ABSTRACT
Familial Hypercholesterolemia (FH) is a common genetic disorder caused most often by mutations in the Low Density Lipoprotein Receptor gene (LDLr) leading to high blood cholesterol levels, and ultimately to development of premature coronary heart disease. Genetic analysis and subsequent cascade screening in relatives allow diagnosis of FH at early stage, especially relevant to diagnose children. So far, more than 2300 LDLr variants have been described but only a minority of them have been functionally analysed to evaluate their pathogenicity in FH. Thus, identifying pathogenic mutations in LDLr is a long-standing challenge in the field. In this study, we investigated in vitro the activity p.(Asp47Asn) and p.(Thr62Met) LDLr variants, both in the LR1 region. We used CHO-ldlA7 transfected cells with plasmids carrying p.(Asp47Asn) or p.(Thr62Met) LDLr variants to analyse LDLr expression by FACS and immunoblotting, LDL binding and uptake was determined by FACS and analysis of mutation effects was assessed in silico. The in vitro activity assessment of p.(Asp47Asn) and p.(Thr62Met) LDLr variants shows a fully functional LDL binding and uptake activities. Therefore indicating that the three of them are non-pathogenic LDLr variants. These findings also emphasize the importance of in vitro functional LDLr activity studies to optimize the genetic diagnosis of FH avoiding the report of non-pathogenic variants and possible misdiagnose in relatives if cascade screening is carried out.
Subject(s)
Hyperlipoproteinemia Type II/pathology , Lipoproteins/metabolism , Mutant Proteins/metabolism , Mutation, Missense , Receptors, LDL/genetics , Receptors, LDL/metabolism , Algorithms , Animals , CHO Cells , Cricetinae , Cricetulus , Humans , Hyperlipoproteinemia Type II/genetics , Hyperlipoproteinemia Type II/metabolism , In Vitro Techniques , Mutant Proteins/geneticsABSTRACT
BACKGROUND AND AIMS: Pathogenic mutations in the Low Density Lipoprotein Receptor gene (LDLR) cause Familial Hypercholesterolemia (FH), one of the most common genetic disorders with a prevalence as high as 1 in 200 in some populations. FH is an autosomal dominant disorder of lipoprotein metabolism characterized by high blood cholesterol levels, deposits of cholesterol in peripheral tissues such as tendon xanthomas and accelerated atherosclerosis. To date, 2500 LDLR variants have been identified in the LDLR gene; however, only a minority of them have been experimentally characterized and proven to be pathogenic. Here we investigated the role of Cys46 located in the first repeat of the LDL receptor binding domain in recognition of apolipoproteins. METHODS: Activity of the p.(Cys46Gly) LDLR variant was assessed by immunoblotting and flow cytometry in CHO-ldlA7 expressing the receptor variant. Affinity of p.(Cys46Gly) for LDL and VLDL was determined by solid-phase immunoassays and in silico analysis was used to predict mutation effects. RESULTS AND CONCLUSION: Functional characterization of p.(Cys46Gly) LDLR variant showed impaired LDL and VLDL binding and uptake activity. Consistent with this, solid-phase immunoassays showed the p.(Cys46Gly) LDLR variant has decreased binding affinity for apolipoproteins. These results indicate the important role of Cys46 in LDL receptor activity and highlight the role of LR1 in LDLr activity modulation. This study reinforces the significance of in vitro functional characterization of LDL receptor activity in developing an accurate approach to FH genetic diagnosis. This is of particular importance because it enables clinicians to tailor personalized treatments for patients' mutation profile.
