Phosphatidylcholine Micelles Research Articles

New Insights into the Loss of Antioxidant Effectiveness of Phenolic Compounds in Vegetable Oils in the Presence of Phosphatidylcholine.

It has been proposed that lipid oxidation reactions in edible oils primarily occur in reverse micelles (RM) of amphiphilic components. While the prooxidative effect of RM has been demonstrated, the mechanism involved is not fully understood. Both reductions and enhancements in the antioxidant efficacy (AE) of α-tocopherol and Trolox have been observed in different studies when phosphatidylcholine (PC) was added and PC RM were formed. However, most of these investigations employed lipid systems consisting of stripped vegetable oil diluted in saturated medium-chain triacylglycerols (MCT) and utilized antioxidant concentrations well below those found in edible oils. These two specific factors were investigated in the present study. The effect of RM of purified egg yolk PC on the AE of 1.16 mmol kg-1 α-tocopherol or Trolox in stripped sunflower oil (SSO) was studied by the Rancimat (100 °C) and oven (50 °C) tests. Increasing PC concentrations (50-1000 ppm) had no significant impact on α-tocopherol, but substantial reductions in AE were observed for Trolox. This phenomenon may be attributed to the partitioning of Trolox into the pre-existing PC micelles, suggesting that primary oxidation reactions occurred in the continuous lipid phase. In addition, the effectiveness of both antioxidants decreased significantly in the presence of PC when a low antioxidant concentration (0.06 mmol kg-1) was assayed in SSO:MCT (1:3, w/w).

Hydrolysis of UV-induced peroxidized phosphatidylcholine initiated by phospholipases of different substrate specificities

The activity of porcine pancreatic phospholipase A2 and the same of cobra venom toward phosphatidylcholine having different supramolecular organization and interfacial charge (micelles with sodium deoxycholate and liposomes) under UV irradiation (180–400 nm) was studied. It was shown that the UV-irradiated lipid phase is characterized by an increased index of phosphatidylcholine oxidation and the absence of a peak with a maximum of 235.5 nm, related to the presence of unsaturated bonds in the UV spectrum of docosahexaenoic acid, but retained in the presence of the antioxidant trolox. The activation of both phospholipases A2 after UV irradiation of the substrate was established, regardless of its supramolecular organization, the charge of the interfacial surface, and the substrate specificity of the enzymes. Using dynamic light scattering, 0.3 % of larger particles were found among the irradiated micelles of phosphatidylcholine. The results obtained indicate that areas of accumulation of hydroperoxidized lipids can be formed in the irradiated model membrane, which serve as a site of intensified attack for phospholipases.

Effects of phosphatidylcholine membrane fluidity on the conformation and aggregation of N-terminally acetylated α-synuclein

Membrane association of α-synuclein (α-syn), a neuronal protein associated with Parkinson's disease (PD), is involved in α-syn function and pathology. Most previous studies on α-syn-membrane interactions have not used the physiologically relevant N-terminally acetylated (N-acetyl) α-syn form nor the most naturally abundant cellular lipid, i.e. phosphatidylcholine (PC). Here, we report on how PC membrane fluidity affects the conformation and aggregation propensity of N-acetyl α-syn. It is well established that upon membrane binding, α-syn adopts an α-helical structure. Using CD spectroscopy, we show that N-acetyl α-syn transitions from α-helical to disordered at the lipid melting temperature (Tm ). We found that this fluidity sensing is a robust characteristic, unaffected by acyl chain length (Tm = 34-55 °C) and preserved in its homologs β- and γ-syn. Interestingly, both N-acetyl α-syn membrane binding and amyloid formation trended with lipid order (1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) > 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC)/sphingomyelin/cholesterol (2:2:1) ≥ DOPC), with gel-phase vesicles shortening aggregation kinetics and promoting fibril formation compared to fluid membranes. Furthermore, we found that acetylation enhances binding to PC micelles and small unilamellar vesicles with high curvature (r ∼16-20 nm) and that DPPC binding is reduced in the presence of cholesterol. These results confirmed that the exposure of hydrocarbon chains (i.e. packing defects) is essential for binding to zwitterionic gel membranes. Collectively, our in vitro results suggest that N-acetyl α-syn localizes to highly curved, ordered membranes inside a cell. We propose that age-related changes in membrane fluidity can promote the formation of amyloid fibrils, insoluble materials associated with PD.

