Biochimica et Biophysica Acta (BBA) - Biomembranes, 2004
Pin2 and Oxki1 are cationic amphipathic peptides that permeate lipid membranes through formation ... more Pin2 and Oxki1 are cationic amphipathic peptides that permeate lipid membranes through formation of pores. Their mechanism of binding to phosphocholine (PC) membranes differs. Spin-probe experiments showed that both Pin2 and Oxki1 penetrate the lipid membrane of small unilamellar vesicles (SUVs). Moreover, the leakage of calcein and dextrans from PC vesicles showed that Pin2 agrees with the accumulation of peptides on lipid membranes and form pores of different size. On the other hand, Oxki1 did not act strictly cooperatively and form pores of limited size.
Two novel antimicrobial peptides have been identified and characterized from venom of the African... more Two novel antimicrobial peptides have been identified and characterized from venom of the African scorpion Pandinus imperator. The peptides, designated pandinin 1 and 2, are αhelical polycationic peptides, with pandinin 1 belonging to the group of antibacterial peptides previously described from scorpions, frogs and insects, and pandinin 2 to the group of short magainin-type helical peptides from frogs. Both peptides demonstrated high antimicrobial activity against a range of Grampositive bacteria (2.4-5.2 µM), but were less active against Gram-negative bacteria (2.4-38.2 µM), and only pandinin 2 affected the yeast Candida albicans. Pandinin 2 also demonstrated strong haemolytic activity (11.1-44.5 µM) against sheep erythrocytes, in contrast with pandinin 1, which was not haemolytic. CD studies and a high-resolution structure of pandinin 2
A mRNA transcript that codes for a phospholipase (PLA2) was isolated from a single venom gland of... more A mRNA transcript that codes for a phospholipase (PLA2) was isolated from a single venom gland of the Bothrops ammodytoides viper. The PLA2 transcript was cloned onto a pCR®2.1-TOPO vector and subsequently expressed heterologously in the E. coli strain M15, using the pQE30 vector. The recombinant phospholipase was named rBamPLA2_1, and is composed of an N-terminal fusion protein of 16 residues, along with 122 residues from the mature protein that includes 14 cysteines that form 7 disulfide bonds. Following bacterial expression, rBamPLA2_1 was obtained from inclusion bodies and extracted using a chaotropic agent. rBamPLA2_1 had an experimental molecular mass of 15,692.5 Da that concurred with its theoretical molecular mass. rBamPLA2_1 was refolded in in vitro conditions and after refolding, three main protein fractions with similar molecular masses, were identified. Although, the three fractions were considered to represent different oxidized cystine isoforms, their secondary structures were comparable. All three recombinant isoforms were active on egg-yolk phospholipid and recognized similar cell membrane phospholipids to be native PLA2s, isolated from B. ammodytoides venom. A mixture of the three rBamPLA2_1 cystine isoforms was used to immunize a horse in order to produce serum antibodies (anti-rBamPLA2_1), which partially inhibited the indirect hemolytic activity of B. ammodytoides venom. Although, anti-rBamPLA2_1 antibodies were not able to recognize crotoxin, a PLA2 from the venom of a related but different viper genus, Crotalus durissus terrificus, they recognized PLA2s in other venoms from regional species of Bothrops.
Cells of Lactobacillus delbrueckii subsp. lactis I produced hydrogen peroxide at 5°C in sodium ph... more Cells of Lactobacillus delbrueckii subsp. lactis I produced hydrogen peroxide at 5°C in sodium phosphate buffer (0.2M, pH 6.5) with or without glucose. However, if the cells were starved by preincubation in buffer alone, glucose or sodium lactate were necessary to cause hydrogen peroxide production at 5°C. Hydrogen peroxide production by nonstarved cells was confirmed to be in part due to a NADH oxidase. The production of hydrogen peroxide by starved cells in buffer plus glucose in the early stage of incubation was associated with the production of a small portion of lactic acid which disappeared upon further incubation. Additional experiments revealed that hydrogen peroxide was produced in buffer containing sodium lactate added in place of glucose. Results suggested the presence of a lactate oxidase in the organism which used D-lactate to produce hydrogen peroxide.