Subject(s)
Apolipoproteins/metabolism , Mutation, Missense , Receptors, LDL/genetics , Receptors, LDL/metabolism , Amino Acid Substitution , Animals , Apolipoprotein E3/metabolism , Binding Sites/genetics , CHO Cells , Computer Simulation , Cricetulus , Humans , Hyperlipoproteinemia Type II/genetics , Hyperlipoproteinemia Type II/metabolism , Lipoproteins, LDL/metabolism , Lipoproteins, VLDL/metabolism , Mutagenesis, Site-Directed , Mutant Proteins/chemistry , Mutant Proteins/genetics , Mutant Proteins/metabolism , Receptors, LDL/chemistry , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/metabolismABSTRACT
Familial hypercholesterolaemia (FH) is an inherited autosomal dominant disorder resulting from defects in the low-density lipoprotein receptor (LDLR), in the apolipoprotein B (APOB) or in the proprotein convertase subtilisin/kexin type 9 (PCSK9) genes. In the majority of the cases FH is caused by mutations occurring within LDLR, while only few mutations in APOB and PCSK9 have been proved to cause disease. p.(Arg3527Gln) was the first mutation in APOB being identified and characterized. Recently two novel pathogenic APOB variants have been described: p.(Arg1164Thr) and p.(Gln4494del) showing impaired LDLR binding capacity, and diminished LDL uptake. The objective of this work was to analyse the structure of p.(Arg1164Thr) and p.(Gln4494del) variants to gain insight into their pathogenicity. Secondary structure of the human ApoB100 has been investigated by infrared spectroscopy (IR) and LDL particle size both by dynamic light scattering (DLS) and electron microscopy. The results show differences in secondary structure and/or in particle size of p.(Arg1164Thr) and p.(Gln4494del) variants compared with wild type. We conclude that these changes underlie the defective binding and uptake of p.(Arg1164Thr) and p.(Gln4494del) variants. Our study reveals that structural studies on pathogenic variants of APOB may provide very useful information to understand their role in FH disease.
Subject(s)
Amino Acid Substitution , Apolipoproteins B/chemistry , Apolipoproteins B/genetics , Codon , Hyperlipoproteinemia Type II/genetics , Mutation , Apolipoprotein B-100/chemistry , Apolipoprotein B-100/genetics , Apolipoprotein B-100/ultrastructure , Apolipoproteins B/metabolism , Apolipoproteins B/ultrastructure , Cell Line , Humans , Hyperlipoproteinemia Type II/metabolism , Lipoproteins, LDL/chemistry , Lipoproteins, LDL/metabolism , Lipoproteins, LDL/ultrastructure , Lymphocytes/metabolism , Particle Size , Protein Binding , Protein Structure, SecondaryABSTRACT
BACKGROUND: The LDL receptor (LDLR) is a Class I transmembrane protein critical for the clearance of cholesterol-containing lipoprotein particles. The N-terminal domain of the LDLR harbours the ligand-binding domain consisting of seven cysteine-rich repeats of approximately 40 amino acids each. Mutations in the LDLR binding domain may result in loss of receptor activity leading to familial hypercholesterolemia (FH). In this study the activity of six mutations located in the cysteine-rich repeats of the LDLR has been investigated. METHODS: CHO-ldlA7 transfected cells with six different LDLR mutations have been used to analyse in vitro LDLR expression, lipoprotein binding and uptake. Immunoblotting of cell extracts, flow cytometry and confocal microscopy have been performed to determine the effects of these mutations. In silico analysis was also performed to predict the mutation effect. RESULTS AND CONCLUSION: From the six mutations, p.Arg257Trp turned out to be a non-pathogenic LDLR variant whereas p.Cys116Arg, p.Asp168Asn, p.Asp172Asn, p.Arg300Gly and p.Asp301Gly were classified as binding-defective LDLR variants whose effect is not as severe as null allele mutations.
