Membrane-perturbing Activities Research Articles

N-Terminally Added Tag Selectively Enhances Heterologous Expression of VacA Cytotoxin Variants from Helicobacter pylori.

Gastric pathogen Helicobacter pylori secretes VacA cytotoxin displaying a high degree of polymorphic variations of which the highest VacA pathogenicity correlates with m1-type variant followed by VacA-m2. To comparatively evaluate expression in Escherichia coli of the mature VacA variants (m1- and m2-types) and their 33- and 55/59-kDa domains fused with His(6) tag at N- or C-terminus. All VacA clones expressed in E. coli TOP10™ were analyzed by SDS-PAGE and Western blotting. VacA inclusions were solubilized under native conditions (~150-rpm shaking at 37°C for 2 h in 20 mM HEPES (pH7.4) and 150 mM NaCl). Membrane-perturbing and cytotoxic activities of solubilized VacA proteins were assessed via liposome-entrapped dye leakage and resazurin- based cell viability assays, respectively. VacA binding to human gastric adenocarcinoma cells was assessed by immunofluorescence microscopy. Side-chain hydrophobicity of VacA was analyzed through modeled structures constructed by homology- and ab initio-based modeling. Both full-length VacA-m1 and 33-kDa domain were efficiently expressed only in the presence of N-terminal extension while its 55-kDa domain was capably expressed with either N- or Cterminal extension. Selectively enhanced expression was also observed for VacA-m2. Protein expression profiles revealed a critical period in IPTG-induced production of the 55-kDa domain with N-terminal extension unlike its C-terminal extension showing relatively stable expression. Both VacA- m1 isolated domains were able to independently bind to cultured gastric cells similar to the full- length toxin, albeit the 33-kDa domain exhibited significantly higher activity of membrane perturbation than others. Membrane-perturbing and cytotoxic activities observed for VacA-m1 appeared to be higher than those of VacA-m2. Homology-based modeling and sequence analysis suggested a potential structural impact of non-polar residues located at the N-terminus of the mature VacA toxin and its 33-kDa domain. Our data provide molecular insights into selective influence of the N-terminally added tag on efficient expression of recombinant VacA variants, signifying biochemical and biological implications of the hydrophobic stretch within the N-terminal domain.

Membrane perturbing activities and structural properties of the frog-skin derived peptide Esculentin-1a(1-21)NH2 and its Diastereomer Esc(1-21)-1c: Correlation with their antipseudomonal and cytotoxic activity

Antimicrobial peptides (AMPs) represent new alternatives to cope with the increasing number of multi-drug resistant microbial infections. Recently, a derivative of the frog-skin AMP esculentin-1a, Esc(1-21), was found to rapidly kill both the planktonic and biofilm forms of the Gram-negative bacterium Pseudomonas aeruginosa with a membrane-perturbing activity as a plausible mode of action. Lately, its diastereomer Esc(1-21)-1c containing two d-amino acids i.e. DLeu14 and DSer17 revealed to be less cytotoxic, more stable to proteolytic degradation and more efficient in eradicating Pseudomonas biofilm. When tested in vitro against the free-living form of this pathogen, it displayed potent bactericidal activity, but this was weaker than that of the all-l peptide. To investigate the reason accounting for this difference, mechanistic studies were performed on Pseudomonas spheroplasts and anionic or zwitterionic membranes, mimicking the composition of microbial and mammalian membranes, respectively. Furthermore, structural studies by means of optical and nuclear magnetic resonance spectroscopies were carried out. Our results suggest that the different extent in the bactericidal activity between the two isomers is principally due to differences in their interaction with the bacterial cell wall components. Indeed, the lower ability in binding and perturbing anionic phospholipid bilayers for Esc(1-21)-1c contributes only in a small part to this difference, while the final effect of membrane thinning once the peptide is inserted into the membrane is identical to that provoked by Esc(1-21). In addition, the presence of two d-amino acids is sufficient to reduce the α-helical content of the peptide, in parallel with its lower cytotoxicity.