The ability ofCeratocystis fimbriata to generate aroma notes from different carbon and nitrogen s... more The ability ofCeratocystis fimbriata to generate aroma notes from different carbon and nitrogen sources was studied in liquid culture. The medium that gave the best sensory results produced a strong banana aroma. Other notes such as pineapple, apple, pear and nuts, with varied, intensities were obtained from other culture media. Biomass and metabolite productions are reported. In solid state fermentation,
Two nonamidated host defense peptides named Pin2[G] and FA1 were evaluated against three types of... more Two nonamidated host defense peptides named Pin2[G] and FA1 were evaluated against three types of pathogenic bacteria: two (Staphylococcus aureus UPD13 and Pseudomonas aeruginosa UPD3) isolated from diabetic foot ulcer patients, and another (Salmonella enterica serovar Typhimurium [ATCC 14028]) from a commercial collection. In vitro experiments showed that the antimicrobial performance of the synthetic peptides Pin2[G] and FA1 was modest, although FA1 was more effective than Pin2[G]. In contrast, Pin2[G] had superior in vivo anti-infective activity to FA1 in rabbit wound infections by the diabetic foot ulcer pathogens S. aureus UPD13 and P. aeruginosa UPD3. Indeed, Pin2[G] reduced bacterial colony counts of both S. aureus UPD13 and P. aeruginosa UPD3 by >100,000-fold after 48 to 72 h on skin wounds of infected rabbits, while in similar infected wounds, FA1 had no major effects at 72 to 96 h of treatment. Ceftriaxone was equally effective versus Pseudomonas but less effective vers...
Bothropic venoms contain enzymes such as metalloproteases, serine-proteases, and phospholipases, ... more Bothropic venoms contain enzymes such as metalloproteases, serine-proteases, and phospholipases, which acting by themselves, or in synergism, are the cause of the envenomation symptoms and death. Here, two mRNA transcripts, one that codes for a metalloprotease and another for a serine-protease, were isolated from a Bothrops ammodytoides venom gland. The metalloprotease and serine-protease transcripts were cloned on a pCR®2.1-TOPO vector and consequently expressed in a recombinant way in E. coli (strains Origami and M15, respectively), using pQE30 vectors. The recombinant proteins were named rBamSP_1 and rBamMP_1, and they were formed by an N-terminal fusion protein of 16 amino acid residues, followed by the sequence of the mature proteins. After bacterial expression, each recombinant enzyme was recovered from inclusion bodies and treated with chaotropic agents. The experimental molecular masses for rBamSP_1 and rBamMP_1 agreed with their expected theoretical ones, and their secondar...
Background and Aims: The production of edible fungi is affected by bacterial, fungal and viral di... more Background and Aims: The production of edible fungi is affected by bacterial, fungal and viral diseases, which very often produces large losses. In the production of mushrooms of the genus Pleurotus, the fungi of the genus Trichoderma represent a serious problem of contamination and although there are some chemical compounds that control the infection, they are not entirely safe for human consumption, so they are looking for alternatives through biotechnology, such as the one presented in this paper. Methods: Strains of fungi of the genus Trichoderma were isolated from the substrate where Pleurotus ostreatus was being cultivated (characteristic contamination of green mold), they were identified morphologically and molecularly, later tests were carried out to inhibit the growth of Trichoderma strains in both agar and wheat straw using a cetonic extract of the fruiting body dehydrated of Pycnoporus sp. Key results: Two strains of Trichoderma (Trichoderma pleuroti and Trichoderma atrob...
The genes of the five disulfide-bonded peptide toxins 1 and 2 (named Oxytoxins or Oxotoxins) from... more The genes of the five disulfide-bonded peptide toxins 1 and 2 (named Oxytoxins or Oxotoxins) from the spider Oxyopes lineatus were cloned into the expression vector pQE30 containing a 6His-tag and a Factor Xa proteolytic cleavage region. These two recombinant vectors were transfected into Escherichia coli BL21 cells and expressed under induction with isopropyl thiogalactoside (IPTG). The product of each gene was named HisrOxyTx1 or HisrOxyTx2, and the protein expression was ca 14 and 6 mg/L of culture medium, respectively. Either recombinant toxin HisrOxyTx1 or HisrOxyTx2 were found exclusively in inclusion bodies, which were solubilized using a chaotropic agent, and then, purified using affinity chromatography and reverse-phase HPLC (RP-HPLC). The HisrOxyTx1 and HisrOxyTx2 products, obtained from the affinity chromatographic step, showed several peptide fractions having the same molecular mass of 9913.1 and 8030.1 Da, respectively, indicating that both HisrOxyTx1 and HisrOxyTx2 were oxidized forming several distinct disulfide bridge arrangements. The isoforms of both HisrOxyTx1 and HisrOxyTx2 after DTT reduction eluted from the column as a single protein component of 9923 and 8040 Da, respectively. In vitro folding of either HisrOxyTx1 or HisrOxyTx2 yielded single oxidized components, which were cleaved independently by the proteolytic enzyme Factor Xa to give the recombinant peptides rOxyTx1 and rOxyTx2. The experimental molecular masses of rOxyTx1 and rOxyTx2 were 8059.0 and 6176.4 Da, respectively, which agree with their expected theoretical masses. The recombinant peptides rOxyTx1 and rOxyTx2 showed lower but comparable toxicity to the native toxins when injected into lepidopteran larvae; furthermore, rOxyTx1 was able to inhibit calcium ion currents on dorsal unpaired median (DUM) neurons from Periplaneta americana.
