Rapid Communications in Mass Spectrometry, Jan 25, 2002
Animal venoms are important sources of novel pharmacological tools, useful in biochemical charact... more Animal venoms are important sources of novel pharmacological tools, useful in biochemical characterization of their receptors. Venom quality control, batch-to-batch homogeneity and high reproducibility of venom fractionation and toxin purification are crucial issues for biochemical and pharmacological studies. To address these issues, a study of the variability of tarantula spider venom samples was undertaken. Venom profiles of samples collected from individuals of different age and sex, and from sibling spiders of the same species, were generated by high-performance liquid chromatography (HPLC) and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS) and analyzed to assess venom variability and method accuracy. Sex-linked venom variation was studied on eight species. Clear qualitative differences were observed for six out of eight species, as well as quantitative differences. Age-related variation studied in Poecilotheria rufilata showed essentially age-related quantitative differences between adults of both sexes and immature juveniles. The venoms of nine siblings and three wild-collected Pterinochilus murinus were studied for individual variation, showing only very minor quantitative differences. On the same samples, the quality of MALDI-TOFMS venom fingerprinting was demonstrated to be highly reproducible. Our results show that tarantula venom peptide fingerprinting is a highly reliable identification method, that pooled batches of venom from several animals can be used for venom purification, that venom composition does not appear to be qualitatively related to ontogenesis in the spiders studied, and that qualitative sex-linked variation occurs across most species and may be important in activity studies.
Advances in mass spectrometry and peptide biochemistry coupled to modern methods in electrophys- ... more Advances in mass spectrometry and peptide biochemistry coupled to modern methods in electrophys- iology have permitted the isolation and identification of numerous novel peptide toxins from animal venoms in re- cent years. These advances have also opened up the field of spider venom research, previously unexplored due to methodological limitations. Many peptide toxins from spider venoms share structural features, amino acid com- position and consensus sequences that allow them to in- teract with related classes of cellular receptors. They have become increasingly useful agents for the study of volt- age-sensitive and ligand-gated ion channels and the dis-
β-defensin host defense peptides are important components of the innate immune system of vertebra... more β-defensin host defense peptides are important components of the innate immune system of vertebrates. Although evidence of their broad antimicrobial, antibiofilm and immunomodulatory activities in mammals have been presented, β-defensins from other vertebrate species, like crocodylians, remain largely unexplored. In this study, five new crocodylian β-defensin variants from Alligator mississippiensis and Crocodylus porosus were selected for synthesis and characterization based on their charge and hydrophobicity values. Linear peptides were synthesized, folded, purified and then evaluated for their antimicrobial and antibiofilm activities against the bacterial pathogens, Salmonella enterica serovar Typhimurium, Staphylococcus aureus, Enterobacter cloacae and Acinetobacter baumannii. The Am23SK variant (SCRFSGGYCIWNWERCRSGHFLVALCPFRKRCCK) from A. mississippiensis displayed promising activity against both planktonic cells and bacterial biofilms, outperforming the human β-defensin 3 under the experimental conditions. Moreover, Am23SK exhibited no cytotoxicity towards mammalian cells and exerted immunomodulatory effects in vitro, moderately suppressing the production of proinflammatory mediators from stimulated human bronchial epithelial cells. Overall, our results have expanded the activity landscape of crocodylian and reptilian β-defensin in general.
Journal of Biomolecular Structure & Dynamics, Jun 22, 2017
Pandinin 2 (Pin2) is an alpha-helical polycationic peptide, identified and characterized from ven... more Pandinin 2 (Pin2) is an alpha-helical polycationic peptide, identified and characterized from venom of the African scorpion Pandinus imperator with high antimicrobial activity against Gram-positive bacteria and less active against Gram-negative bacteria, however it has demonstrated strong hemolytic activity against sheep red blood cells. In the chemically synthesized Pin2GVG analog, the GVG motif grants it low hemolytic activity while keeping its antimicrobial activity. In this work, we performed 12 μs all-atom molecular dynamics simulation of the antimicrobial peptides (AMPs) Pin2 and Pin2GVG to explore their adsorption mechanism and the role of their constituent amino acid residues when interacting with pure POPC and pure POPG membrane bilayers. Starting from an α-helical conformation, both AMPs are attracted at different rates to the POPC and POPG bilayer surfaces due to the electrostatic interaction between the positively charged amino acid residues and the charged moieties of the membranes. Since POPG is an anionic membrane, the PAMs adhesion is stronger to the POPG membrane than to the POPC membrane and they are stabilized more rapidly. This study reveals that, before the insertion begins, Pin2 and Pin2GVG remained partially folded in the POPC surface during the first 300 and 600 ns, respectively, while they are mostly unfolded in the POPG surface during most of the simulation time. The unfolded structures provide for a large number of intermolecular hydrogen bonds and stronger electrostatic interactions with the POPG surface. The results show that the aromatic residues at the N-terminus of Pin2 initiate the insertion process in both POPC and POPG bilayers. As for Pin2GVG in POPC the C-terminus residues seem to initiate the insertion process while in POPG this process seems to be slowed down due to a strong electrostatic attraction. The membrane conformational effects upon PAMs binding are measured in terms of the area per lipid and the contact surface area. Several replicas of the systems lead to the same observations.