Subject(s)
Hyperlipoproteinemia Type II/genetics , Lipoproteins/metabolism , Mutation, Missense , Receptors, LDL/genetics , Amino Acid Motifs , Animals , CHO Cells , Computer Simulation , Cricetulus , Cysteine , Flow Cytometry , Genetic Predisposition to Disease , Hyperlipoproteinemia Type II/metabolism , Microscopy, Confocal , Models, Molecular , Mutagenesis, Site-Directed , Phenotype , Protein Binding , Protein Interaction Domains and Motifs , Receptors, LDL/metabolism , Repetitive Sequences, Amino Acid , Structure-Activity Relationship , TransfectionABSTRACT
Escherichia coli alpha-hemolysin (HlyA) can lyse both red blood cells (RBC) and liposomes. However, the cells are lysed at HlyA concentrations 1-2 orders of magnitude lower than liposomes (large unilamellar vesicles). Treatment of RBC with trypsin, but not with chymotrypsin, reduces the sensitivity of RBC toward HlyA to the level of the liposomes. Since glycophorin, one of the main proteins in the RBC surface, can be hydrolyzed by trypsin much more readily than by chymotrypsin, the possibility was tested of a specific binding of HlyA to glycophorin. With this purpose, a number of experiments were performed. (a) HlyA was preincubated with purified glycophorin, after which it was found to be inactive against both RBC and liposomes. (b) Treatment of RBC with an anti-glycophorin antibody protected the cells against HlyA lysis. (c) Immobilized HlyA was able to bind glycophorin present in a detergent lysate of RBC ghosts. (d) Incorporation of glycophorin into pure phosphatidylcholine liposomes increased notoriously the sensitivity of the vesicles toward HlyA. (e) Treatment of the glycophorin-containing liposomes with trypsin reverted the vesicles to their original low sensitivity. The above results are interpreted in terms of glycophorin acting as a receptor for HlyA in RBC. The binding constant of HlyA for glycophorin was estimated, in RBC at sublytic HlyA concentrations, to be 1.5 x 10(-9) m.
Subject(s)
Bacterial Proteins/blood , Bacterial Toxins/blood , Erythrocyte Membrane/physiology , Escherichia coli Proteins , Glycophorins/metabolism , Hemolysin Proteins/blood , Animals , Bacterial Proteins/chemistry , Bacterial Toxins/chemistry , Binding Sites , Escherichia coli , Glycophorins/chemistry , Hemolysin Proteins/chemistry , Horses , Kinetics , Lipid Bilayers/chemistry , Proteolipids/chemistry , Proteolipids/metabolism , Receptors, Cell Surface/blood , TrypsinABSTRACT
alpha-Haemolysin is an extracellular protein toxin (approximately 107 kDa) secreted by Escherichia coli that acts at the level of the plasma membranes of target eukaryotic cells. The nature of the toxin interaction with the membrane is not known at present, although it has been established that receptor-mediated binding is not essential. In this work, we have studied the perturbation produced by purified alpha-haemolysin on pure phosphatidylcholine bilayers in the form of large unilamellar vesicles, under conditions in which the toxin has been shown to induce vesicle leakage. The bilayer systems containing bound protein have been examined by differential scanning calorimetry, fluorescence spectroscopy, differential solubilization by Triton X-114, and freeze-fracture electron microscopy. All the data concur in indicating that alpha-haemolysin, under conditions leading to cell lysis, becomes inserted in the target membrane in the way of intrinsic or integral proteins. In addition, the experimental results support the idea that inserted alpha-haemolysin occupies only one of the membrane phospholipid monolayers, i.e. it is not a transmembrane protein. The experimental data are complemented by structure prediction studies according to which as many as ten amphipathic alpha-helices, appropriate for protein-lipid interaction, but no hydrophobic transmembrane helices are predicted in alpha-haemolysin. These observations and predictions have important consequences for the mechanism of cell lysis by alpha-haemolysin; in particular, a non-transmembrane arrangement of the toxin in the target membrane is not compatible with the concept of alpha-haemolysin as a pore-forming toxin.
Subject(s)
Bacterial Proteins/chemistry , Escherichia coli Proteins , Escherichia coli/chemistry , Hemolysin Proteins/chemistry , Lipid Bilayers/chemistry , Amino Acid Sequence , Calorimetry, Differential Scanning , Fluorescence Polarization , Freeze Fracturing , Microscopy, Electron , Models, Molecular , Molecular Sequence Data , Octoxynol , Phosphatidylcholines/chemistry , Polyethylene Glycols , Protein Conformation , Protein Structure, Secondary , Solubility , Spectrometry, FluorescenceABSTRACT
A synthetic peptide (23 residues) that includes the antibacterial and lipopolysaccharide-binding regions of human lactoferricin, an antimicrobial sequence of lactoferrin, was used to study its action on cytoplasmic membrane of Escherichia coli 0111 and E. coli phospholipid vesicles. The peptide caused a depolarization of the bacterial cytoplasmic membrane, loss of the pH gradient, and a bactericidal effect on E. coli. Similarly, the binding of the peptide to liposomes dissipated previously created transmembrane electrical and pH gradients. The dramatic consequences of the transmembrane ion flux during the peptide exposure indicate that the adverse effect on bacterial cells occurs at the bacterial inner membrane.