Advances in NMR and mass spectrometry as well as in peptide biochemistry coupled to modern method... more Advances in NMR and mass spectrometry as well as in peptide biochemistry coupled to modern methods in electrophysiology have permitted the isolation and identification of numerous products from spider venoms, previously unexplored due to technical limitations. The chemical composition of spider venoms is diverse, ranging from low molecular weight organic compounds such acylpolyamines to complex peptides. First, acylpolyamines (<1000 Da) have an aromatic moiety linked to a hydrophilic lateral chain. They were characterized for the first time in spider venoms, and are ligand-gated ion channel antagonists, which block mainly postsynaptic glutamate receptors in invertebrate and vertebrate nervous systems. Acylpolyamines represent the vast majority of organic components from the spider venom. Acylpolyamine analogues have proved to suppress hippocampal epileptic discharges. Moreover, acylpolyamines could suppress excitatory postsynaptic currents inducing Ca 2+ accumulation in neurons leading to protection against a brain ischemic insult. Second, short spider peptides (<6000 Da) modulate ionic currents in Ca 2+ , Na + , or K + voltage-gated ion channels. Such peptides may contain from three to four disulfide bridges. Some spider peptides act specifically to discriminate among Ca 2+ , Na + , and K + ion channel subtypes. Their selective affinities for ion channel subfamilies are functional for mapping excitable cells. Furthermore, several of these peptides have proved to hyperpolarize peripheral neurons, which are associated with supplying sensation to the skin and skeletal muscles. Some spider N-type calcium ion channel blockers may be important for the treatment of chronic pain. A special group of spider peptides are the amphipathic and positively charged peptides. Their secondary structure is a-helical and they insert into the lipid cell membrane of eukaryotic or prokaryotic cells leading to the formation of pores and subsequently depolarizing the cell membrane. Acylpolyamines and peptides from spider venoms represent an interesting source of molecules for the design of novel pharmaceutical drugs. 1 Introduction 2 Spider acylpolyamines 2.1 Acylpolyamines and glutamate receptors 2.2 Acylpolyamines and the polyamine catabolism pathway 2.3 Acylpolyamines as neuroprotective agents 2.4 Acylpolyamines and pain 2.5 Acylpolyamines and nervous diseases 2.6 Synthetic analogues of acylpolyamines 3 Spider peptides 3.1 Spider peptides and potassium channels 3.2 Spider peptides and calcium channels 3.3 Spider peptides and sodium channels 3.4 Glutamate receptors 3.5 Peptides acting on acid-sensing ion channels (ASIC) and mechanosensitive ion channels (MS) 3.6 Non-selective spider peptides 3.7 Pore-forming peptides
Two antimicrobial peptides (AMPs), named La47 and Css54, were isolated from the venom of the spid... more Two antimicrobial peptides (AMPs), named La47 and Css54, were isolated from the venom of the spider Lachesana sp. and from the scorpion Centruroides suffusus suffusus, respectively. The primary structures of both La47 and Css54 were determined using N-terminal sequencing and mass spectrometry. La47 is identical to the AMP latarcin 3a obtained previously from the venom of the spider Lachesana tarabaevi, but the primary structure of Css54 is unique having 60% identities to the AMP ponericin-W2 from the venom of the ant Pachycondyla goeldii. Both La47 and Css54 have typical a-helix secondary structures in hydrophobic mimicking environments. The biological activities of both La47 and Css54 were compared with the AMP Pin2 isolated from the venom of the scorpion Pandinus imperator. La47 has lower antimicrobial and hemolytic activities compared with Css54 and Pin2. In addition, La47 and Pin2 were evaluated in the presence of the commercial antibiotics, chloramphenicol, ampicillin, novobiocin, streptomycin and kanamycin. Interestingly, the best antimicrobial combinations were obtained with mixtures of La47 and Pin2 with the antibiotics chloramphenicol, streptomycin and kanamycin, respectively. Furthermore, the novel peptide Css54 was evaluated in the presence of antibiotics used for the treatment of tuberculosis, isoniazid, rifampicin, pyrazinamide and ethambutol. Although the mixtures of Css54 with isoniazid, pyrazinamide or ethambutol inhibit the growth of Staphylococcus aureus, the best effect was found with rifampicin. Overall, these data show a motivating outlook for potential clinical treatments of bacterial infections using AMPs and commercial antibiotics.
