membranes, at a protein concentration of 1–2 g/l, were solubilized with 0.12 M cholate in the presence of 0.06 M phospholipid (egg yolk phospholipids or phosphatidylcholine). More than 40% of the protein was solubilized. Cholate was removed by molecular sieve chromatography, whereby liposomes formed. These liposomes exchanged D-glucose faster than L-glucose. The recovery of glucose transport activity in the reconstituted system was estimated to be higher than 16%.The liposomes were heterogeneous in size, as shown by molecular sieve chromatography on Sepharose 4B, and small liposomes predominated. In liposomes formed with phosphatidylcholine, the distribution of glucose transport activity did not parallel the distribution of protein or phospholipid, and the activity was found mainly in the smallest liposomes. The proteins were incorporated mainly into the liposomes that eluted at the lowest ionic strength upon ion exchange chromatography.The glucose transport activity separated into three main peaks upon ion exchange chromatography of egg yolk phospholipid liposomes. The activity eluted at low ionic strength. The liposomes contained proteins mainly from the 3- and 4.5-regions (nomenclature according to Steck, T.L. (1974) J. Cell Biol. 62, 1–19). The activity peaks were highest in the first part of the chromatogram. The protein distribution did not coincide with the variation in activity over each peak. Therefore, it cannot be excluded that a minor component not seen in the electrophoretic analyses might be responsible for the glucose transport activity.     

Liposomal membranes. VII. Fusion and aggregation of egg lecithin liposomes as promoted by polysaccharides

Pullulan, one of polysaccharides, induces aggregation of egg lecithin multilamellar liposomes as detected by turbidity of the solution. The turbidity disappears upon treatment with pullulanase, indicating that the aggregation induced by pullulan is reversible. On the other hand, the turbidity induced on single-walled liposomes is not completely reversed by incubation with the enzyme. The phenomenon is interpreted as the involvement of irreversible fusion of liposomes. Thus, the enzyme digestion method provides a novel simple means to distinguish fusion of liposomes from aggregation.     

Protein dolichylation in Plasmodium falciparum

We performed reverse-phase thin-layer chromatography and reverse-phase high-performance liquid chromatography (RP-HPLC) analysis of polyisoprenoids released by sulfonium-salt cleavage with methyl iodide from Plasmodium falciparum proteins labeled with [3H]FPP or [3H]GGPP and showed that a dolichol of 11 isoprene units is bound to 21-28-kDa protein clusters from trophozoite and schizont stages. The dolichol structure was confirmed by electrospray-ionization mass spectrometry analysis. Treatment with protein synthesis inhibitors and RP-HPLC analysis of the proteolytic digestion products from parasite proteins labeled with [35S]cysteine and [3H]FPP showed that the attachment of dolichol to protein is a post-translational event and probably occurs via a covalent bond to cysteine residues.     

Interaction of dolichol and dolichyl phosphate with phospholipid bilayers

The thermotropic phase transition of dipalmitoylphosphatidylcholine vesicles reconstituted with dolichol or dolichyl phosphate was investigated as a function of the lipid-to-polyisoprenoid ratio by means of differential scanning calorimetry and fluorescence depolarization of the embedded probe 1,6-diphenyl-1,3,5-hexatriene. At the concentrations studied, dolichol and dolichyl phosphate lowered and broadened the transition temperature of dipalmitoylphosphatidylcholine bilayers. Dolichol was found to increase the motional freedom of the bilayer both below and above the transition temperature as determined by fluorescence depolarization. In contrast, low concentrations of dolichyl phosphate decreased the bilayer motional freedom below the transition temperature while high concentrations increased the motional freedom. Above the transition temperature, dolichyl phosphate decreased bilayer 'fluidity' at all concentrations. The data suggest that these polyisoprenoids perturb the bilayer lattice, with the neutral species dolichol increasing membrane 'fluidity', while dolichyl phosphate acts to 'stiffen' the membrane.     

Sugar availability modulates polyisoprenoid and phytosterol profiles in Arabidopsis thaliana hairy root culture