Subject(s)
Lactoferrin/analogs & derivatives , Lactoferrin/pharmacology , Membranes, Artificial , Anti-Bacterial Agents/pharmacology , Electrophysiology , Escherichia coli/chemistry , Escherichia coli/growth & development , Escherichia coli/metabolism , Humans , Hydrogen-Ion Concentration/drug effects , Liposomes/drug effects , Liposomes/metabolism , Membrane Potentials/drug effects , Permeability/drug effects , Protein Binding/drug effects , Spectrometry, Fluorescence , Time Factors , Tryptophan/metabolism , Valinomycin/pharmacologyABSTRACT
alpha-Hemolysin is synthesized as a 1024-amino acid polypeptide, then intracellularly activated by specific fatty acylation. A second activation step takes place in the extracellular medium through binding of Ca2+ ions. Even in the absence of fatty acids and Ca2+ HlyA is an amphipathic protein, with a tendency to self-aggregation. However, Ca(2+)-binding appears to expose hydrophobic patches on the protein surface, facilitating both self-aggregation and irreversible insertion into membranes. The protein may somehow bind membranes in the absence of divalent cations, but only when Ca2+ (or Sr2+, or Ba2+) is bound to the toxin in aqueous suspensions, i.e., prior to its interaction with bilayers, can alpha-hemolysin bind irreversibly model or cell membranes in such a way that the integrity of the membrane barrier is lost, and cell or vesicle leakage ensues. Leakage is not due to the formation of proteinaceous pores, but rather to the transient disruption of the bilayer, due to the protein insertion into the outer membrane monolayer, and subsequent perturbations in the bilayer lateral tension. Protein or glycoprotein receptors for alpha-hemolysin may exist on the cell surface, but the toxin is also active on pure lipid bilayers.
Subject(s)
Bacterial Proteins/physiology , Bacterial Toxins , Cell Membrane/physiology , Escherichia coli Proteins , Hemolysin Proteins/physiologyABSTRACT
Previous studies from this laboratory had shown that calcium ions were essential for the membrane lytic activity of E. coli alpha-haemolysin (HlyA), while zinc ions did not sustain such a lytic activity. The present data indicate that calcium-binding does not lead to major changes in the secondary structure, judging from circular dichroism spectra. However binding to Ca2+ exposes new hydrophobic residues at the protein surface, as indicated by the increased binding of the fluorescent probe aniline naphtholsulphonate (ANS), and by the increased tendency of the Ca2+-bound protein to self-aggregate. In addition zinc ions are seen to decrease the thermal stability of HlyA which, according to intrinsic fluorescence and differential scanning calorimetry data, is stable below 95 degrees C when bound to calcium, while it undergoes irreversible denaturation above 60 degrees C in the zinc-bound form. Binding to phosphatidylcholine bilayers is quantitatively similar in the presence of both cations, but about one-third of the zinc-bound HlyA is released in the presence of 2 M NaCl. Differential scanning calorimetry of dimyristoylglycerophosphocholine large unilamellar vesicles reveals that Zn2+-HlyA interaction with the lipid bilayer has a strong polar component, while Ca2+-HlyA appears to interact mainly through hydrophobic forces. Experiments in which HIyA transfer is measured from phospholipid vesicles to red blood cells demonstrate that Ca2+ ions promote the irreversible binding of the toxin to bilayers. All these data can be interpreted in terms of a specific Ca2+ effect that increases the surface hydrophobicity of the protein, thus facilitating its irreversible bilayer insertion in the fashion of intrinsic membrane proteins.