The interaction of phospholipase A2 with oxidized phospholipids at the lipid-water surface with different structural organization

Phospholipase A2 (PLA2 IB) activity towards UV-irradiated (λ=180–400nm) phospholipids in comparison to non-irradiated ones was investigated using phosphatidylcholine (PC) liposomes and mixed micelles of phosphatidylcholine and sodium deoxycholate as a membrane model. PLA2 activity, determined by spectral changes of hemoglobin (Hb) under the interaction with fatty acids (product of the phospholipolysis), correlated well with the phospholipid peroxidation degree. The present work is the first study that determines the degree of oxidation of non-fragmented OxPCs, on the base of PLA2 activity. Fragmented OxPLs have been determined as usually by analysis of MDA using spectroscopy at 532nm.Antioxidant Trolox and human blood serum reduced observed exceeding of PLA2 activity toward OxPLs, what makes this model perspective for determining the total antioxidant activity of biological liquids.

Encapsulation of sesamol in phosphatidyl choline micelles: Enhanced bioavailability and anti-inflammatory activity.

Sesamol, the phenolic degradation product of sesamolin, although recognised for its anti-inflammatory effects, has low bioavailability. In this manuscript, we attempted to improve its bioavailability by encapsulation in mixed phosphatidylcholine micelles. Sesamol could be solubilised and entrapped in phosphatidylcholine mixed micelles (PCS) with 96.8% efficiency (particle size 3.0±0.06nm). Fluorescence spectra of PCS revealed lower relative fluorescence intensity (RFI 112) compared to 'free' sesamol (FS) (RFI 271). The bioaccessibility, transport across a monolayer of cells and cellular uptake of PCS was 8.58%, 1.5-fold and 1.2-fold better, respectively, compared to FS. The anti-inflammatory effects of FS and PCS were compared using LPS treated RAW 264.7 cell line and lipoxygenase inhibition. PCS effected downregulation of iNOS protein expression (27%), NO production (20%), ROS (32%) and lipoxygenase inhibition (IC50=31.24μM) compared to FS.

Novel self-assembled nano-tubular mixed micelles of Pluronics P123, Pluronic F127 and phosphatidylcholine for oral delivery of nimodipine: In vitro characterization, ex vivo transport and in vivo pharmacokinetic studies

Subarachnoid hemorrhage (SAH) is a major cause of death in patients suffering from stroke. Nimodipine (NM) is the only FDA-approved drug for treating SAH-induced vasospasm. However, NM suffers from poor oral bioavailability (5–13%) due to its low aqueous solubility, extensive first pass metabolism and short elimination half-life (1–2h). The objective of this study was to develop NM-loaded Pluronic/phosphatidylcholine/polysorbate 80 mixed micelles (PPPMM) that can solubilize NM in aqueous media even after dilution, prolong its circulation time, improve its bioavailability and eventually help in targeting it to the brain tissue. PPPMM formulations were prepared using the thin film hydration technique, and evaluated for drug payload, solubilization efficiency (SE), micellar size, zeta potential, transmission electron microscopy (TEM) and ex vivo transport through rat intestine. The selected NM-loaded PPPMM, containing PC to Pluronics® molar ratio of 75:25, showed a drug payload, SE, micellar size and zeta potential of 1.06±0.03mg/mL, 99.2±2.01%, 571.5±11.87nm and −31.2±0.06mv, respectively. The selected formulation had a much larger hydrophobic core volume for solubilization of NM and exhibited the highest NM transport. TEM micrographs illustrated the formation of highly flexible nano-tubular mixed micelles (NTMM). The in vivo pharmacokinetic study showed greater bioavailability of NM in plasma (232%) and brain (208%) of rats from NM-loaded PPPMM compared to that of the drug solution due to the efficiency of flexible NTMM to enhance absorption of NM from the intestinal mucosa. The significant increase in drug solubility, enhanced drug absorption and the long circulation time of the NTMM could be promising to improve oral and parenteral delivery of NM.

Viscoelasticity and interface bending properties of lecithin reverse wormlike micelles studied by diffusive wave spectroscopy in hydrophobic environment.

Upon the addition of minute quantities of water into a phosphatidylcholine (PC) solution in certain organic solvents, PC micelles elongate into giant reverse wormlike micelles that entangle and form highly viscous microemulsions, called lecithin organogels. We investigated the microrheological properties of concentrated PC-cyclohexane reverse wormlike micellar systems by diffusive wave spectroscopy (DWS) in apolar medium, combined with bulk shear rheology. We applied DWS to our oil-continuous system by using hydrophobic poly(hydroxystearic acid)-grafted PMMA particles as monodisperse tracer particles. Relevant parameters such as the micellar scission energy and persistence length were extracted from the microrheology data and interpreted according to the sphere-to-rod-to-sphere structural transition. On the basis of these quantities, we calculated the bending and saddle-splay moduli of the PC-covered water-cyclohexane interface. This approach represents a new method for the quantitative estimation of these fundamental parameters, which are thought to underpin the self-assembly of surfactants.

