Azolectin

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Joaquim Procopio - One of the best experts on this subject based on the ideXlab platform.

  • influence of the bilayer composition on the membrane disruption effect of polybia mp1 a mastoparan peptide with antimicrobial and leukemic cell selectivity
    Biophysical Journal, 2009
    Co-Authors: Marcia Perez Dos Santos Cabrera, Manoel Arcisiomiranda, Bibiana Monson De Souza, Joao Ruggiero Neto, Mario Sergio Palma, Renata Gorjao, Natalia Bueno Leite, Rui Cury, Joaquim Procopio
    Abstract:

    Unlike other mastoparans, Polybia-MP1 (IDWKKLLDAAKQIL), from the venom Polybia paulista (wasp), is highly selective for bacterial cells. By flow cytometry, we also found out this selective behavior: Polybia-MP1 promoted a decrease of 60 % cell viability at 25 μM in Jurkat (leukemic) cells, while it was not altered in primary human lymphocytes. The mechanism of selectivity was studied in the interaction with different bilayers. Ion channel-like activity was detected at 0.12 μM peptide concentration with anionic lipid membranes of Azolectin, showing conductance in the range of 250 pS. On zwitterionic diphytanoylphosphatidylcholine-(DPhPC) it required 0.18 μM for the same conductance level. Further experiments with DPhPC bilayers containing 30% phophatidylserine or cardiolipin required higher peptide concentration to induce single channel events at slightly lower conductance levels. However, the presence of 20 mol% cholesterol in the mixture significantly reduced the ion channel-like activity, dropped the average conductance to around 120 pS and required 0.30 μM. On vesicles the activity of Polybia-MP1 also shows greater rate of leakage on the anionic over the zwitterionic, impaired by the presence of cholesterol; the lytic activity is characterized by a threshold peptide to lipid molar ratio that depends on the phospholipid composition. Preliminary results of changes in DPH anisotropy and acrylamide quenching of Trp fluorescence show a slight decrease in the anisotropy, and a significant quenching of the Trp fluorescence, indicating small influence on the lipid packing associated to preferential interaction with the lipid head group region. Results suggest that the selectivity of Polybia-MP1 is a consequence of a shallow interaction with zwitterionic bilayers, favored by the presence and position of negatively charged Asp residues, which is not possible for other mastoparan peptides.Support: CAPES, CNPq, FAPESP

  • effects of the cationic antimicrobial peptide eumenitin from the venom of solitary wasp eumenes rubronotatus in planar lipid bilayers surface charge and pore formation activity
    Toxicon, 2008
    Co-Authors: Manoel Arcisiomiranda, Marcia Perez Dos Santos Cabrera, Katsuhiro Konno, Marisa Rangel, Joaquim Procopio
    Abstract:

    Abstract Eumenitin, a novel cationic antimicrobial peptide from the venom of solitary wasp Eumenes rubronotatus , was characterized by its effects on black lipid membranes of negatively charged (Azolectin) and zwitterionic (1,2-diphytanoyl- sn -glycero-3-phosphocholine (DPhPC) or DPhPC-cholesterol) phospholipids: surface potential changes, single-channel activity, ion selectivity, and pore size were studied. We found that eumenitin binds preferentially to charged lipid membranes as compared with zwitterionic ones. Eumenitin is able to form pores in Azolectin ( G 1 =118.00±3.67 pS or G 2 =160.00±7.07 pS) and DPhPC membranes ( G =61.13±7.57 pS). Moreover, cholesterol addition to zwitterionic DPhPC membranes inhibits pore formation activity but does not interfere with the binding of peptide. Open pores presented higher cation (K + ) over anion (Cl − ) selectivity. The pore diameter was estimated at between 8.5and 9.8 A in Azolectin membranes and about 4.3 A in DPhPC membranes. The results are discussed based on the toroidal pore model for membrane pore-forming activity and ion selectivity.

Roland Benz - One of the best experts on this subject based on the ideXlab platform.