Six peptide toxins (Magi 1^6) were isolated from the Hexathelidae spider Macrothele gigas. The am... more Six peptide toxins (Magi 1^6) were isolated from the Hexathelidae spider Macrothele gigas. The amino acid sequences of Magi 1, 2, 5 and 6 have low similarities to the amino acid sequences of known spider toxins. The primary structure of Magi 3 is similar to the structure of the palmitoylated peptide named PlTx-II from the North American spider Plectreurys tristis (Plectreuridae). Moreover, the amino acid sequence of Magi 4, which was revealed by cloning of its cDNA, displays similarities to the Na + channel modi¢er N N-atracotoxin from the Australian spider Atrax robustus (Hexathelidae). Competitive binding assays using several 125 I-labelled peptide toxins clearly demonstrated the speci¢c binding a⁄nity of Magi 1^5 to site 3 of the insect sodium channel and also that of Magi 5 to site 4 of the rat sodium channel. Only Magi 6 did not compete with the scorpion toxin LqhK KIT in binding to site 3 despite high toxicity on lepidoptera larvae of 3.1 nmol/g. The K i s of other toxins were between 50 pM for Magi 4 and 1747 nM for Magi 1. In addition, only Magi 5 binds to both site 3 in insects (K i = 267 nM) and site 4 in rat brain synaptosomes (K i = 1.2 nM), whereas it showed no a⁄nities for either mammal binding site 3 or insect binding site 4. Magi 5 is the ¢rst spider toxin with binding a⁄nity to site 4 of a mammalian sodium channel.
Delta-palutoxins from the spider Paracoelotes luctuosus (Araneae: Amaurobiidae) are 36-37 residue... more Delta-palutoxins from the spider Paracoelotes luctuosus (Araneae: Amaurobiidae) are 36-37 residue long peptides that show preference for insect sodium channels (NaChs) and modulate their function. Although they slow NaCh inactivation in a fashion similar to that of receptor site 3 modifiers, such as scorpion alpha-toxins, they actually bind with high affinity to the topologically distinct receptor site 4 of scorpion beta-toxins. To resolve this riddle, we scanned by Ala mutagenesis the surface of delta-PaluIT2, a delta-palutoxin variant with the highest affinity for insect NaChs, and compared it to the bioactive surface of a scorpion beta-toxin. We found three regions on the surface of delta-PaluIT2 important for activity: the first consists of Tyr-22 and Tyr-30 (aromatic), Ser-24 and Met-28 (polar), and Arg-8, Arg-26, Arg-32, and Arg-34 (basic) residues; the second is made of Trp-12; and the third is made of Asp-19, whose substitution by Ala uncoupled the binding from toxicity to lepidopteran larvae. Although spider delta-palutoxins and scorpion beta-toxins have developed from different ancestors, they show some commonality in their bioactive surfaces, which may explain their ability to compete for an identical receptor (site 4) on voltage-gated NaChs. Yet, their different mode of channel modulation provides a novel perspective about the structural relatedness of receptor sites 3 and 4, which until now have been considered topologically distinct.
Biochimica et Biophysica Acta (BBA) - Proteins and Proteomics, 2009
Soluble venom and purified fractions of the theraposid spider Brachypelma albiceps were screened ... more Soluble venom and purified fractions of the theraposid spider Brachypelma albiceps were screened for insecticidal peptides based on toxicity to crickets. Two insecticidal peptides, named Ba1 and Ba2, were obtained after the soluble venom was separated by high performance liquid chromatography and cation exchange chromatography. The two insecticidal peptides contain 39 amino acid residues and three disulfide bonds, and based on their amino acid sequence, they are highly identical to the insecticidal peptides from the theraposid spiders Aphonopelma sp. from the USA and Haplopelma huwenum from China indicating a relationship among these genera. Although Ba1 and Ba2 were not able to modify currents in insect and vertebrate cloned voltage-gated sodium ion channels, they have noteworthy insecticidal activities compared to classical arachnid insecticidal toxins indicating that they might target unknown receptors in insect species. The most abundant insecticidal peptide Ba2 was submitted to NMR spectroscopy to determine its 3-D structure; a remarkable characteristic of Ba2 is a cluster of basic residues, which might be important for receptor recognition.