Lyso-myristoyl phosphatidylcholine micelles sustain the activity of Dengue non-structural (NS) protein 3 protease domain fused with the full-length NS2B

Dengue virus (DENV), a member of the flavivirus genus, affects 50–100million people in tropical and sub-tropical regions. The DENV protease domain is located at the N-terminus of the NS3 protease and requires for its enzymatic activity a hydrophilic segment of the NS2B that acts as a cofactor. The protease is an important antiviral drug target because it plays a crucial role in virus replication by cleaving the genome-coded polypeptide into mature functional proteins. Currently, there are no drugs to inhibit DENV protease activity. Most structural and functional studies have been conducted using protein constructs containing the NS3 protease domain connected to a soluble segment of the NS2B membrane protein via a nine-residue linker. For in vitro structural and functional studies, it would be useful to produce a natural form of the DENV protease containing the NS3 protease domain and the full-length NS2B protein. Herein, we describe the expression and purification of a natural form of DENV protease (NS2BFL–NS3pro) containing the full-length NS2B protein and the protease domain of NS3 (NS3pro). The protease was expressed and purified in detergent micelles necessary for its folding. Our results show that this purified protein was active in detergent micelles such as lyso-myristoyl phosphatidylcholine (LMPC). These findings should facilitate further structural and functional studies of the protease and will facilitate drug discovery targeting DENV.

The Homotrimeric HIV-1 Viral Coat Protein GP41 is Highly Dynamic

A solution NMR study on the structure and dynamics of the homotrimeric gp41 complex, reconstituted in dodecyl phosphatidylcholine (DPC) micelles, is presented. Observed resonances were assigned to the fusion peptide (FP), N-terminal heptad repeat (NHR) and immuno-dominant loop region (IL), whereas the C-terminal heptad repeat (CHR) region, membrane proximal external region (MPER) and transmembrane helix (TM) remain completely invisible to solution NMR. 15N relaxation data reveal a high degree of intrinsic mobility: The a-helical fusion peptide (FP) exhibits high amplitude motion on the fast nanosecond time scale relative to the NHR region. The linker between NHR and CHR shows both fast and slow (microsecond) motions.Figure 1: Homotrimeric gp41 shows a high degree of intrinsic mobility. Our data are compatible with the protein switching on a microsecond time scale between the pre-hairpin intermediate, three-helical bundle state, and the late-fusion, anti-parallel six-helical bundle. MPER and TM are not shown since their conformation cannot be evaluated from the available solution NMR data. While MPER and TM are absent from the spectra, the FP shows the most intense resonances, which excludes a strong interaction between FP and TM region.View Large Image | View Hi-Res Image | Download PowerPoint Slide

1B1512 Decay kinetics of the M photointermediate of bacteriorhodopsin in short chain phosphatidylcholine micelles(Membrane Proteins 1,The 49th Annual Meeting of the Biophysical Society of Japan)

1B1512 Decay kinetics of the M photointermediate of bacteriorhodopsin in short chain phosphatidylcholine micelles(Membrane Proteins 1,The 49th Annual Meeting of the Biophysical Society of Japan)

High resolution NMR conformational studies of new bivalent NOP receptor antagonists in model membrane systems

The interaction of new bivalent NOP receptor antagonists with dodecyl phosphatidylcholine micelles and DMPC/cholesterol liposomes was investigated in solution by high resolution NMR. The ligands are structurally related to the NOP antagonist JTC-801 plus a propanediamine or heptanediamine spacer between the pharmacophoric units . Ligand internuclear distances were derived from 2D NOESY data and applied to molecular modelling calculations as conformational restraints. NMR experiments on micelles evidenced that the ligands closely approached the micelles but gave no hints on the preferential conformations of the interacting ligands. Results from NMR experiments in the presence of liposomes clearly indicated that both ligands strongly interacted with the bilayer assuming a preferential folded conformation with the quinoline arms superimposing on each other. The finding suggested that these strongly lipophilic pharmacophores could localize in the native receptorial membrane in the form of a depot, gaining access to the recognition site via the lipid bilayer.