Subject(s)
Bacterial Proteins/chemistry , Calcium/pharmacology , Escherichia coli Proteins , Escherichia coli/chemistry , Hemolysin Proteins/chemistry , Lipid Bilayers/chemistry , Protein Conformation , Cations, Divalent , Hot Temperature , Peptide Fragments/analysis , Phosphatidylcholines , Protein Denaturation , Protein Structure, Secondary , Spectrometry, Fluorescence , Trypsin , ZincABSTRACT
The relative weight of electrostatic interactions and hydrophobic forces in the process of membrane disruption caused by E. coli alpha-haemolysin (HlyA) has been studied with a purified protein preparation and a model system consisting of large unilamellar vesicles loaded with water-soluble fluorescent probes. Vesicles were prepared in buffers of different ionic strengths, or pHs, and the net surface charge of the bilayers was also modified by addition of negatively (e.g., phosphatidylinositol) or positively (e.g., stearylamine) charged lipids. The results can be interpreted in terms of a multiple equilibrium in which alpha-haemolysin may exist: aggregated HlyA <==> monomeric HlyA <==> membrane-bound HlyA. In these equilibria both electrostatic and hydrophobic forces are significant. Electrostatic forces become substantial under certain circumstances, e.g., membrane binding when bilayer and protein have opposite electric charges. Protein adsorption to the bilayer is more sensitive to electrostatic forces than membrane disruption itself. In the latter case, the irreversible nature of protein insertion may overcome electrostatic repulsions. Also of interest is the complex effect of pH on the degree of aggregation of an amphipathic toxin like alpha-haemolysin, since pH changes are not only influencing the net protein charge but may also be inducing protein conformational transitions shown by changes in the protein intrinsic fluorescence and in its susceptibility to protease digestion, that appear to regulate the presence of hydrophobic patches at the surface of the molecule, thus modifying the ability of the toxin to either aggregate or become inserted in membranes.
Subject(s)
Bacterial Proteins/toxicity , Escherichia coli Proteins , Hemolysin Proteins/toxicity , Membranes, Artificial , Binding Sites , Hydrogen-Ion Concentration , Lipid Bilayers/chemistry , Osmolar Concentration , Static Electricity , Surface Properties , WaterABSTRACT
Alpha-Hemolysin is an extracellular protein toxin (107 kDa) produced by some pathogenic strains of Escherichia coli. Although stable in aqueous medium, it can bind to lipid bilayers and produce membrane disruption in model and cell membranes. Previous studies had shown that toxin binding to the bilayer did not always lead to membrane lysis. In this paper, we find that alpha-hemolysin may bind the membranes in at least two ways, a reversible adsorption and an irreversible insertion. Reversibility is detected by the ability of liposome-bound toxin to induce hemolysis of added horse erythrocytes; insertion is accompanied by an increase in the protein intrinsic fluorescence. Toxin insertion does not necessarily lead to membrane lysis. Studies of alpha-hemolysin insertion into bilayers formed from a variety of single phospholipids, or binary mixtures of phospholipids, or of phospholipid and cholesterol, reveal that irreversible insertion is favored by fluid over gel states, by low over high cholesterol concentrations, by disordered liquid phases over gel or ordered liquid phases, and by gel over ordered liquid phases. These results are relevant to the mechanism of action of alpha-hemolysin and provide new insights into the membrane insertion of large proteins.
Subject(s)
Bacterial Proteins/chemistry , Escherichia coli Proteins , Hemolysin Proteins/chemistry , Lipid Bilayers , Phosphatidylcholines/chemistry , 1,2-Dipalmitoylphosphatidylcholine/chemistry , Adsorption , Bacterial Toxins/chemistry , Cholesterol/chemistry , Dimyristoylphosphatidylcholine/chemistry , Escherichia coli , Fluorescent Dyes , Kinetics , Protein Binding , Spectrometry, FluorescenceABSTRACT
Pro-haemolysin (approximately 110 kDa), the inactive precursor of the membrane-lytic toxin alpha-haemolysin, has been purified from an overproducing strain of Escherichia coli. Pro-haemolysin forms aggregates in aqueous media, like the mature protein, suggesting an amphipathic structure. Direct measurements of protein binding to liposomal membranes, following a novel procedure, show that pro-haemolysin can bind the lipid bilayers to a similar extent as alpha-haemolysin. This is confirmed by the observed changes in the intrinsic fluorescence emission of the protein upon binding the bilayers. However, pro-haemolysin is totally unable to induce liposomal membrane lysis. Binding of Ca2+, that is essential for the lytic activity of alpha-haemolysin, is greatly diminished in the precursor protein, as shown both by direct measurements of 45Ca(2+)-binding and by fluorescence measurements. The results suggest that binding of a fatty acyl residue in the activation step brings about an important conformational change in the protein that involves the Ca(2+)-binding domain.
