Schistosoma mansoni: Phospholipid nature of the “agglutinin”

The haemagglutinating activity of the membrane-associated Schistosoma mansoni “agglutinin” is mainly due to acidic phospholipids, particularly phosphatidylinositol and phosphatidylserine. The role of these phospholipids in possible lipid-protein interactions in the host-parasite relationship is discussed.     

The plasma membrane lipid composition affects fusion between cells and model membranes

Investigations were carried out on the effect of plasma membrane lipid modifications on the fusogenic capacity of control and ras-transformed fibroblasts. The plasma membrane lipid composition was modified by treatment of cells with exogenous phospholipases C and D, sphingomyelinase and cyclodextrin. The used enzymes hydrolyzed definite membrane lipids thus inducing specific modifications of the lipid composition while cyclodextrin treatment reduced significantly the level of cholesterol. The cells with modified membranes were used for assessment of their fusogenic capacity with model membranes with a constant lipid composition. Treatment with phospholipases C and D stimulated the fusogenic potential of both cell lines whereas the specific reduction of either sphingomyelin or cholesterol induced the opposite effect. The results showed that all modifications of the plasma membrane lipid composition affected the fusogenic capacity irrespective of the initial differences in the membrane lipid composition of the two cell lines. These results support the notion that the lipid composition plays a significant role in the processes of membrane-membrane fusion. This role could be either direct or through modulation of the activity of specific proteins which regulate membrane fusion.     

Expression cloning of cholesterol alpha-glucosyltransferase, a unique enzyme that can be inhibited by natural antibiotic gastric mucin O-glycans, from Helicobacter pylori

Helicobacter pylori infects over half the world's population, but only 3% of those infected develop peptic ulcer, gastric cancer, and mucosa-associated lymphoid tissue (MALT) lymphoma. In H. pylori, alpha-glucosyl cholesterol constitutes more than 25% of cell wall lipids, and it has been suggested that alpha-glucosyl cholesterol is essential for H. pylori viability. Here, we identified cholesterol alpha-glucosyltransferase (CHLalphaGcT) using an expression cloning strategy and showed that this enzyme is distinctively inhibited by mucin-type O-glycans similar to those present in deeper portions of the gastric mucosa. Moreover, inactivation of CHLalphaGcT by homologous recombination led to H. pylori lethality. These results indicate that H. pylori CHLalphaGcT is a unique enzyme targeted by a natural antibiotic mucin and constitutes an excellent therapeutic target to prevent H. pylori-induced peptic ulcer, gastric carcinoma, and MALT lymphoma.     

The proton gradient of secretory granules and glutamate transport in blood platelets during cholesterol depletion of the plasma membrane by methyl-β-cyclodextrin

Glutamate transport in blood platelets resembles that in brain nerve terminals because platelets contain neuronal Na⁺-dependent glutamate transporters, glutamate receptors in the plasma membrane, vesicular glutamate transporters in secretory granules, which use the proton gradient as a driving force, and can release glutamate during aggregation/activation. The acidification of secretory granules and glutamate transport were assessed during acute treatment of isolated platelets with cholesterol-depleting agent methyl-β-cyclodextrin (MβCD). Confocal imaging with the cholesterol-sensitive fluorescent dye filipin showed a quick reduction of cholesterol level in platelets. Using pH-sensitive fluorescent dye acridine orange, we demonstrated that the acidification of secretory granules of human and rabbit platelets was decreased by ∼15% and 51% after the addition of 5 and 15 mM MβCD, respectively. The enrichment of platelet plasma membrane with cholesterol by the application of complex MβCD-cholesterol (1:0.2) led to the additional accumulation of acridine orange in secretory granules indicating an increase in the proton pumping activity of vesicular H⁺-ATPase. MβCD did not evoke release of glutamate from platelets that was measured with glutamate dehydrogenase assay. Flow cytometric analysis did not reveal alterations in platelet size and granularity in the presence of MβCD. These data showed that the dissipation of the proton gradient of secretory granules rather than their exocytosis caused MβCD-evoked decrease in platelet acidification. Thus, the depletion of plasma membrane cholesterol in the presence of MβCD changed the functional state of platelets affecting storage capacity of secretory granules but did not evoke glutamate release from platelets.     