Cholesteryl ester hydrolase activity is abolished in HSL macrophages but unchanged in macrophages lacking KIAA1363

Cholesteryl ester (CE) accumulation in macrophages represents a crucial event during foam cell formation, a hallmark of atherogenesis. Here we investigated the role of two previously described CE hydrolases, hormone-sensitive lipase (HSL) and KIAA1363, in macrophage CE hydrolysis. HSL and KIAA1363 exhibited marked differences in their abilities to hydrolyze CE, triacylglycerol (TG), diacylglycerol (DG), and 2-acetyl monoalkylglycerol ether (AcMAGE), a precursor for biosynthesis of platelet-activating factor (PAF). HSL efficiently cleaved all four substrates, whereas KIAA1363 hydrolyzed only AcMAGE. This contradicts previous studies suggesting that KIAA1363 is a neutral CE hydrolase. Macrophages of KIAA1363−/− and wild-type mice exhibited identical neutral CE hydrolase activity, which was almost abolished in tissues and macrophages of HSL−/− mice. Conversely, AcMAGE hydrolase activity was diminished in macrophages and some tissues of KIAA1363−/− but unchanged in HSL−/− mice. CE turnover was unaffected in macrophages lacking KIAA1363 and HSL, whereas cAMP-dependent cholesterol efflux was influenced by HSL but not by KIAA1363. Despite decreased CE hydrolase activities, HSL−/− macrophages exhibited CE accumulation similar to wild-type (WT) macrophages. We conclude that additional enzymes must exist that cooperate with HSL to regulate CE levels in macrophages. KIAA1363 affects AcMAGE hydrolase activity but is of minor importance as a direct CE hydrolase in macrophages.

Molecular Determinants of Sphingomyelin Specificity of a Eukaryotic Pore-forming Toxin

Sphingomyelin (SM) is abundant in the outer leaflet of the cell plasma membrane, with the ability to concentrate in so-called lipid rafts. These specialized cholesterol-rich microdomains not only are associated with many physiological processes but also are exploited as cell entry points by pathogens and protein toxins. SM binding is thus a widespread and important biochemical function, and here we reveal the molecular basis of SM recognition by the membrane-binding eukaryotic cytolysin equinatoxin II (EqtII). The presence of SM in membranes drastically improves the binding and permeabilizing activity of EqtII. Direct binding assays showed that EqtII specifically binds SM, but not other lipids and, curiously, not even phosphatidylcholine, which presents the same phosphorylcholine headgroup. Analysis of the EqtII interfacial binding site predicts that electrostatic interactions do not play an important role in the membrane interaction and that the two most important residues for sphingomyelin recognition are Trp(112) and Tyr(113) exposed on a large loop. Experiments using site-directed mutagenesis, surface plasmon resonance, lipid monolayer, and liposome permeabilization assays clearly showed that the discrimination between sphingomyelin and phosphatidylcholine occurs in the region directly below the phosphorylcholine headgroup. Because the characteristic features of SM chemistry lie in this subinterfacial region, the recognition mechanism may be generic for all SM-specific proteins.

Role of Glycosphingolipid Conformational Change in Membrane Pore Forming Activity of Cobra Cardiotoxin

The major cardiotoxin from Taiwan cobra (CTX A3) is a pore forming beta-sheet polypeptide that requires sulfatide (sulfogalactosylceramide, SGC) on the plasma membrane of cardiomyocytes for CTX-induced membrane leakage and cell internalization. Herein, we demonstrate by fluorescence spectroscopic studies that sulfatides induce CTX A3 oligomerization in sulfatide containing phosphatidylcholine (PC) vesicles to form transient pores with pore size and lifetime in the range of about 30 A and 10(-2) s, respectively. These values are consistent with the CTX A3-induced conductance and mean lifetime determined previously by using patch-clamp electrophysiological experiments on the plasma membrane of H9C2 cells. We also derived the peripheral binding structural model of CTX A3-sulfatide complex in sulfatide containing PC micelles by NMR and molecular docking method and compared with other CTX A3-sulfatide complex structure determined previously by X-ray in membrane-like environment. The NMR results indicate that sulfatide head group conformation changes from a bent shovel (-sc/ap) to an extended (sc/ap) conformation upon initial binding of CTX A3. An additional global reorientation of sulfatide molecule is also needed for CTX A3 dimer formation as inferred by the difference between the X-ray and NMR complex structure. Since the overall folding of CTX A3 molecules remained the same, sulfatide in phospholipid bilayer is proposed to play an active role by involving its local and global conformational changes to promote both the oligomerization and reorientation of CTX A3 molecule for its transient pore formation and cell internalization.