  • pore forming activity of the potent rtx toxin produced by pediatric pathogen kingella kingae characterization and comparison to other rtx family members
    Biochimica et Biophysica Acta, 2015
    Co-Authors: Ivan Barcenauribarri, Eleonora Zakharian, Roland Benz, Mathias Winterhalter, Nataliya V Balashova
    Abstract:

    Pediatric septic arthritis in patients under age of four is frequently caused by the oral Gram-negative bacterium Kingella kingae. This organism may be responsible for a severe form of infective endocarditis in otherwise healthy children and adults. A major virulence factor of K. kingae is RtxA, a toxin that belongs to the RTX (Repeats-in-ToXin) group of secreted pore forming toxins. To understand the RtxA effects on host cell membranes, the toxin activity was studied using planar lipid bilayers. K. kingae strain PYKK081 and its isogenic RtxA-deficient strain, KKNB100, were tested for their ability to form pores in artificial membranes of asolectin/n-decane. RtxA, purified from PYKK081, was able to rapidly form pores with an apparent diameter of 1.9 nm as measured by the partition of nonelectrolytes in the pores. The RtxA channels are cation-selective and showed strong voltage-dependent gating. In contrast to supernatants of PYKK081, those of KKNB100 did not show any pore forming activity. We concluded that RtxA toxin is the only secreted protein from K. kingae forming large channels in host cell membranes where it induces cation flux leading to programmed cell death. Furthermore, our findings suggested that the planar lipid bilayer technique can effectively be used to test possible inhibitors of RTX toxin activity and to investigate the mechanism of the toxin binding to the membrane.

  • adenylate cyclase toxin cyaa of bordetella pertussis evidence for the formation of small ion permeable channels and comparison with hlya of escherichia coli
    Journal of Biological Chemistry, 1994
    Co-Authors: Roland Benz, E Maier, Peter Sebo
    Abstract:

    Abstract The interaction between the adenylate cyclase toxin (CyaA) of Bordetella pertussis and lipid was studied using the lipid bilayer assay. The addition of CyaA to the aqueous phase bathing lipid bilayer membranes composed of different lipids resulted in the increase of the membrane conductance. This increase was rather small for membranes formed of pure lipids as compared with lipid mixtures such as asolectin. The toxin formed in asolectin membranes small transient ion-permeable channels with a single-channel conductance of 27 pS in 1 M KCl, which is considerably smaller than that of the alpha-hemolysin (HlyA) of Escherichia coli (1500 pS). Experiments with different salts suggested that the CyaA-induced channels were exclusively cation-selective because of negative charges localized at the channel mouth. The single-channel conductance of channels initiated by CyaA was independent of whether the toxin was purified from B. pertussis or from recombinant E. coli. However, the channel-forming activity of the CyaA expressed in B. pertussis was substantially higher than that of the recombinant toxin. Experiments with mutant forms of CyaA suggested that both the activation of CyaA by CyaC and the hemeolytic part of the toxin, but not the repeats and the cyclase activity, are required for channel formation in lipid bilayer membranes.

  • pore formation in artificial membranes by the secreted hemolysins of proteus vulgaris and morganella morganii
    FEBS Journal, 1994
    Co-Authors: Roland Benz, Kim R Hardie, Colin Hughes
    Abstract:

    Lipid-bilayer experiments were performed with the related hemolysins from Proteus vulgaris and Morganella morganii (HlyA). The addition of the toxins to the aqueous phase bathing lipidbilayer membranes composed of different lipids resulted in the formation of transient ion-permeable channels. Membranes formed of pure lipids were rather inactive targets for the hemolysins as compared with lipid mixtures such as asolectin. The channels had several different substates. The major open state had single-channel conductances of 500 pS in 0.15 M KC1 at small transmembrane voltages. Experiments with different salts suggested that the hemolysin-induced channels of l? vulgaris and M. morganii were exclusively cation selective at neutral pH, caused by negative charges localized at the channel mouth. The mobility sequence of the cations within the channels was similar if not identical to their mobility sequence in the aqueous phase. The single-channel data were consistent with wide, water-filled channels with estimated minimal diameters of about 1 nm since the large organic cation Tris' can permeate the channels without any detectable interaction with its interior. Pore-forming properties of these hemolysins were compared with those of HlyA of Escherichia coli. All these toxins share common features, oligomerize probably to form pores in lipidbilayer membranes and form channels with similar properties which suggests that their structures are more or less identical.

Marisa Rangel - One of the best experts on this subject based on the ideXlab platform.