Ovalbumin with Glycated Carboxyl Groups Shows Membrane-Damaging Activity.

The aim of the present study was to investigate whether glycated ovalbumin (OVA) showed novel activity at the lipid-water interface. Mannosylated OVA (Man-OVA) was prepared by modification of the carboxyl groups with p-aminophenyl α-dextro (d)-mannopyranoside. An increase in the number of modified carboxyl groups increased the membrane-damaging activity of Man-OVA on cell membrane-mimicking vesicles, whereas OVA did not induce membrane permeability in the tested phospholipid vesicles. The glycation of carboxyl groups caused a notable change in the gross conformation of OVA. Moreover, owing to their spatial positions, the Trp residues in Man-OVA were more exposed, unlike those in OVA. Fluorescence quenching studies suggested that the Trp residues in Man-OVA were located on the interface binds with the lipid vesicles, and their microenvironment was abundant in positively charged residues. Although OVA and Man-OVA showed a similar binding affinity for lipid vesicles, the lipid-interacting feature of Man-OVA was distinct from that of OVA. Chemical modification studies revealed that Lys and Arg residues, but not Trp residues, played a crucial role in the membrane-damaging activity of Man-OVA. Taken together, our data suggest that glycation of carboxyl groups causes changes in the structural properties and membrane-interacting features of OVA, generating OVA with membrane-perturbing activities at the lipid-water interface.

Synthesis, spectroscopy, theoretical and biological studies of new gramine-steroids salts and conjugates

New gramine connections with bile acids (lithocholic, deoxycholic, cholic) and sterols (cholesterol, cholestanol) were synthesized. The structures of products were confirmed by spectral (NMR, FT-IR) analysis, mass spectrometry (ESI-MS) as well as PM5 semiempirical methods. Unexpectedly, the products of the reaction of gramine with cholesterol and cholestanol were symmetrical compounds consisting of two molecules of sterols connected by N(CH3)2 group. All new synthesized compounds interact in vitro with the human erythrocyte membrane and alter discoid erythrocyte shape inducing stomatocytosis or echinocytosis. Increase in the incorporation of the fluorescent dye merocyanine 540 (MC540) into the erythrocyte membrane indicates that new compounds at sublytic concentrations are capable of disturbing membrane phospholipids asymmetry and loosening the molecular packing of phospholipids in the bilayer. Gramine significantly decreases the membrane partitioning properties as well as haemolytic activity of lithocholic acid in its new salt. Moreover, both deoxycholic and cholic acids completely lost their membrane perturbing activities in the gramine salts. On the other hand, the capacity of new gramine-sterols connections to alter the erythrocyte membrane structure and its permeability is much higher in comparison with sterols alone. The dual effect of gramine on the bile acid and sterols cell membrane partitioning activity observed in our study should not be neglected in vivo.

Bovine serum albumin with glycated carboxyl groups shows membrane-perturbing activities

The aim of the present study aimed to investigate whether glycated bovine serum albumin (BSA) showed novel activities on the lipid–water interface. Mannosylated BSA (Man-BSA) was prepared by modification of the carboxyl groups with p-aminophenyl α-d-mannopyranoside. In contrast to BSA, Man-BSA notably induced membrane permeability of egg yolk phosphatidylcholine (EYPC)/egg yolk sphingomyelin (EYSM)/cholesterol (Chol) and EYPC/EYSM vesicles. Noticeably, Man-BSA induced the fusion of EYPC/EYSM/Chol vesicles, but not of EYPC/EYSM vesicles. Although BSA and Man-BSA showed similar binding affinity for lipid vesicles, the lipid-bound conformation of Man-BSA was distinct from that of BSA. Moreover, Man-BSA adopted distinct structure upon binding with the EYPC/EYSM/Chol and EYPC/EYSM vesicles. Man-BSA could induce the fusion of EYPC/EYSM/Chol vesicles with K562 and MCF-7 cells, while Man-BSA greatly induced the leakage of Chol-depleted K562 and MCF-7 cells. The modified BSA prepared by conjugating carboxyl groups with p-aminophenyl α-d-glucopyranoside also showed membrane-perturbing activities. Collectively, our data indicate that conjugation of carboxyl groups with monosaccharide generates functional BSA with membrane-perturbing activities on the lipid–water interface.