Sugars are recognized as signaling molecules regulating the biosynthesis of secondary metabolites in plants. Here, a modulatory effect of sugars on dolichol and phytosterol profiles was noted in the hairy roots of Arabidopsis thaliana. Arabidopsis roots contain a complex dolichol mixture comprising three groups (‘families’) of dolichols differing in the chain-length. These dolichols, especially the longest ones are accompanied by considerable amounts of polyprenols of the same length. The spectrum of polyisoprenoid alcohols, i.e. dolichols and polyprenols, was dependent on sugar type (glucose or sucrose) and its concentration in the medium. Among the long-chain dolichols Dol/Pren-20 (dolichol or prenol molecule composed of 20 isoprene residues) and Dol/Pren-23 were the main components at 0.5% and 2% glucose, respectively. Moreover, the ratio of polyprenols versus respective dolichols was also modulated by sugar in this group of polyisoprenoids, with polyprenols dominating at 3% sucrose and dolichols at 2% glucose. Glucose concentration affected the expression level of genes encoding cis-prenyltransferases, enzymes responsible for elongation of the polyisoprenoid chain. The most abundant phytosterols of the A. thaliana roots, β-sitosterol, stigmasterol and campesterol, were accompanied by corresponding stanols and traces of brassicasterol, stigmast-4,22-dien-3-one and stigmast-4-en-3-one. Similar to the polyisoprenoids, sterol profile responded to the sugar present in the medium, β-sitosterol dominating in roots grown on 3% or lower glucose concentrations and stigmasterol in 3% sucrose. These results indicate an involvement of sugar signaling in the regulation of cis-prenyltransferases and phytosterol pathway enzymes.     

A fluorescent sterol probe study of cholesterol/phospholipid membranes

The behavior of dehydroergosterol in -α-dimyristoylphosphatidylcholine (DMPC) unsonicated multilamellar liposomes was characterized by absorption spectroscopy and fluorescence measurements. Dehydroergosterol exhibited a lowered absorption coefficient in multilamellar liposomes whiel the steady-state fluorescence anisotropy of dehydroergosterol in these membranes decreased significantly with increasing dehydroergosterol concentration, suggesting membrane sterol-sterol interactions. The comparative steady-state anisotropy of 0.9 mole percent dehydroergosterol in multilamellar liposomes was lower than in small unilamellar vesicles suggesting different sterol environments for dehydroergosterol. Dehydroergosterol fluorescence lifetime was relatively independent of membrane sterol content and yielded similar values in sonicated and unsonicated model membranes. In multilamellar liposomes containing 5 mole percent cholesterol, the gel-to-liqui crystalline phase transition of DMPC detected by 0.9 mole percent dehydroergosterol was significantly broadened when compared to the phase transition detected by dehydroergosterol in the absence of membrane cholesterol (Smutzer, G. et al. (1986) Biochim. Biophys. Acta 862, 361–371). In multilamellar liposomes containing 10 mole percent cholesterol, the major fluorescence lifetime of dehydroergosterol did not detect the gel-to-liquid crystalline phase transition of DMPC. Time-correlated fluorescence anisotropy decays of dehydroergosterol in DMPC multilamellar liposomes in the absence and presence of 5 mole percent cholesterol exhibited a single rotational correlation time near one nanosecond that was relatively independent of temperature and low concentrations of membrane cholesterol. The limiting anisotropy of 0.9 mole percent dehydroergosterol decreased above the gel-to-liquid crystalline phase transition in membranes without cholesterol and was not significantly affected by the phase transition in membranes containing 5 mole percent cholesterol. These results suggested hindered rotational diffusion of dehydroergosterol in multilamellar liposomes. Lifetime and time-correlated fluorescence measurements of 0.9 mole percent dehydroergosterol in multilamellar liposomes further suggested this fluorophore was detecting physical properties of the bulk membrane phospholipids in membranes devoid of cholesterol and was detecting sterol-rich regions in membranes of low sterol concentration.     

Nuclear magnetic resonance studies of polyisoprenols in model membranes

2H-NMR investigation of polyisoprenols (PIs) in model membranes has revealed information about their motions, relative order, and locale within the membrane. Initial 2H-NMR studies of the organization of the shorter chain homologues geraniol (C10), farnesol (C15), and solanesol (C45) were carried out by incorporating 2H-acetyl esters of the alcohol or the di-perdeuterome-thylated derivatives of the omega-labeled prenols into multilamellar phosphatidylcholine (PC) vesicles. 2H-NMR powder patterns interpretable in terms of quadrupole splittings and spin-lattice relaxation times were obtained. Similar experiments have now been carried out with the labeled free alcohol, acetyl ester, and phosphate ester of dolichol (C95) and undecaprenol (C55). 2H-NMR results show that the head and tail 2H-labeled sites of C55 and C95 exhibit a fast motion isotropic signal only; no slower motion anisotropy, as exhibited by the short chain PIs, was observed. These data suggest that C55 and C95 either have substantially different (faster) motions and/or conformations relative to the shorter chain PIs within the membrane, and that the longer PIs alter the membrane host packing matrix. This conclusion was supported by 31P-NMR studies of C55 and C95 derivatives in PC and PE/PC membranes, which showed new pronounced spectral changes relative to the results obtained with the shorter chain PIs. These spectral changes indicate that undecaprenol and dolichol derivatives appear to induce a non-bilayer (isotropic) organization of phospholipid molecules in PE/PC (2:1) vesicles. The possible physiological consequences of this perturbation remains to be determined.     