  • Peptidomic analysis of the venom of the solitary bee Xylocopa appendiculata circumvolans
    Journal of Venomous Animals and Toxins including Tropical Diseases, 2017
    Co-Authors: Kohei Kazuma, Marisa Rangel, Kenji Ando, Ken-ichi Nihei, Xiaoyu Wang, Marcia Regina Franzolin, Kanami Mori-yasumoto, Setsuko Sekita, Makoto Kadowaki, Motoyoshi Satake
    Abstract:

    Background Among the hymenopteran insect venoms, those from social wasps and bees – such as honeybee, hornets and paper wasps – have been well documented. Their venoms are composed of a number of peptides and proteins and used for defending their nests and themselves from predators. In contrast, the venoms of solitary wasps and bees have not been the object of further research. In case of solitary bees, only major peptide components in a few venoms have been addressed. Therefore, the aim of the present study was to explore the peptide component profile of the venom from the solitary bee Xylocopa appendiculata circumvolans by peptidomic analysis with using LC-MS. Methods A reverse-phase HPLC connected to ESI-OrbiTrap MS was used for LC-MS. On-line mass fingerprinting was made from TIC, and data-dependent tandem mass spectrometry gave MSMS spectra. A major peptide component was isolated by reverse-phase HPLC by conventional way, and its sequence was determined by Edman degradation, which was finally corroborated by solid phase synthesis. Using the synthetic specimen, biological activities (antimicrobial activity, mast cell devaluation, hemolysis, leishmanicidal activity) and pore formation in artificial lipid bilayer were evaluated. Results On-line mass fingerprinting revealed that the crude venom contained 124 components. MS/MS analysis gave 75 full sequences of the peptide components. Most of these are related to the major and novel peptide, xylopin. Its sequence, GFVALLKKLPLILKHLH-NH_2, has characteristic features of linear cationic α-helical peptides; rich in hydrophobic and basic amino acids with no disulfide bond, and accordingly, it can be predicted to adopt an amphipathic α-helix secondary structure. In biological evaluation, xylopin exhibited broad-spectrum antimicrobial activity, and moderate mast cell degranulation and leishmanicidal activities, but showed virtually no hemolytic activity. Additionally, the peptide was able to incorporate pores in artificial lipid bilayers of Azolectin, confirming the mechanism of the cytolytic activity by pore formation in biological membranes. Conclusions LC-ESI-MS and MS/MS analysis of the crude venom extract from a solitary bee Xylocopa appendiculata circumvolans revealed that the component profile of this venom mostly consisted of small peptides. The major peptide components, xylopin and xylopinin, were purified and characterized in a conventional manner. Their chemical and biological characteristics, belonging to linear cationic α-helical peptides, are similar to the known solitary bee venom peptides, melectin and osmin. Pore formation in artificial lipid bilayers was demonstrated for the first time with a solitary bee peptide.

  • Peptidomic analysis of the venom of the solitary bee Xylocopa appendiculata circumvolans.
    Journal of Venomous Animals and Toxins Including Tropical Diseases, 2017
    Co-Authors: Kohei Kazuma, Marisa Rangel, Kenji Ando, Ken-ichi Nihei, Xiaoyu Wang, Marcia Regina Franzolin, Kanami Mori-yasumoto, Setsuko Sekita, Makoto Kadowaki, Motoyoshi Satake
    Abstract:

    Among the hymenopteran insect venoms, those from social wasps and bees – such as honeybee, hornets and paper wasps – have been well documented. Their venoms are composed of a number of peptides and proteins and used for defending their nests and themselves from predators. In contrast, the venoms of solitary wasps and bees have not been the object of further research. In case of solitary bees, only major peptide components in a few venoms have been addressed. Therefore, the aim of the present study was to explore the peptide component profile of the venom from the solitary bee Xylocopa appendiculata circumvolans by peptidomic analysis with using LC-MS. A reverse-phase HPLC connected to ESI-OrbiTrap MS was used for LC-MS. On-line mass fingerprinting was made from TIC, and data-dependent tandem mass spectrometry gave MSMS spectra. A major peptide component was isolated by reverse-phase HPLC by conventional way, and its sequence was determined by Edman degradation, which was finally corroborated by solid phase synthesis. Using the synthetic specimen, biological activities (antimicrobial activity, mast cell devaluation, hemolysis, leishmanicidal activity) and pore formation in artificial lipid bilayer were evaluated. On-line mass fingerprinting revealed that the crude venom contained 124 components. MS/MS analysis gave 75 full sequences of the peptide components. Most of these are related to the major and novel peptide, xylopin. Its sequence, GFVALLKKLPLILKHLH-NH2, has characteristic features of linear cationic α-helical peptides; rich in hydrophobic and basic amino acids with no disulfide bond, and accordingly, it can be predicted to adopt an amphipathic α-helix secondary structure. In biological evaluation, xylopin exhibited broad-spectrum antimicrobial activity, and moderate mast cell degranulation and leishmanicidal activities, but showed virtually no hemolytic activity. Additionally, the peptide was able to incorporate pores in artificial lipid bilayers of Azolectin, confirming the mechanism of the cytolytic activity by pore formation in biological membranes. LC-ESI-MS and MS/MS analysis of the crude venom extract from a solitary bee Xylocopa appendiculata circumvolans revealed that the component profile of this venom mostly consisted of small peptides. The major peptide components, xylopin and xylopinin, were purified and characterized in a conventional manner. Their chemical and biological characteristics, belonging to linear cationic α-helical peptides, are similar to the known solitary bee venom peptides, melectin and osmin. Pore formation in artificial lipid bilayers was demonstrated for the first time with a solitary bee peptide.