Evidence for the Presence of "Metabolic Sterols" in Pneumocystis: Identification and Initial Characterization of Pneumocystis carinii Sterols

Mixed life cycle stages of rat-derived Pneumocystis carinii were isolated from host lungs and their sterols were compared with those present in lungs from normal and immunosuppressed uninfected rats. Gas-liquid chromatography consistently detected, resolved, and quantified 9, 10, and 20 sterol components in the total nonsaponifiable neutral lipid fraction of lungs from normal rats, lungs from immunosuppressed uninfected rats, and P. carinii preparations, respectively. In all samples, cholesterol was the most abundant sterol present, comprising 97%, 93%, and 78% of total sterols in lungs from normal rats, lungs from immunosuppressed uninfected rats, and P. carinii , respectively. Tentative identifications of several rat lung and P. carinii minor sterols were made based on gas-liquid chromatogram retention times and fragmentation patterns from mass spectral analyses. Campesterol (ergost-5-en-3-ol), cholest-5-en-3-one, and β -sitosterol (stigmast-5-en-3-ol) were among the minor components present in both types of lung controls, and were also components of P. carinii sterols. In contrast to lung controls, the sterols of P. carinii were enriched in C₂₈ and C₂₉ sterols with one or two double bonds, and a hydroxyl group at C-3 (ergost-5-en-3-ol, ergost-7-en-3-ol, ergosta-dien-3-ol, stigmast-5-en-3-ol, stigmast-7-en-3-ol and stigmasta-dien-3-ol). Steryl esters of P. carinii , probably stored in cytoplasmic lipid droplets, were dominated by those present in the host lung. In separate studies. 3-hydroxy-3-methylglutaryl coenzyme A activity, a key enzyme in the regulation of sterol biosynthesis, was detected in purified P. carinii preparations and incorporation of radiolabeled squalene and mevalonate was observed. Together, these results suggest that the parasite readily takes up and incorporates host sterols, and that the organism synthesizes some of its own "metabolic sterols"     

Cholesterol depletion induces ANTXR2-dependent activation of MMP-2 via ERK1/2 phosphorylation in neuroglioma U251 cell

Cholesterol is a critical component of lipid rafts implicated in regulating multiple signal transduction. The anthrax toxin receptor 2 (ANTXR2) is a type I membrane protein acting as the second receptor for the anthrax toxin. In this study, we first investigated the association between cholesterol and ANTXR2. We provided the evidence that cholesterol depletion by methyl-beta-cyclodextrin (MβCD) promoted ANTXR2 expression in U251 neuroglioma cell, which was reversed by cholesterol supplement. MβCD-induced ANTXR2 up-regulation contributed to ERK1/2 phosphorylation, which was responsible for MT1-MMP and MMP-2 activation. Our data suggested that cellular cholesterol regulated ANTXR2-dependent activation of MMP-2 via ERK1/2 phosphorylation in neuroglioma U251 cell.     

Assay of microsomal oxysterol 7α-hydroxylase activity in the hamster liver by a sensitive method: in vitro modulation by oxysterols