Conformation and Membrane Orientation of Amphiphilic Helical Peptides by Oriented Circular Dichroism

Oriented circular dichroism (OCD) was used to characterize and compare in a quantitative manner the secondary structure and concentration dependent realignment of the antimicrobial peptides PGLa and MSI-103, and of the structurally related cell-penetrating peptide MAP in aligned phospholipid bilayers. All these peptides adopt an amphiphilic α-helical conformation, and from solid-state NMR analysis they are known to bind to membranes in two distinct orientations depending on their concentration. At low peptide/lipid (P/L) ratio the helices are aligned parallel to membrane surface (S-state), but with increasing concentration they realign to a tilted orientation (T-state), getting immersed into the membrane with an oblique angle supposedly as a result of dimer-formation. In macroscopically aligned liquid crystalline 1,2-dimyristoyl-sn-glycero-3-phosphatidylcholine bilayers the two limiting states are represented by distinct OCD spectra, and all spectra at intermediate peptide concentrations can be described by a linear combination of these two line shapes. The corresponding fraction of molecules occupying the T-state was determined by fitting the intermediate spectra with a superposition of the two extreme line shapes. By plotting this fraction versus 1/(P/L), the threshold P/L* ratio for realignment was extracted for each of the three related peptides. Despite their structural similarity distinctly different thresholds were obtained, namely for MSI-103 realignment starts already at a low P/L of ∼1:236, for a MAP derivative (using a nonaggregating analog containing a D-amino acid) the transition begins at P/L ∼1:156, whereas PGLa needs the highest concentration to flip into T-state at P/L ∼1:85. Analysis of the original MAP sequence (containing only L-amino acids) gave OCD spectra compatible with β-pleated conformation, suggesting that this peptide starts to aggregate with increasing concentration, unlike the other helical peptides. All these changes in peptide conformation and membrane alignment observed here by OCD seem to be functionally relevant, as they can be correlated with the membrane perturbing activities of the three antimicrobial and cell-penetrating sequences.

Modulation of the physical properties of dielaidoylphosphatidylethanolamine membranes by a dirhamnolipid biosurfactant produced by Pseudomonas aeruginosa

Rhamnolipids are bacterial biosurfactants produced by Pseudomonas spp. These compounds have been shown to present several interesting biological activities, restricting the growth of Bacillus subtilis and showing zoosporicidal activity on zoosporic phytopathogens. It has been suggested that the interaction with the membrane could be the ultimate responsible for these actions. Therefore, it is of great interest to get insight into the molecular mechanism of the interaction of purified rhamnolipids with the various phospholipid components of biological membranes. In this paper we report on the phase behaviour of mixtures of dielaidoylphosphatidylethanolamine (DEPE) with a purified dirhamnolipid (DiRL) fraction from Pseudomonas aeruginosa, as studied by a number of physical techniques such as differential scanning calorimetry, FTIR, small angle X-ray (SAX) diffraction and dynamic light scattering. Our data indicate that the presence of DiRL counteracts the tendency of DEPE to form vesicular aggregates of large size, forming vesicles of smaller diameter which most probably have a lower lamellarity index. The partial phase diagram obtained from calorimetric data shows a complex behaviour with a solid-phase immiscibility. X-ray diffraction shows that DiRL has a bilayer stabilizing effect, impeding formation of the inverted hexagonal-H II phase of DEPE. The presented data are discussed focussing into how DiRL/DEPE interactions could help to explain the membrane perturbing activities of this biosurfactant.

An optimized amphiphilic cationic peptide as an efficient non-viral gene delivery vector.