  • effects of the cationic antimicrobial peptide eumenitin from the venom of solitary wasp eumenes rubronotatus in planar lipid bilayers surface charge and pore formation activity
    Toxicon, 2008
    Co-Authors: Manoel Arcisiomiranda, Marcia Perez Dos Santos Cabrera, Katsuhiro Konno, Marisa Rangel, Joaquim Procopio
    Abstract:

    Abstract Eumenitin, a novel cationic antimicrobial peptide from the venom of solitary wasp Eumenes rubronotatus , was characterized by its effects on black lipid membranes of negatively charged (Azolectin) and zwitterionic (1,2-diphytanoyl- sn -glycero-3-phosphocholine (DPhPC) or DPhPC-cholesterol) phospholipids: surface potential changes, single-channel activity, ion selectivity, and pore size were studied. We found that eumenitin binds preferentially to charged lipid membranes as compared with zwitterionic ones. Eumenitin is able to form pores in Azolectin ( G 1 =118.00±3.67 pS or G 2 =160.00±7.07 pS) and DPhPC membranes ( G =61.13±7.57 pS). Moreover, cholesterol addition to zwitterionic DPhPC membranes inhibits pore formation activity but does not interfere with the binding of peptide. Open pores presented higher cation (K + ) over anion (Cl − ) selectivity. The pore diameter was estimated at between 8.5and 9.8 A in Azolectin membranes and about 4.3 A in DPhPC membranes. The results are discussed based on the toroidal pore model for membrane pore-forming activity and ion selectivity.

Peter Sebo - One of the best experts on this subject based on the ideXlab platform.

  • Residues 529 to 549 participate in membrane penetration and pore-forming activity of the Bordetella adenylate cyclase toxin.
    Scientific reports, 2019
    Co-Authors: Jana Roderova, Peter Sebo, Adriana Osickova, Radovan Fiser, Anna Sukova, Gabriela Mikušová, Radim Osicka
    Abstract:

    The adenylate cyclase toxin-hemolysin (CyaA, ACT or AC-Hly) of pathogenic Bordetellae delivers its adenylyl cyclase (AC) enzyme domain into the cytosol of host cells and catalyzes uncontrolled conversion of cellular ATP to cAMP. In parallel, the toxin forms small cation-selective pores that permeabilize target cell membrane and account for the hemolytic activity of CyaA on erythrocytes. The pore-forming domain of CyaA is predicted to consist of five transmembrane α-helices, of which the helices I, III, IV and V have previously been characterized. We examined here the α-helix II that is predicted to form between residues 529 to 549. Substitution of the glycine 531 residue by a proline selectively reduced the hemolytic capacity but did not affect the AC translocating activity of the CyaA-G531P toxin. In contrast, CyaA toxins with alanine 538 or 546 replaced by diverse residues were selectively impaired in the capacity to translocate the AC domain across cell membrane but remained fully hemolytic. Such toxins, however, formed pores in planar asolectin bilayer membranes with a very low frequency and with at least two different conducting states. The helix-breaking substitution of alanine 538 by a proline residue abolished the voltage-activated increase of membrane activity of CyaA in asolectin bilayers. These results reveal that the predicted α-helix comprising the residues 529 to 549 plays a key role in CyaA penetration into the target plasma membrane and pore-forming activity of the toxin.

  • adenylate cyclase toxin cyaa of bordetella pertussis evidence for the formation of small ion permeable channels and comparison with hlya of escherichia coli
    Journal of Biological Chemistry, 1994
    Co-Authors: Roland Benz, E Maier, Peter Sebo
    Abstract:

    Abstract The interaction between the adenylate cyclase toxin (CyaA) of Bordetella pertussis and lipid was studied using the lipid bilayer assay. The addition of CyaA to the aqueous phase bathing lipid bilayer membranes composed of different lipids resulted in the increase of the membrane conductance. This increase was rather small for membranes formed of pure lipids as compared with lipid mixtures such as asolectin. The toxin formed in asolectin membranes small transient ion-permeable channels with a single-channel conductance of 27 pS in 1 M KCl, which is considerably smaller than that of the alpha-hemolysin (HlyA) of Escherichia coli (1500 pS). Experiments with different salts suggested that the CyaA-induced channels were exclusively cation-selective because of negative charges localized at the channel mouth. The single-channel conductance of channels initiated by CyaA was independent of whether the toxin was purified from B. pertussis or from recombinant E. coli. However, the channel-forming activity of the CyaA expressed in B. pertussis was substantially higher than that of the recombinant toxin. Experiments with mutant forms of CyaA suggested that both the activation of CyaA by CyaC and the hemeolytic part of the toxin, but not the repeats and the cyclase activity, are required for channel formation in lipid bilayer membranes.

Manoel Arcisiomiranda - One of the best experts on this subject based on the ideXlab platform.

  • influence of the bilayer composition on the membrane disruption effect of polybia mp1 a mastoparan peptide with antimicrobial and leukemic cell selectivity
    Biophysical Journal, 2009
    Co-Authors: Marcia Perez Dos Santos Cabrera, Manoel Arcisiomiranda, Bibiana Monson De Souza, Joao Ruggiero Neto, Mario Sergio Palma, Renata Gorjao, Natalia Bueno Leite, Rui Cury, Joaquim Procopio
    Abstract:

    Unlike other mastoparans, Polybia-MP1 (IDWKKLLDAAKQIL), from the venom Polybia paulista (wasp), is highly selective for bacterial cells. By flow cytometry, we also found out this selective behavior: Polybia-MP1 promoted a decrease of 60 % cell viability at 25 μM in Jurkat (leukemic) cells, while it was not altered in primary human lymphocytes. The mechanism of selectivity was studied in the interaction with different bilayers. Ion channel-like activity was detected at 0.12 μM peptide concentration with anionic lipid membranes of Azolectin, showing conductance in the range of 250 pS. On zwitterionic diphytanoylphosphatidylcholine-(DPhPC) it required 0.18 μM for the same conductance level. Further experiments with DPhPC bilayers containing 30% phophatidylserine or cardiolipin required higher peptide concentration to induce single channel events at slightly lower conductance levels. However, the presence of 20 mol% cholesterol in the mixture significantly reduced the ion channel-like activity, dropped the average conductance to around 120 pS and required 0.30 μM. On vesicles the activity of Polybia-MP1 also shows greater rate of leakage on the anionic over the zwitterionic, impaired by the presence of cholesterol; the lytic activity is characterized by a threshold peptide to lipid molar ratio that depends on the phospholipid composition. Preliminary results of changes in DPH anisotropy and acrylamide quenching of Trp fluorescence show a slight decrease in the anisotropy, and a significant quenching of the Trp fluorescence, indicating small influence on the lipid packing associated to preferential interaction with the lipid head group region. Results suggest that the selectivity of Polybia-MP1 is a consequence of a shallow interaction with zwitterionic bilayers, favored by the presence and position of negatively charged Asp residues, which is not possible for other mastoparan peptides.Support: CAPES, CNPq, FAPESP

  • effects of the cationic antimicrobial peptide eumenitin from the venom of solitary wasp eumenes rubronotatus in planar lipid bilayers surface charge and pore formation activity
    Toxicon, 2008
    Co-Authors: Manoel Arcisiomiranda, Marcia Perez Dos Santos Cabrera, Katsuhiro Konno, Marisa Rangel, Joaquim Procopio
    Abstract:

    Abstract Eumenitin, a novel cationic antimicrobial peptide from the venom of solitary wasp Eumenes rubronotatus , was characterized by its effects on black lipid membranes of negatively charged (Azolectin) and zwitterionic (1,2-diphytanoyl- sn -glycero-3-phosphocholine (DPhPC) or DPhPC-cholesterol) phospholipids: surface potential changes, single-channel activity, ion selectivity, and pore size were studied. We found that eumenitin binds preferentially to charged lipid membranes as compared with zwitterionic ones. Eumenitin is able to form pores in Azolectin ( G 1 =118.00±3.67 pS or G 2 =160.00±7.07 pS) and DPhPC membranes ( G =61.13±7.57 pS). Moreover, cholesterol addition to zwitterionic DPhPC membranes inhibits pore formation activity but does not interfere with the binding of peptide. Open pores presented higher cation (K + ) over anion (Cl − ) selectivity. The pore diameter was estimated at between 8.5and 9.8 A in Azolectin membranes and about 4.3 A in DPhPC membranes. The results are discussed based on the toroidal pore model for membrane pore-forming activity and ion selectivity.