Biochimica et Biophysica Acta (BBA) - Biomembranes, 2004
Pin2 and Oxki1 are cationic amphipathic peptides that permeate lipid membranes through formation ... more Pin2 and Oxki1 are cationic amphipathic peptides that permeate lipid membranes through formation of pores. Their mechanism of binding to phosphocholine (PC) membranes differs. Spin-probe experiments showed that both Pin2 and Oxki1 penetrate the lipid membrane of small unilamellar vesicles (SUVs). Moreover, the leakage of calcein and dextrans from PC vesicles showed that Pin2 agrees with the accumulation of peptides on lipid membranes and form pores of different size. On the other hand, Oxki1 did not act strictly cooperatively and form pores of limited size.
Two novel antimicrobial peptides have been identified and characterized from venom of the African... more Two novel antimicrobial peptides have been identified and characterized from venom of the African scorpion Pandinus imperator. The peptides, designated pandinin 1 and 2, are αhelical polycationic peptides, with pandinin 1 belonging to the group of antibacterial peptides previously described from scorpions, frogs and insects, and pandinin 2 to the group of short magainin-type helical peptides from frogs. Both peptides demonstrated high antimicrobial activity against a range of Grampositive bacteria (2.4-5.2 µM), but were less active against Gram-negative bacteria (2.4-38.2 µM), and only pandinin 2 affected the yeast Candida albicans. Pandinin 2 also demonstrated strong haemolytic activity (11.1-44.5 µM) against sheep erythrocytes, in contrast with pandinin 1, which was not haemolytic. CD studies and a high-resolution structure of pandinin 2
A mRNA transcript that codes for a phospholipase (PLA2) was isolated from a single venom gland of... more A mRNA transcript that codes for a phospholipase (PLA2) was isolated from a single venom gland of the Bothrops ammodytoides viper. The PLA2 transcript was cloned onto a pCR®2.1-TOPO vector and subsequently expressed heterologously in the E. coli strain M15, using the pQE30 vector. The recombinant phospholipase was named rBamPLA2_1, and is composed of an N-terminal fusion protein of 16 residues, along with 122 residues from the mature protein that includes 14 cysteines that form 7 disulfide bonds. Following bacterial expression, rBamPLA2_1 was obtained from inclusion bodies and extracted using a chaotropic agent. rBamPLA2_1 had an experimental molecular mass of 15,692.5 Da that concurred with its theoretical molecular mass. rBamPLA2_1 was refolded in in vitro conditions and after refolding, three main protein fractions with similar molecular masses, were identified. Although, the three fractions were considered to represent different oxidized cystine isoforms, their secondary structures were comparable. All three recombinant isoforms were active on egg-yolk phospholipid and recognized similar cell membrane phospholipids to be native PLA2s, isolated from B. ammodytoides venom. A mixture of the three rBamPLA2_1 cystine isoforms was used to immunize a horse in order to produce serum antibodies (anti-rBamPLA2_1), which partially inhibited the indirect hemolytic activity of B. ammodytoides venom. Although, anti-rBamPLA2_1 antibodies were not able to recognize crotoxin, a PLA2 from the venom of a related but different viper genus, Crotalus durissus terrificus, they recognized PLA2s in other venoms from regional species of Bothrops.
Cells of Lactobacillus delbrueckii subsp. lactis I produced hydrogen peroxide at 5°C in sodium ph... more Cells of Lactobacillus delbrueckii subsp. lactis I produced hydrogen peroxide at 5°C in sodium phosphate buffer (0.2M, pH 6.5) with or without glucose. However, if the cells were starved by preincubation in buffer alone, glucose or sodium lactate were necessary to cause hydrogen peroxide production at 5°C. Hydrogen peroxide production by nonstarved cells was confirmed to be in part due to a NADH oxidase. The production of hydrogen peroxide by starved cells in buffer plus glucose in the early stage of incubation was associated with the production of a small portion of lactic acid which disappeared upon further incubation. Additional experiments revealed that hydrogen peroxide was produced in buffer containing sodium lactate added in place of glucose. Results suggested the presence of a lactate oxidase in the organism which used D-lactate to produce hydrogen peroxide.