Due to their chemical definition and reduced size, the use of peptides as gene delivery systems is gaining interest as compared to the more common polymeric non-viral vectors. To achieve gene transfer efficiencies that would make peptides a realistic alternative to existing methods, we have evaluated and attempted to concert those properties with a direct impact on the activity of the system. These considerations have led to the design, synthesis and characterization of a 23-residue cationic peptide which we term RAWA. We have characterized RAWA biophysically and functionally. Biophysical studies include evaluation of DNA condensation and membrane perturbing activities. DNA transfer activity has been evaluated in cell culture at controlled DNA-to-peptide stoichiometries, using a luciferase gene as reporter. Requirements for additional effectors such as chloroquine and peptide cofactors have also been considered. RAWA displays in vitro DNA condensing activity similar to that of protamines, reaching maximum effect at a peptide-to-DNA molar charge ratio (CR) of 4 (+/-). The reduced membrane perturbing activity diminishes its cytotoxic potential. In COS-7 cells, transfection efficiency with RAWA peptiplexes, compares favorably with well-recognized systems, including Lipofectamine Plus, Superfect, GenePorter and FuGene. The peptide-associated activity between free and DNA-bound species has been mapped by analyzing dependency on chloroquine treatment. The lack of significant serum inhibition and low toxicity make this system advantageous for potential in vivo application. A ternary complex including the acid-triggered fusogenic JTS-1 peptide is presented as a potential strategy for further in vivo studies. We have developed a gene delivery system based on an amphipathic cationic peptide with improved DNA condensation ability and reduced cytotoxicity, which maintains membrane binding and perturbing activities. Observed efficiency with this molecule is very high and compares favorably with currently available transfection systems.

Structural properties of the putative fusion peptide of fertilin, a protein active in sperm-egg fusion, upon interaction with the lipid bilayer.

We recently demonstrated that a peptide representing the putative fusion domain of fertilin, a surface membrane protein of sperm involved in sperm-egg fusion, induces fusion of large unilamellar vesicles containing negatively charged lipids [Martin, I., and Ruysschaert, J. M. (1997) FEBS Lett. 405, 351-355]. In the present work, we demonstrate that increasing the concentration in negatively charged lipids strongly enhances the binding of the fertilin fusion peptide to the membrane, suggesting that electrostatic attractions play a crucial role in the binding process. While no significant change of the secondary structure content is observed by increasing the amounts of negatively charged lipids in the bilayer, the orientation of the alpha-helix changes from a parallel to an oblique orientation in the membrane. This topological change is confirmed by amide II hydrogen/deuterium exchange measurements that monitor the accessibility of the peptide to the water medium. Differential scanning calorimetry data also suggest that the fertilin fusion peptide lowers the bilayer to hexagonal phase transition temperature of model membranes composed of mixtures of dipalmitoleoylphosphatidylethanolamine and 1-palmitoyl-2-oleoylphosphatidylserine and therefore promotes negative curvature in lipid vesicles. A comparison of the biophysical properties and the membrane-perturbing activities of fertilin and of viral fusion peptides is discussed in terms of sperm-egg fusion and virus cell fusion.

Enhanced Membrane-Perturbing Activities of Bundled Amphiphilic α-Helix Polypeptides on Interaction with Phospholipid Bilayer

Abstract The 24-peptide 46, designed to take an amphiphilic α-helix structure, had strong activity toward phospholipid membranes and could form ion-channels selective for cations on a planar membrane [J. Biol. Chem., 266, 20218 (1991)]. Four, six, and eight segments of the peptide 46 were bundled (4α-46, 6α-46, and 8α-46) on dendrimers consisting of Lys residues. These bundled peptides had highly α-helical structures with an enhanced α-helicity compared with the original 46 peptide. These peptides had hydrophobic pockets inside the bundle structures in aqueous solution in which the fluorescent hydrophobic probe, 1-anilino-8-naphthalenesulfonate (ANS), bound strongly. The peptides caused much leakage of carboxyfluorescein from small unilamellar vesicles of phospholipids at much lower concentrations of peptides than 46 did. Furthermore, the peptides induced the vesicle fusion at lower concentrations than those of the leakage. Four segments of the analogous 24-peptide 46S, in which six Ser residues were introduced instead of Ala and Leu residues in 46, were bundled by the same method (4α-46S). The fluorescent properties of the Trp residues incorporated at the 1 and 12 positions in 4α-46S, respectively, indicated that the centers of the helices were in more hydrophobic conditions than the N-terminal was. These facts caused us to conclude that the bundled conformation increased the perturbation of the phospholipid membrane, and as a result, the peptides were embedded in the membrane.