Subject(s)
Bacterial Proteins/metabolism , Escherichia coli Proteins , Escherichia coli/chemistry , Hemolysin Proteins/metabolism , Protein Precursors/metabolism , Amino Acid Sequence , Bacterial Proteins/chemistry , Bacterial Proteins/isolation & purification , Calcium/metabolism , Electrophoresis, Polyacrylamide Gel , Hemolysin Proteins/chemistry , Hemolysin Proteins/isolation & purification , Hemolysis , Lipid Bilayers/metabolism , Liposomes/metabolism , Molecular Sequence Data , Phosphatidylcholines/metabolism , Protein Binding , Protein Precursors/chemistry , Protein Precursors/isolation & purification , Spectrometry, FluorescenceABSTRACT
Recombinant Nef-protein of HIV-1 Bru derived from Escherichia coli revealed heparin-binding activity. This property was used to purify the Nef-protein by a one-step procedure, yielding about 90% homogenous Nef-protein as evaluated by silver staining. The Nef-protein was soluble without denaturing agents. Native folding of Nef was demonstrated with antibodies against conformational epitopes of Nef by a slot blot assay under native conditions. Despite its affinity to heparin and its nuclear localization in persistently HIV-1 infected glioblastoma cells (Kohleisen et al., 1992), Nef did not show DNA-binding properties by slot blot/hybridization assay and South/Western blot. In nucleotide-binding assays a strong autophosphorylation activity with [gamma-32P]ATP was observed. Nef-protein was not a substrate for ADP-ribosylation by bacterial toxins arguing against G-protein-like activities of Nef. Recombinant Nef did not interact with membranes as shown by the lack of increased fluorescence emission of Nef in the presence of liposomes. The recombinant Nef-protein obtained by one-step heparin-based purification shares immunological properties with native Nef and should prove useful for further studies of Nef function and immunogenicity.
Subject(s)
Gene Products, nef/metabolism , HIV-1/metabolism , Heparin/metabolism , Animals , Antibodies, Monoclonal/immunology , Cell Membrane/metabolism , DNA/metabolism , Gene Products, nef/genetics , Gene Products, nef/isolation & purification , HIV Antibodies/immunology , Humans , Mice , Rabbits , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/isolation & purification , Recombinant Fusion Proteins/metabolism , Tumor Cells, Cultured , nef Gene Products, Human Immunodeficiency VirusABSTRACT
alpha-Haemolysin interaction with model membranes has been investigated by a 2-fold procedure. First, protein binding has been measured, by a direct method as well as through changes in the intrinsic fluorescence of the protein when incubated with liposomes and divalent cations. Then, the above results have been correlated with the protein lytic activity. The extent of protein binding is not significantly modified by the presence or absence of Ca2+, or by changes in lipid composition, although these factors influence greatly the membrane lytic activity of the protein. Moreover, Ca2+ binding to the toxin must occur prior to protein binding to the bilayer, for a lytic effect to take place.
Subject(s)
Bacterial Proteins/metabolism , Bacterial Toxins/metabolism , Escherichia coli Proteins , Hemolysin Proteins/metabolism , Liposomes/metabolism , Cations, Divalent , Membrane Lipids/metabolism , Protein BindingABSTRACT
alpha-haemolysin, an extracellular protein toxin of Escherichia coli, is known to disrupt eukaryotic cell membranes. In spite of genetic evidence of Ca(2+)-binding motifs in its sequence, conflicting results are found in the literature on the requirement of divalent cations for the membranolytic activity of the toxin. Moreover, Ca(2+)-binding sites have not been characterized to date in the native protein. The results in this paper show that when Ca2+ levels are kept sufficiently low during bacterial growth and toxin purification, membrane lysis does not occur in the absence of added divalent cations. Ca2+ and, at higher concentrations, Sr2+ and Ba2+, support the lytic activity, but Mg2+, Mn2+, Zn2+ and Cd2+ appear to be inactive in this respect. Binding of metal ions can be followed by changes in the intrinsic fluorescence of alpha-haemolysin; ions supporting lytic activity produce changes in the intrinsic fluorescence that are not caused by the inactive ones. Scatchard analysis of 45Ca2+ binding reveals three equivalent, independent sites, with Kd approximately 0.11 mM. No 45Ca2+ binding is observed when the protein is incubated with Zn2+; conversely, incubation with Ca2+ prevents subsequent binding of 65Zn2+. In the light of three-dimensional data available for a structurally related protein, alkaline protease of Pseudomonas aeruginosa [Baumann, U., Wu, S., Flaherty, K. M. & McKay, D. B. (1993) EMBO J. 12, 3357-3364] it is suggested that alpha-haemolysin may bind a larger number of Ca2+ than the three that are more easily exchangeable and are thus detected in the 45Ca(2+)-binding experiments. In addition, structural similarities and conservation of ion-binding motifs support the hypothesis that His 859 is involved in the mutually exclusive binding of Zn2+ and Ca2+.