The ability ofCeratocystis fimbriata to generate aroma notes from different carbon and nitrogen s... more The ability ofCeratocystis fimbriata to generate aroma notes from different carbon and nitrogen sources was studied in liquid culture. The medium that gave the best sensory results produced a strong banana aroma. Other notes such as pineapple, apple, pear and nuts, with varied, intensities were obtained from other culture media. Biomass and metabolite productions are reported. In solid state fermentation,
Two nonamidated host defense peptides named Pin2[G] and FA1 were evaluated against three types of... more Two nonamidated host defense peptides named Pin2[G] and FA1 were evaluated against three types of pathogenic bacteria: two (Staphylococcus aureus UPD13 and Pseudomonas aeruginosa UPD3) isolated from diabetic foot ulcer patients, and another (Salmonella enterica serovar Typhimurium [ATCC 14028]) from a commercial collection. In vitro experiments showed that the antimicrobial performance of the synthetic peptides Pin2[G] and FA1 was modest, although FA1 was more effective than Pin2[G]. In contrast, Pin2[G] had superior in vivo anti-infective activity to FA1 in rabbit wound infections by the diabetic foot ulcer pathogens S. aureus UPD13 and P. aeruginosa UPD3. Indeed, Pin2[G] reduced bacterial colony counts of both S. aureus UPD13 and P. aeruginosa UPD3 by >100,000-fold after 48 to 72 h on skin wounds of infected rabbits, while in similar infected wounds, FA1 had no major effects at 72 to 96 h of treatment. Ceftriaxone was equally effective versus Pseudomonas but less effective vers...
Bothropic venoms contain enzymes such as metalloproteases, serine-proteases, and phospholipases, ... more Bothropic venoms contain enzymes such as metalloproteases, serine-proteases, and phospholipases, which acting by themselves, or in synergism, are the cause of the envenomation symptoms and death. Here, two mRNA transcripts, one that codes for a metalloprotease and another for a serine-protease, were isolated from a Bothrops ammodytoides venom gland. The metalloprotease and serine-protease transcripts were cloned on a pCR®2.1-TOPO vector and consequently expressed in a recombinant way in E. coli (strains Origami and M15, respectively), using pQE30 vectors. The recombinant proteins were named rBamSP_1 and rBamMP_1, and they were formed by an N-terminal fusion protein of 16 amino acid residues, followed by the sequence of the mature proteins. After bacterial expression, each recombinant enzyme was recovered from inclusion bodies and treated with chaotropic agents. The experimental molecular masses for rBamSP_1 and rBamMP_1 agreed with their expected theoretical ones, and their secondar...
Background and Aims: The production of edible fungi is affected by bacterial, fungal and viral di... more Background and Aims: The production of edible fungi is affected by bacterial, fungal and viral diseases, which very often produces large losses. In the production of mushrooms of the genus Pleurotus, the fungi of the genus Trichoderma represent a serious problem of contamination and although there are some chemical compounds that control the infection, they are not entirely safe for human consumption, so they are looking for alternatives through biotechnology, such as the one presented in this paper. Methods: Strains of fungi of the genus Trichoderma were isolated from the substrate where Pleurotus ostreatus was being cultivated (characteristic contamination of green mold), they were identified morphologically and molecularly, later tests were carried out to inhibit the growth of Trichoderma strains in both agar and wheat straw using a cetonic extract of the fruiting body dehydrated of Pycnoporus sp. Key results: Two strains of Trichoderma (Trichoderma pleuroti and Trichoderma atrob...
The genes of the five disulfide-bonded peptide toxins 1 and 2 (named Oxytoxins or Oxotoxins) from... more The genes of the five disulfide-bonded peptide toxins 1 and 2 (named Oxytoxins or Oxotoxins) from the spider Oxyopes lineatus were cloned into the expression vector pQE30 containing a 6His-tag and a Factor Xa proteolytic cleavage region. These two recombinant vectors were transfected into Escherichia coli BL21 cells and expressed under induction with isopropyl thiogalactoside (IPTG). The product of each gene was named HisrOxyTx1 or HisrOxyTx2, and the protein expression was ca 14 and 6 mg/L of culture medium, respectively. Either recombinant toxin HisrOxyTx1 or HisrOxyTx2 were found exclusively in inclusion bodies, which were solubilized using a chaotropic agent, and then, purified using affinity chromatography and reverse-phase HPLC (RP-HPLC). The HisrOxyTx1 and HisrOxyTx2 products, obtained from the affinity chromatographic step, showed several peptide fractions having the same molecular mass of 9913.1 and 8030.1 Da, respectively, indicating that both HisrOxyTx1 and HisrOxyTx2 were oxidized forming several distinct disulfide bridge arrangements. The isoforms of both HisrOxyTx1 and HisrOxyTx2 after DTT reduction eluted from the column as a single protein component of 9923 and 8040 Da, respectively. In vitro folding of either HisrOxyTx1 or HisrOxyTx2 yielded single oxidized components, which were cleaved independently by the proteolytic enzyme Factor Xa to give the recombinant peptides rOxyTx1 and rOxyTx2. The experimental molecular masses of rOxyTx1 and rOxyTx2 were 8059.0 and 6176.4 Da, respectively, which agree with their expected theoretical masses. The recombinant peptides rOxyTx1 and rOxyTx2 showed lower but comparable toxicity to the native toxins when injected into lepidopteran larvae; furthermore, rOxyTx1 was able to inhibit calcium ion currents on dorsal unpaired median (DUM) neurons from Periplaneta americana.