Latrotoxin-induced fusion of liposomes with bilayer phospholipid membranes

Liposomes containing amphotericin B as ionophoric marker were used to investigate the fusion of bilayer phospholipid membranes with liposomes. It was found that latrotoxin isolated from black widow spider venom induced the fusion of liposomes with planar bilayer when liposomes and latrotoxin were administered at opposite sides of the membrane.     

Dolichol biosynthesis and its effects on the unfolded protein response and abiotic stress resistance in Arabidopsis

Dolichols are long-chain unsaturated polyisoprenoids with multiple cellular functions, such as serving as lipid carriers of sugars used for protein glycosylation, which affects protein trafficking in the endoplasmic reticulum. The biological functions of dolichols in plants are largely unknown. We isolated an Arabidopsis thaliana mutant, lew1 (for leaf wilting1), that showed a leaf-wilting phenotype under normal growth conditions. LEW1 encoded a cis-prenyltransferase, which when expressed in Escherichia coli catalyzed the formation of dolichol with a chain length around C(80) in an in vitro assay. The lew1 mutation reduced the total plant content of main dolichols by approximately 85% and caused protein glycosylation defects. The mutation also impaired plasma membrane integrity, causing electrolyte leakage, lower turgor, reduced stomatal conductance, and increased drought resistance. Interestingly, drought stress in the lew1 mutant induced higher expression of the unfolded protein response pathway genes BINDING PROTEIN and BASIC DOMAIN/LEUCINE ZIPPER60 as well as earlier expression of the stress-responsive genes RD29A and COR47. The lew1 mutant was more sensitive to dark treatment, but this dark sensitivity was suppressed by drought treatment. Our data suggest that LEW1 catalyzes dolichol biosynthesis and that dolichol is important for plant responses to endoplasmic reticulum stress, drought, and dark-induced senescence in Arabidopsis.     

The Role of the Target Membrane Structure in Fusion with Sendai Virus

Fusion between membranes of Sendai virus and liposomes or human erythrocytes ghosts was studied using an assay for lipid mixing based on the relief of self-quenching of octadecylrhodamine (R18) fluorescence. We considered only viral fusion that reflects the biological activity of the viral spike glycoproteins. The liposomes were made of phosphatidylcholine, and the effects of including cholesterol, the sialoglycolipid GD1a, and/or the sialoglycoprotein glycophorin as receptors were tested. Binding of Sendai virus to those liposomes at 37 ?C was very weak. Fusion with the erythrocyte membranes occurred at a 30-fold faster rate than with the liposomes. Experiments with biological and liposomal targets of different size indicated that size did not account for differences in fusion efficiency.     

The influence of dolichols on fluidity of mouse synaptic plasma membranes

Dolichols are isoprenologues which constitute an important component of biological membranes. However, an understanding of the effects of dolichols on the organization and dynamics of biological membranes has not been forthcoming. The experiments reported here are aimed at understanding the effects of dolichols on the physical properties of mouse brain synaptic plasma membranes. The effect of dolichols incorporated into mouse brain synaptic plasma membranes on fluorescent and electron spin resonance probes sensing the hydrophobic core differed from that of probes reporting closer to the surface of membrane bilayers. Dolichols significantly (P less than 0.01) lowered the polarization, limiting anisotropy, and order parameter of diphenylhexatriene in synaptic plasma membranes and liposomes extracted from synaptic plasma membranes, without changing the rotational relaxation time. Similarly, dolichol increased the fluidity reported by 16-doxylstearic acid in synaptic plasma membranes or liposomes extracted from synaptic plasma membranes. In contrast, dolichols exerted no effect on those properties for trans-parinaric acid or 5-doxylstearic acid in synaptic plasma membranes or liposomes derived therefrom. Dolichols can dramatically alter the structure and dynamics of lipid motion in synaptic plasma membranes and these effects are dependent on the location of the probe in the membrane.     

Induction of apoptosis in macrophages by cationic liposomes

The effects of liposomes on apoptosis in macrophages were evaluated from DNA content and DNA fragmentation. Cationic liposomes composed of different kinds of cationic lipids induced apoptosis in mouse splenic macrophages and the macrophage-like cell line, RAW264.7 cells. Generation of reactive oxygen radicals from macrophages treated with cationic liposomes was detected using flow cytometry, and further apoptosis was inhibited by the addition of oxidant scavenger, N-acetylcysteine. From these findings, the production of reactive oxygen species may be important in the regulation of apoptosis induced by cationic liposomes.     