Selective Effects of Mastoparan Analogs: Separation of G-Protein-Directed and Membrane-Perturbing Activities

Mastoparan (MP), a wasp venom peptide, is known both to interact with G-proteins and to alter membrane structure and function. To determine the structural requirements for these two aspects of MP action, we constructed several analogs of the peptide and characterized them using Swiss 3T3 cell membranes. The effects of these peptides were measured on: i) G-protein-mediated stimulation of phospholipase-C activity by GTPγS and bombesin and ii) the membrane enzyme activities, calcium-activated phospholipase-C and Na,K-ATPase. MP strongly inhibited all the above activities and caused membrane permeabilization. Substitution of one Lys residue by Gly at either the N- or C-terminal of the MP molecule resulted in peptides which selectively inhibited G-protein stimulated phospholipase-C with no or very slight membrane-perturbing effects. Introduction of additional Lys residues to MP led to the opposite effect. Thus, G-protein modulating and membrane disrupting actions of MP appear to be not necessarily linked, and may be separated.

Steroid-induced perturbations of membranes and its relevance to sperm acrosome reaction

The interaction of progesterone, 17-α-hydroxyprogesterone, testosterone and estradiol with membrane vesicles prepared from phosphatidylserine (PS), from the total lipids of human and hamster spermatozoa, from the lipids of hamster spermatozoal plasma and acrosomal membrane and with the native membranes of hamster spermatozoa have been investigated by 90° light scattering and fluorescence spectroscopy. The results indicate that progesterone decreases the fluidity of membranes, aggregates membrane vesicles, induces fusiionn of membrane vesicles and also renders them permeable to hydrophilic molecules like car☐yfluorescein. But, testosterone and estradiol at the same concentration had very little effect on membrane fluidity, membrane aggregation, fusion and leakage. The above membrane perturbing activities of the steroids is discussed in light of the recent findings that progesterone induces acrosome reaction in human and hamster spermatozoa [11, 18].

Physicochemical determinants for the interactions of magainins 1 and 2 with acidic lipid bilayers

Permeability enhancement of acidic lipid small unilamellar vesicles (dioleoylphosphatidylglycerol, DOPG; dipalmitoyl-phosphatidylglycerol, DPPG; bovine brain phosphatidylserine, PS) induced by magainins 1 and 2, basic antimicrobial peptides from Xenopus skin, was investigated at 30°C based on leakage of calcein, an entrapped fluorescent marker. Both the peptide concentration and the lipid concentration dependencies of the leakage rate were analyzed to obtain the binding isotherms of the peptides to the membranes and the ‘membrane-perturbing activities’ of the membrane-bound peptides. For both peptides, the binding affinity was in the order DOPG > PPG > PS, which coincided with the zeta potential order (−54, −39, and −9 mV, respectively). An increase in salt concentration of the medium reduced binding and leakage. Electrostatic interactions play a crucial role in the binding process. On the other hand, the membrane-perturbing activity is regulated by membrane fluidity: The fluid membranes (DOPG and PS) were leakier. A circular dichroism study suggested that at least 14 positively charged residues in the N-terminal regions can form amphiphilic helices which interact with the membranes. An even stronger binding of magainin 2 can be explained in terms of more positive charges in its N-terminal region. A tentative model for the magainin-lipid interactions is hypothesized.