Characterization of phospholipid mixed micelles by translational diffusion.

The concentration dependence of the translational self diffusion rate, D (s), has been measured for a range of micelle and mixed micelle systems. Use of bipolar gradient pulse pairs in the longitudinal eddy current delay experiment minimizes NOE attenuation and is found critical for optimizing sensitivity of the translational diffusion measurement of macromolecules and aggregates. For low volume fractions Phi (Phi\\ le 15% v/v) of the micelles, experimental measurement of the concentration dependence, combined with use of the D (s)= D (o)(1-3.2lambdaPhi) relationship, yields the hydrodynamic volume. For proteins, the hydrodynamic volume, derived from D (s) at infinitely dilute concentration, is found to be about 2.6 times the unhydrated molecular volume. Using the data collected for hen egg white lysozyme as a reference, diffusion data for dihexanoyl phosphatidylcholine (DHPC) micelles indicate approximately 27 molecules per micelle, and a critical micelle concentration of 14 mM. Differences in translational diffusion rates for detergent and long chain phospholipids in mixed micelles are attributed to rapid exchange between free and micelle-bound detergent. This difference permits determination of the free detergent concentration, which, for a high detergent to long chain phospholipid molar ratio, is found to depend strongly on this ratio. The hydrodynamic volume of DHPC/POPC bicelles, loaded with an M2 channel peptide homolog, derived from translational diffusion, predicts a rotational correlation time that slightly exceeds the value obtained from peptide (15)N relaxation data.

Transformation of Phospholipids by Cabbage Phospholipase D in Mixed Micelles Containing 3-[(3-Cholamidopropyl)dimethylammonio]-1-propanesulfonate

We compared the activities of cabbage phospholipase D during hydrolysis and transesterification of phosphatidylcholine in mixed micelles of surface-active compounds with various physicochemical properties. Mixed micelles of phospholipids and 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (ratio, 1:2) were among the best substrates. Hydrolysis and transphosphatidylation were studied in micelles containing 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate. Mixed micelles of phosphatidylcholine and 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate may serve as a new substrate for measurements of phospholipase D activity and preparative isolation of phospholipids using this enzyme.

Role of Helices and Loops in the Ability of Apolipophorin-III To Interact with Native Lipoproteins and Form Discoidal Lipoprotein Complexes

The structure of Locusta migratoria apolipophorin-III consists of a five-helix bundle connected by four short loops. The role of the conformational flexibility of helices and loops on the lipid-binding activity of this apolipoprotein was investigated by disulfide mediated tethering experiments. One disulfide mutant tethering the second and fourth loops (L2-L4), and two disulfide mutants restricting the flexibility of the neighboring alpha-helices 3 and 4 (H3-H4) and 1 and 5 (H1-H5), were studied. The ability of the disulfide mutants to interact with phospholipid vesicles, mixed micelles of phosphatidylcholine and cholate, and in vivo with native spherical lipoprotein particles was studied. The L2-L4 mutant was active with native lipoproteins as well as being able to form discoidal lipoproteins upon incubation with either liposomes or discoidal micelles. The H3-H4 mutant was not able to interact with liposomes or native lipoproteins but interacted with discoidal micelles. The H1-H5 mutant was unable to interact with lipid in any of the three systems. Three conclusions were reached: (1) opening of the helix bundle does not require the separation of loops 2 and 4 as recently proposed by others and (2) alpha-helices 3 and/or 4 are involved in the insertion of apoLp-III in both phospholipid bilayers and monolayers. The conformational flexibility of helices 3 and 4 is required for the lipid-binding activity of apoLp-III. (3) Interaction of helices 1 and/or 5 with the lipid surface is required to the formation of stable lipoprotein complexes of any kind.

Optimizing the Interfacial Binding and Activity of a Bacterial Phosphatidylinositol-specific Phospholipase C