Advances in NMR and mass spectrometry as well as in peptide biochemistry coupled to modern method... more Advances in NMR and mass spectrometry as well as in peptide biochemistry coupled to modern methods in electrophysiology have permitted the isolation and identification of numerous products from spider venoms, previously unexplored due to technical limitations. The chemical composition of spider venoms is diverse, ranging from low molecular weight organic compounds such acylpolyamines to complex peptides. First, acylpolyamines (<1000 Da) have an aromatic moiety linked to a hydrophilic lateral chain. They were characterized for the first time in spider venoms, and are ligand-gated ion channel antagonists, which block mainly postsynaptic glutamate receptors in invertebrate and vertebrate nervous systems. Acylpolyamines represent the vast majority of organic components from the spider venom. Acylpolyamine analogues have proved to suppress hippocampal epileptic discharges. Moreover, acylpolyamines could suppress excitatory postsynaptic currents inducing Ca 2+ accumulation in neurons leading to protection against a brain ischemic insult. Second, short spider peptides (<6000 Da) modulate ionic currents in Ca 2+ , Na + , or K + voltage-gated ion channels. Such peptides may contain from three to four disulfide bridges. Some spider peptides act specifically to discriminate among Ca 2+ , Na + , and K + ion channel subtypes. Their selective affinities for ion channel subfamilies are functional for mapping excitable cells. Furthermore, several of these peptides have proved to hyperpolarize peripheral neurons, which are associated with supplying sensation to the skin and skeletal muscles. Some spider N-type calcium ion channel blockers may be important for the treatment of chronic pain. A special group of spider peptides are the amphipathic and positively charged peptides. Their secondary structure is a-helical and they insert into the lipid cell membrane of eukaryotic or prokaryotic cells leading to the formation of pores and subsequently depolarizing the cell membrane. Acylpolyamines and peptides from spider venoms represent an interesting source of molecules for the design of novel pharmaceutical drugs. 1 Introduction 2 Spider acylpolyamines 2.1 Acylpolyamines and glutamate receptors 2.2 Acylpolyamines and the polyamine catabolism pathway 2.3 Acylpolyamines as neuroprotective agents 2.4 Acylpolyamines and pain 2.5 Acylpolyamines and nervous diseases 2.6 Synthetic analogues of acylpolyamines 3 Spider peptides 3.1 Spider peptides and potassium channels 3.2 Spider peptides and calcium channels 3.3 Spider peptides and sodium channels 3.4 Glutamate receptors 3.5 Peptides acting on acid-sensing ion channels (ASIC) and mechanosensitive ion channels (MS) 3.6 Non-selective spider peptides 3.7 Pore-forming peptides
Two antimicrobial peptides (AMPs), named La47 and Css54, were isolated from the venom of the spid... more Two antimicrobial peptides (AMPs), named La47 and Css54, were isolated from the venom of the spider Lachesana sp. and from the scorpion Centruroides suffusus suffusus, respectively. The primary structures of both La47 and Css54 were determined using N-terminal sequencing and mass spectrometry. La47 is identical to the AMP latarcin 3a obtained previously from the venom of the spider Lachesana tarabaevi, but the primary structure of Css54 is unique having 60% identities to the AMP ponericin-W2 from the venom of the ant Pachycondyla goeldii. Both La47 and Css54 have typical a-helix secondary structures in hydrophobic mimicking environments. The biological activities of both La47 and Css54 were compared with the AMP Pin2 isolated from the venom of the scorpion Pandinus imperator. La47 has lower antimicrobial and hemolytic activities compared with Css54 and Pin2. In addition, La47 and Pin2 were evaluated in the presence of the commercial antibiotics, chloramphenicol, ampicillin, novobiocin, streptomycin and kanamycin. Interestingly, the best antimicrobial combinations were obtained with mixtures of La47 and Pin2 with the antibiotics chloramphenicol, streptomycin and kanamycin, respectively. Furthermore, the novel peptide Css54 was evaluated in the presence of antibiotics used for the treatment of tuberculosis, isoniazid, rifampicin, pyrazinamide and ethambutol. Although the mixtures of Css54 with isoniazid, pyrazinamide or ethambutol inhibit the growth of Staphylococcus aureus, the best effect was found with rifampicin. Overall, these data show a motivating outlook for potential clinical treatments of bacterial infections using AMPs and commercial antibiotics.