A method of assaying hepatic cytochrome P-450, oxysterol 7α-hydroxylase (CYP7B), was developed by combining the use of 25-[26,27-³H]hydroxycholesterol as a substrate and hydroxypropyl-β-cyclodextrin as a substrate vehicle. When these assay conditions were tested, an undesirable transformation was observed of the reaction product, 7α,25-dihydroxycholesterol, into 3-oxo-7α,25-dihydroxy-4-cholesten by the activity of 3β-hydroxy-Δ⁵-C₂₇ steroid oxydoreductase, a microsomal NAD⁺ and NADP⁺ dependent enzyme of bile acid metabolism. A great improvement was reached by using a continuous NADPH generating system which constantly re-transforms NADP⁺ into NADPH, thus inhibiting this activity. This improved CYP7B assay, comparable to our previously described assay for cholesterol 7α-hydroxylase (CYP7A), allowed a 3-fold increase of the apparent enzyme activity. The possibility to simultaneously measure CYP7A and CYP7B activities on the same microsomal preparation was investigated. A marked decrease (?33%) in the CYP7B activity was noticed, while that of CYP7A remained unchanged. The CYP7B activity was observed to be inhibited by cholesterol (?30%) and also by the oxysterols 7α-hydroxycholesterol (?21%), 7β-hydroxycholesterol (?25%) and epicoprostanol (?20%), and by cyclosporin A (?26%). It can be concluded that this sensible and easy to perform CYP7B assay allows to observe, at least in vitro, a modulation of the enzyme activity by oxysterols.     

Study of thermodynamic parameters for solubilization of plant sterol and stanol in bile salt micelles

We investigated the difference between the molecular structures of plant sterols and stanols that affect the solubilization of cholesterol in bile salt micelles (in vitro study). First, the aqueous solubility of beta-sitosterol, beta-sitostanol, and campesterol was determined by considering the specific radioactivity by using a fairly small quantity of each radiolabeled compound. The order of their aqueous solubilities was as follows: cholesterol > campesterol > beta-sitostanol > beta-sitosterol. The maximum solubility of cholesterol and the above mentioned sterol/stanol in sodium taurodeoxycholate and sodium taurocholate solutions (single solubilizate system) was measured. Moreover, the preferential solubilization of cholesterol in bile salt solutions was systematically studied by using different types of plant sterols/stanols. The solubilization results showed that the cholesterol-lowering effect was similar for sterols and stanol. Thermodynamic analysis was applied to these experimental results. The Gibbs energy change (Delta G degrees ) for the solubilization of plant sterols/stanols showed a negative value larger than that for cholesterol.     

Glycine transporter 1 associates with cholesterol-rich membrane raft microdomains

Membrane rafts, the highly-ordered, cholesterol-rich microdomains of the plasma membrane play important roles in cellular functions. In this study, GLYT1-CFP and GLYT2-CFP were constructed, followed by investigation of whether the tagged transporters associate with a fluorescence probe that labels membrane rafts (DilC16) by using Fluorescence Resonance Energy Transfer. A close association was observed between DiIC16 and GLYT1-CFP, but not for GLYT2-CFP. The glycine transport ability of GLYT1 is also highly dependent on the integrity of this area. Together, the results suggest that GLYT1 and membrane rafts are co-localized in the membrane, and that this influences the rate of glycine transport.     

Pregnane X receptor-agonists down-regulate hepatic ATP-binding cassette transporter A1 and scavenger receptor class B type I

Pregnane X receptor (PXR) is the molecular target for a wide variety of endogenous and xenobiotic compounds. It regulates the expression of genes central to the detoxification (cytochrome P-450 enzymes) and excretion (xenobiotic transporters) of potentially harmful compounds. The aim of the present investigation was to determine the role of PXR in regulation of high-density lipoprotein (HDL) cholesterol metabolism by studying its impact on ATP-binding cassette transporter A1 (ABCA1) and scavenger receptor class B type I (SR-BI) expression in hepatocytes. ABCA1 and SR-BI are major factors in the exchange of cholesterol between cells and HDL. Expression analyses were performed using Western blotting and quantitative real time RT-PCR. Luciferase reporter gene assays were used to measure promoter activities. Total cholesterol was measured enzymatically after lipid extraction (Folch's method). The expression of ABCA1 and SR-BI was inhibited by the PXR activators rifampicin and lithocholic acid (LCA) in HepG2 cells and pregnenolone 16alpha-carbonitrile (PCN) in primary rat hepatocytes. Thus, PXR appears to be a regulator of hepatic cholesterol transport by inhibiting genes central to cholesterol uptake (SR-BI) and efflux (ABCA1).