Enhancement of immune responses by DNA vaccination through targeted gene delivery using mannosylated cationic liposome formulations following intravenous administration in mice

The present study investigated the potency of the mannosylated cationic liposomes (Man liposomes) that we have developed in novel DNA vaccine carrier. Ovalbumin (OVA) was selected as a model antigen for vaccination; accordingly, OVA-encoding pDNA (pCMV-OVA) was constructed to evaluate DNA vaccination. The potency of the Man liposome/pCMV-OVA complex was compared with naked pCMV-OVA and that complexed with DC-Chol liposomes. In cultured mouse peritoneal macrophages, MHC class I-restricted antigen presentation of the Man liposome/pCMV-OVA complex was significantly higher than that of naked pCMV-OVA and that complexed with DC-Chol liposomes. After intravenous administration, OVA mRNA expression and MHC class I-restricted antigen presentation on CD11c+ cells and inflammatory cytokines, such as TNF-alpha, IL-12, and IFN-gamma, that can enhance the Th1 response of the Man liposome/pCMV-OVA complex were higher than that of naked pCMV-OVA and that complexed with DC-Chol liposomes. Also, the spleen cells from mice immunized by intravenous administration of the Man liposome/pCMV-OVA complex showed the highest proliferation response and IFN-gamma secretion. These findings suggest that the targeted delivery of DNA vaccine by Man liposomes is a potent vaccination method for DNA vaccine therapy.     

Transfer of cholesterol between liposomal membranes.

The transfer of cholesterol between liposomal membranes was examined. On incubation of liposomes compsoed of egg yolk phosphatidylcholine, phosphatidic acid and cholesterol (molar percentage, 65.8 : 1.3 : 32.9 or 65.5 : 6.3 : 31.2), almost complete equilibration of the cholesterol pools was achieved within 6 to 8 h at 37 degrees C. The rate of transfer of cholesterol from the liposomes, in which cholesterol was introduced by 'the exchange reaction', was not significantly different from that from liposomes prepared in the presence of cholesterol, in which the cholesterol was distributed homogenously. These findings indicate that half life for 'flip-flop' of cholesterol molecules in egg yolk phosphatidylcholine liposomes is less than 6 h at 37 degrees C. The transfer of cholesterol between liposomes was strongly dependent on temperature and was affected by the fatty acid composition of the phospholipid, suggesting that the 'fluidity' of the membranes strongly influences the transfer rate. A preferential distribution of cholesterol molecules was observed in heterogeneous liposomes with different classes of phospholipids. The 'affinity order' of cholesterol for phospholipid deduced from the present experiments is as follows: beef brain sphingomyelin greater than dipalmitoylglycerophosphocholine = dimyristoylglycerophosphocholine greater than egg yolk phosphatidylcholine.     

Rescue of retroviral envelope fusion deficiencies by cationic liposomes

Background

Retroviral particles that are inappropriately enveloped can transduce target cells if pre‐associated with cationic liposomes. This study optimises and addresses the mechanism of liposome‐enhanced gene delivery, and explores the potential for such agents to compensate for fusion deficiency associated with chimaeric envelope proteins.

Methods

Particles bearing wild‐type, chimaeric or no envelope proteins were complexed with DOTAP or DC‐Chol/DOPE cationic liposomes and added to target cells for various times. Particle binding was determined by detection of cell‐associated capsid protein and infectivity was measured histochemically.

Results

Stable association of cationic liposomes with retrovirus particles significantly enhanced their binding rate to target cells in proportion to the increase of transduction kinetics for infectious virus. Binding of virus was equivalent with or without envelope protein and/or virus receptor, indicating that a non‐specific interaction precedes receptor recognition. Non‐infectious combinations were rescued by the intrinsic fusogenicity of the cationic liposomes, which enabled entry of the viral core, but left subsequent events unaltered. The optimised transduction rate with non‐enveloped particles and DOTAP approached that of amphotropic‐enveloped virus in some cases, although the effect was target‐cell‐dependent. DC‐Chol/DOPE was less potent at direct fusion but was able to enhance 600‐fold the receptor‐dependent action of chimaeric envelopes that were deficient in fusion by virtue of the addition of targeting domains.

Conclusions

These data have implications for the development of retroviral vector targeting strategies from the perspectives of the specificity of target cell interaction and compensating for chimaeric envelope fusion deficiency. Copyright © 2002 John Wiley & Sons, Ltd.