Six peptide toxins (Magi 1^6) were isolated from the Hexathelidae spider Macrothele gigas. The am... more Six peptide toxins (Magi 1^6) were isolated from the Hexathelidae spider Macrothele gigas. The amino acid sequences of Magi 1, 2, 5 and 6 have low similarities to the amino acid sequences of known spider toxins. The primary structure of Magi 3 is similar to the structure of the palmitoylated peptide named PlTx-II from the North American spider Plectreurys tristis (Plectreuridae). Moreover, the amino acid sequence of Magi 4, which was revealed by cloning of its cDNA, displays similarities to the Na + channel modi¢er N N-atracotoxin from the Australian spider Atrax robustus (Hexathelidae). Competitive binding assays using several 125 I-labelled peptide toxins clearly demonstrated the speci¢c binding a⁄nity of Magi 1^5 to site 3 of the insect sodium channel and also that of Magi 5 to site 4 of the rat sodium channel. Only Magi 6 did not compete with the scorpion toxin LqhK KIT in binding to site 3 despite high toxicity on lepidoptera larvae of 3.1 nmol/g. The K i s of other toxins were between 50 pM for Magi 4 and 1747 nM for Magi 1. In addition, only Magi 5 binds to both site 3 in insects (K i = 267 nM) and site 4 in rat brain synaptosomes (K i = 1.2 nM), whereas it showed no a⁄nities for either mammal binding site 3 or insect binding site 4. Magi 5 is the ¢rst spider toxin with binding a⁄nity to site 4 of a mammalian sodium channel.
Delta-palutoxins from the spider Paracoelotes luctuosus (Araneae: Amaurobiidae) are 36-37 residue... more Delta-palutoxins from the spider Paracoelotes luctuosus (Araneae: Amaurobiidae) are 36-37 residue long peptides that show preference for insect sodium channels (NaChs) and modulate their function. Although they slow NaCh inactivation in a fashion similar to that of receptor site 3 modifiers, such as scorpion alpha-toxins, they actually bind with high affinity to the topologically distinct receptor site 4 of scorpion beta-toxins. To resolve this riddle, we scanned by Ala mutagenesis the surface of delta-PaluIT2, a delta-palutoxin variant with the highest affinity for insect NaChs, and compared it to the bioactive surface of a scorpion beta-toxin. We found three regions on the surface of delta-PaluIT2 important for activity: the first consists of Tyr-22 and Tyr-30 (aromatic), Ser-24 and Met-28 (polar), and Arg-8, Arg-26, Arg-32, and Arg-34 (basic) residues; the second is made of Trp-12; and the third is made of Asp-19, whose substitution by Ala uncoupled the binding from toxicity to lepidopteran larvae. Although spider delta-palutoxins and scorpion beta-toxins have developed from different ancestors, they show some commonality in their bioactive surfaces, which may explain their ability to compete for an identical receptor (site 4) on voltage-gated NaChs. Yet, their different mode of channel modulation provides a novel perspective about the structural relatedness of receptor sites 3 and 4, which until now have been considered topologically distinct.
Biochimica et Biophysica Acta (BBA) - Proteins and Proteomics, 2009
Soluble venom and purified fractions of the theraposid spider Brachypelma albiceps were screened ... more Soluble venom and purified fractions of the theraposid spider Brachypelma albiceps were screened for insecticidal peptides based on toxicity to crickets. Two insecticidal peptides, named Ba1 and Ba2, were obtained after the soluble venom was separated by high performance liquid chromatography and cation exchange chromatography. The two insecticidal peptides contain 39 amino acid residues and three disulfide bonds, and based on their amino acid sequence, they are highly identical to the insecticidal peptides from the theraposid spiders Aphonopelma sp. from the USA and Haplopelma huwenum from China indicating a relationship among these genera. Although Ba1 and Ba2 were not able to modify currents in insect and vertebrate cloned voltage-gated sodium ion channels, they have noteworthy insecticidal activities compared to classical arachnid insecticidal toxins indicating that they might target unknown receptors in insect species. The most abundant insecticidal peptide Ba2 was submitted to NMR spectroscopy to determine its 3-D structure; a remarkable characteristic of Ba2 is a cluster of basic residues, which might be important for receptor recognition.
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Papers by Elba Villegas