In this study we cloned a chitinase gene (SmchiC), from Serratia marcescens isolated from the cor... more In this study we cloned a chitinase gene (SmchiC), from Serratia marcescens isolated from the corpse of a Diatraea magnifactella lepidopteran, which is an important sugarcane pest. The chitinase gene SmchiC amplified from the S. marcescens genome was cloned into the transformation vector p2X35SChiC and used to transform tobacco (Nicotiana tabacum L. cv Petit Havana SR1). The resistance of these transgenic plants to the necrotrophic fungus Botrytis cinerea and to the pest Spodoptera frugiperda was evaluated: both the activity of chitinase as well as the resistance against B. cinerea and S. frugiperda was significantly higher in transgenic plants compared to the wild-type.
Phage display and directed evolution have made it possible to generate recombinant antibodies in ... more Phage display and directed evolution have made it possible to generate recombinant antibodies in the format of single chain variable fragments (scFvs) capable of neutralizing different toxins and venoms of Mexican scorpions. Despite having managed to neutralize a significant number of venoms, some others have not yet been completely neutralized, due to the diversity of the toxic components present in them. An example is the venom of the scorpion Centruroides limpidus, which contains three toxins of medical importance, called Cll1, Cll2 and Cl13. The first two are neutralized by scFv 10FG2, while Cl13, due to its sequence divergence, was not even recognized. For this reason, the aim of the present work was the generation of a new scFv capable of neutralizing Cl13 toxin and thereby helping to neutralize the whole venom of this scorpion. By hybridoma technology, a monoclonal antibody (mAb B7) was generated, which was able to recognize and partially neutralize Cl13 toxin. From mAb B7, its scFv format was obtained, named scFv B7 and subjected to three cycles of directed evolution. At the end of these processes, scFv 11F which neutralized Cl13 toxin was obtained. This scFv, administered in conjunction with scFv 10FG2, allowed to fully neutralize the whole venom of Centruroides limpidus scorpion.
Rapid Communications in Mass Spectrometry, Jan 25, 2002
Animal venoms are important sources of novel pharmacological tools, useful in biochemical charact... more Animal venoms are important sources of novel pharmacological tools, useful in biochemical characterization of their receptors. Venom quality control, batch-to-batch homogeneity and high reproducibility of venom fractionation and toxin purification are crucial issues for biochemical and pharmacological studies. To address these issues, a study of the variability of tarantula spider venom samples was undertaken. Venom profiles of samples collected from individuals of different age and sex, and from sibling spiders of the same species, were generated by high-performance liquid chromatography (HPLC) and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS) and analyzed to assess venom variability and method accuracy. Sex-linked venom variation was studied on eight species. Clear qualitative differences were observed for six out of eight species, as well as quantitative differences. Age-related variation studied in Poecilotheria rufilata showed essentially age-related quantitative differences between adults of both sexes and immature juveniles. The venoms of nine siblings and three wild-collected Pterinochilus murinus were studied for individual variation, showing only very minor quantitative differences. On the same samples, the quality of MALDI-TOFMS venom fingerprinting was demonstrated to be highly reproducible. Our results show that tarantula venom peptide fingerprinting is a highly reliable identification method, that pooled batches of venom from several animals can be used for venom purification, that venom composition does not appear to be qualitatively related to ontogenesis in the spiders studied, and that qualitative sex-linked variation occurs across most species and may be important in activity studies.
Advances in mass spectrometry and peptide biochemistry coupled to modern methods in electrophys- ... more Advances in mass spectrometry and peptide biochemistry coupled to modern methods in electrophys- iology have permitted the isolation and identification of numerous novel peptide toxins from animal venoms in re- cent years. These advances have also opened up the field of spider venom research, previously unexplored due to methodological limitations. Many peptide toxins from spider venoms share structural features, amino acid com- position and consensus sequences that allow them to in- teract with related classes of cellular receptors. They have become increasingly useful agents for the study of volt- age-sensitive and ligand-gated ion channels and the dis-
β-defensin host defense peptides are important components of the innate immune system of vertebra... more β-defensin host defense peptides are important components of the innate immune system of vertebrates. Although evidence of their broad antimicrobial, antibiofilm and immunomodulatory activities in mammals have been presented, β-defensins from other vertebrate species, like crocodylians, remain largely unexplored. In this study, five new crocodylian β-defensin variants from Alligator mississippiensis and Crocodylus porosus were selected for synthesis and characterization based on their charge and hydrophobicity values. Linear peptides were synthesized, folded, purified and then evaluated for their antimicrobial and antibiofilm activities against the bacterial pathogens, Salmonella enterica serovar Typhimurium, Staphylococcus aureus, Enterobacter cloacae and Acinetobacter baumannii. The Am23SK variant (SCRFSGGYCIWNWERCRSGHFLVALCPFRKRCCK) from A. mississippiensis displayed promising activity against both planktonic cells and bacterial biofilms, outperforming the human β-defensin 3 under the experimental conditions. Moreover, Am23SK exhibited no cytotoxicity towards mammalian cells and exerted immunomodulatory effects in vitro, moderately suppressing the production of proinflammatory mediators from stimulated human bronchial epithelial cells. Overall, our results have expanded the activity landscape of crocodylian and reptilian β-defensin in general.