Subject(s)
Bacterial Proteins/metabolism , Calcium/metabolism , Escherichia coli Proteins , Hemolysin Proteins/metabolism , Amino Acid Sequence , Bacterial Toxins/metabolism , Binding Sites , Binding, Competitive , Fluorescence , Molecular Sequence Data , Zinc/metabolismABSTRACT
alpha-Haemolysin is a protein toxin (107 kDa) secreted by some pathogenic strains of E. coli. It binds to mammalian cell membranes, disrupting cellular activities and lysing cells. This paper describes the mechanism of alpha-haemolysin-induced membrane leakage, from experiments in which extrusion large unilamellar vesicles, loaded with fluorescent solutes, are treated with purified toxin. The results show that the toxin does not require of any membrane receptor to exert its activity, that vesicles become leaky following an 'all-or-none' mechanism, and that leakage occurs through a non-osmotic detergent-like bilayer disruption induced by the protein. Small pores formed by monomeric alpha-haemolysin, as described by other authors, do not appear to be related to the process of membrane disruption. Instead, the experimental data would be in agreement with the idea of oligomeric assemblies being required to produce release of solutes from a single vesicle.
Subject(s)
Bacterial Proteins/pharmacology , Bacterial Toxins/pharmacology , Escherichia coli Proteins , Hemolysin Proteins/pharmacology , Liposomes/metabolism , Membrane Lipids/metabolism , Cell Membrane/chemistry , Cell Membrane/drug effects , Detergents/pharmacology , Dextrans/metabolism , Fluorescein-5-isothiocyanate/metabolism , Lipid Bilayers/chemistry , Membrane Lipids/analysis , Receptors, Cell Surface/physiologyABSTRACT
An improved, straightforward purification procedure for E.coli alpha-haemolysin has been developed. The protein exists in the form of large aggregates, held together mainly by hydrophobic forces. In the presence of urea or other chaotropic agents, the size of the aggregates decreases, while the specific activity is increased.
Subject(s)
Bacterial Proteins/isolation & purification , Bacterial Toxins/isolation & purification , Escherichia coli Proteins , Escherichia coli/analysis , Hemolysin Proteins , Bacterial Proteins/chemistry , Binding Sites , Chromatography , Solubility , UreaABSTRACT
The effects of a variety of detergents (non-ionic, ionic and bile derivatives) on B16 melanoma cells have been examined. Two main effects can be clearly differentiated: loss of cell viability and cell lysis. Under our conditions, cell-surfactant interaction is highly dependent on the nature of the amphiphile (more specifically, on its critical micellar concentration). Loss of cell viability occurs at surfactant concentrations below the critical micellar concentration, i.e. the incorporation of detergent monomers into the cell membranes is enough to impair their barrier function, so that Trypan Blue is no longer actively secreted outside the cell. On the other hand, cell lysis only occurs at or near the critical micellar concentration of the detergent, i.e. when the bilayer-micelle transition may take place. Comparative studies using B16 cells and phospholipid vesicles indicate that the amount of detergent required to induce cell lysis is the same that produces disruption of the lipid bilayer. Thus, our results suggest that membranes are the primary target for the toxicologic effects of surfactants on cells. Moreover, they provide a rationale for the interpretation of other studies in this field: previous results from different laboratories are shown to fit very well our data.