The phosphatidylinositol-specific phospholipase C from Bacillus thuringiensis can be activated by nonsubstrate interfaces such as phosphatidylcholine micelles or bilayers. This activation corresponds with partial insertion into the interface of two tryptophans, Trp-47 in helix B and Trp-242 in a loop, in the rim of the alphabeta-barrel. Both W47A and W242A have much weaker binding to interfaces and considerably lower kinetic interfacial activation. Tryptophan rescue mutagenesis, reinsertion of a tryptophan at a different place in helix B in the W47A mutant or in the loop (residues 232-244) of the W242A mutant, has been used to determine the importance and orientation of a tryptophan in these two structural features. Phosphotransferase and phosphodiesterase assays, and binding to phosphatidylcholine vesicles were used to assess both orientation and position of tryptophans needed for interfacial activity. Of the helix B double mutants, only one mutant, I43W/W47A, has tryptophan in the same orientation as Trp-47. I43W/W47A shows recovery of phosphatidylinositol-specific phospholipase C (PC) activation of d-myo-inositol 1,2-cyclic phosphate hydrolysis. However, the specific activity toward phosphatidylinositol is still lower than wild type enzyme and high activity with phosphatidylinositol solubilized in 30% isopropyl alcohol (a hallmark of the native enzyme) is lost. Reinserting a tryptophan at several positions in the loop composed of residues 232-244 partially recovers PC activation and affinity of the enzyme for lipid interfaces as well as activation by isopropyl alcohol. G238W/W242A shows an enhanced activation and affinity for PC interfaces above that of wild type. These results provide constraints on how this bacterial phosphatidylinositol-specific phospholipase C binds to activating PC interfaces.

Plasmodium falciparum histidine-rich protein II binds to actin, phosphatidylinositol 4,5-bisphosphate and erythrocyte ghosts in a pH-dependent manner and undergoes coil-to-helix transitions in anionic micelles.

The recombinant histidine-rich protein II (HRPII) from Plasmodium falciparum was shown to bind actin and phosphatidylinositol 4,5-bisphosphate (PIP(2)) in vitro in a pH-dependent manner, very similar to hisactophilin, an actin-binding protein from ameba. Binding of HRPII to actin and PIP(2) occurred at pH 6.0 and 6.5, but not above pH 7.0. Circular dichroism (CD) spectroscopy confirmed that HRPII interacts with actin at pH below 7.0, as judged by the changes induced in the secondary structure of the HRPII/actin mixture. Further CD analysis demonstrated that HRPII adopts a predominantly alpha-helical conformation with anionic micelles of PIP(2) and SDS, but not with neutral micelles of phosphatidylcholine (PC), a feature that is common to many actin-binding proteins involved in cytoskeleton remodeling. Similarly to hisactophilin, a GFP-HRPII fusion protein shuttled from the cytoplasm to the nucleus of HeLa cells as the cellular pH was lowered from 8.0 to 6.0. HeLa cells transfected with the HRPII gene showed increased levels of histidine-rich proteins (HRPs) in the soluble cell fraction at pH 8.0. At pH 6.0, however, HRPs were detected mainly in the insoluble cell fraction. Interestingly, we found that HRPII binds to human erythrocyte membranes at pH 6.0 and 6.5 but not at pH above 7.0. Our results point to remarkable similarities between HRPII, hisactophilin, and actin-binding proteins. Possible roles of the HRPII during Plasmodium infection are discussed in the light of these findings.

Characterization of Anti-Phosphatidylcholine Polyreactive Natural Autoantibodies from Normal Human Subjects

In 1990 our group reported a patient with autoimmune hemolytic anemia and high titers of IgM anticardiolipin antibodies that cross-reacted with phosphatidylcholine (PTC). These autoantibodies also recognized bromelain-treated erythrocytes (BrE) and in vitro aged erythrocytes. The epitope exposed with this treatment is PTC. To detect and characterize antiphosphatidylcholine antibodies (anti-PTC) in a normal human population, we studied by ELISA the presence of serum anti-PTC (IgG and IgM) in clinically healthy human subjects. The most representative samples were also studied for IgG or IgM activity against BrE by flow cytometry, rheumatoid factor activity, anti-dsDNA, anti-ssDNA by ELISA and by indirect immunofluorecence (IIF) using HEp-2 line and a healthy human fibroblast strain as substratum. Eighty five percent of sera had IgM anti-PTC and none had IgG. IgM antibodies against BrE were inhibited by PTC micelles (mPTC). Anti-PTC were also inhibited by phosphorylcholine and phosphatidic acid. Aggregated gammaglobulin (AGG) reactivity was inhibited by dsDNA and mPTC. The IgM anti-dsDNA activity was inhibited by soluble dsDNA, AGG and mPTC. All sera gave intermediate filaments pattern by IIF and reacted against purified vimentin by dot blot and Western blot.Our study shows hemolytic IgM anti-PTC present in normal human serum. The main epitope recognized by these autoantibodies is phosphorylcholine. The physicochemical characteristics, crossreactivity with self-antigens and functional properties are typical features of natural autoantibodies.