Journal of Biomolecular Structure & Dynamics, Jun 22, 2017
Pandinin 2 (Pin2) is an alpha-helical polycationic peptide, identified and characterized from ven... more Pandinin 2 (Pin2) is an alpha-helical polycationic peptide, identified and characterized from venom of the African scorpion Pandinus imperator with high antimicrobial activity against Gram-positive bacteria and less active against Gram-negative bacteria, however it has demonstrated strong hemolytic activity against sheep red blood cells. In the chemically synthesized Pin2GVG analog, the GVG motif grants it low hemolytic activity while keeping its antimicrobial activity. In this work, we performed 12 μs all-atom molecular dynamics simulation of the antimicrobial peptides (AMPs) Pin2 and Pin2GVG to explore their adsorption mechanism and the role of their constituent amino acid residues when interacting with pure POPC and pure POPG membrane bilayers. Starting from an α-helical conformation, both AMPs are attracted at different rates to the POPC and POPG bilayer surfaces due to the electrostatic interaction between the positively charged amino acid residues and the charged moieties of the membranes. Since POPG is an anionic membrane, the PAMs adhesion is stronger to the POPG membrane than to the POPC membrane and they are stabilized more rapidly. This study reveals that, before the insertion begins, Pin2 and Pin2GVG remained partially folded in the POPC surface during the first 300 and 600 ns, respectively, while they are mostly unfolded in the POPG surface during most of the simulation time. The unfolded structures provide for a large number of intermolecular hydrogen bonds and stronger electrostatic interactions with the POPG surface. The results show that the aromatic residues at the N-terminus of Pin2 initiate the insertion process in both POPC and POPG bilayers. As for Pin2GVG in POPC the C-terminus residues seem to initiate the insertion process while in POPG this process seems to be slowed down due to a strong electrostatic attraction. The membrane conformational effects upon PAMs binding are measured in terms of the area per lipid and the contact surface area. Several replicas of the systems lead to the same observations.
In this study we cloned a chitinase gene (SmchiC), from Serratia marcescens isolated from the cor... more In this study we cloned a chitinase gene (SmchiC), from Serratia marcescens isolated from the corpse of a Diatraea magnifactella lepidopteran, which is an important sugarcane pest. The chitinase gene SmchiC amplified from the S. marcescens genome was cloned into the transformation vector p2X35SChiC and used to transform tobacco (Nicotiana tabacum L. cv Petit Havana SR1). The resistance of these transgenic plants to the necrotrophic fungus Botrytis cinerea and to the pest Spodoptera frugiperda was evaluated: both the activity of chitinase as well as the resistance against B. cinerea and S. frugiperda was significantly higher in transgenic plants compared to the wild-type.
Phage display and directed evolution have made it possible to generate recombinant antibodies in ... more Phage display and directed evolution have made it possible to generate recombinant antibodies in the format of single chain variable fragments (scFvs) capable of neutralizing different toxins and venoms of Mexican scorpions. Despite having managed to neutralize a significant number of venoms, some others have not yet been completely neutralized, due to the diversity of the toxic components present in them. An example is the venom of the scorpion Centruroides limpidus, which contains three toxins of medical importance, called Cll1, Cll2 and Cl13. The first two are neutralized by scFv 10FG2, while Cl13, due to its sequence divergence, was not even recognized. For this reason, the aim of the present work was the generation of a new scFv capable of neutralizing Cl13 toxin and thereby helping to neutralize the whole venom of this scorpion. By hybridoma technology, a monoclonal antibody (mAb B7) was generated, which was able to recognize and partially neutralize Cl13 toxin. From mAb B7, its scFv format was obtained, named scFv B7 and subjected to three cycles of directed evolution. At the end of these processes, scFv 11F which neutralized Cl13 toxin was obtained. This scFv, administered in conjunction with scFv 10FG2, allowed to fully neutralize the whole venom of Centruroides limpidus scorpion.
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Papers by Gerardo Corzo