Peptidomic investigation of Neoponera villosa venom by high-resolution mass spectrometry: seasonal and nesting habitat variations

Background: Advancements in proteomics, including the technological improvement in instrumentation, have turned mass spectrometry into an indispensable tool in the study of venoms and toxins. In addition, the advance of nanoscale liquid chromatography coupled to nanoelectrospray mass spectrometry allows, due to its high sensitivity, the study of venoms from species previously left aside, such as ants. Ant venoms are a complex mixture of compounds used for defense, predation or communication purposes. The venom from Neoponera ants, a genus restricted to Neotropical regions, is known to have cytolytic, hemolytic, antimicrobial and insecticidal activities. Moreover, venoms from several Neoponera species have been compared and differences in their toxicity related to nesting habitat variation were reported. Therefore, the present study aimed to perform a deep peptidomic analysis of Neoponera villosa venom and a comparison of seasonal and nesting habitat variations using high-resolution mass spectrometry. Methods: Specimens of N. villosa ants were captured in Panga Natural Reserve (Uberlândia, MG, Brazil) from arboreal and ground-dwelling nests during summer and winter time. The venom glands were dissected, pooled and disrupted by ultra-sonic waves. The venom collected from different habitats (arboreal and ground-dwelling) and different seasons (summer and winter) was injected into a nanoACQUITY ULPC hyphened to a Q-Exactive Orbitrap mass spectrometer. The raw data were analyzed using PEAKS 7. Results: The results showed a molecular diversity of more than 500 peptides among these venoms, mostly in the mass range of 8004000 Da. Mutations and post-translational modifications were described and differences among the venoms were observed. Part of the peptides matched with ponericins, a well-known antimicrobial peptide family...(AU)

Peptidomic investigation of Neoponera villosa venom by high-resolution mass spectrometry: seasonal and nesting habitat variations

Background: Advancements in proteomics, including the technological improvement in instrumentation, have turned mass spectrometry into an indispensable tool in the study of venoms and toxins. In addition, the advance of nanoscale liquid chromatography coupled to nanoelectrospray mass spectrometry allows, due to its high sensitivity, the study of venoms from species previously left aside, such as ants. Ant venoms are a complex mixture of compounds used for defense, predation or communication purposes. The venom from Neoponera ants, a genus restricted to Neotropical regions, is known to have cytolytic, hemolytic, antimicrobial and insecticidal activities. Moreover, venoms from several Neoponera species have been compared and differences in their toxicity related to nesting habitat variation were reported. Therefore, the present study aimed to perform a deep peptidomic analysis of Neoponera villosa venom and a comparison of seasonal and nesting habitat variations using high-resolution mass spectrometry. Methods: Specimens of N. villosa ants were captured in Panga Natural Reserve (Uberlândia, MG, Brazil) from arboreal and ground-dwelling nests during summer and winter time. The venom glands were dissected, pooled and disrupted by ultra-sonic waves. The venom collected from different habitats (arboreal and ground-dwelling) and different seasons (summer and winter) was injected into a nanoACQUITY ULPC hyphened to a Q-Exactive Orbitrap mass spectrometer. The raw data were analyzed using PEAKS 7. Results: The results showed a molecular diversity of more than 500 peptides among these venoms, mostly in the mass range of 8004000 Da. Mutations and post-translational modifications were described and differences among the venoms were observed. Part of the peptides matched with ponericins, a well-known antimicrobial peptide family...

Identification, expression and characterization of the recombinant Sol g 4. 1 protein from the venom of the tropical fire ant Solenopsis geminata

Fire ant venom is a complex mixture consisting of basic piperidine alkaloids, various biologically active peptides and protein components, including a variety of major allergenic proteins. Tropical fire ant Solenopsis geminata is an important stinging ant species that causes anaphylaxis and serious medical problems. Although the biological activities of allergenic venom proteins that are unique to ant venom, particularly Solenopsis 2 and 4, are still unknown, these proteins are believed to play important roles in mediating the effects of the piperidine derivatives in the venom. Methods: In the present study, the cDNA cloning, sequencing and three-dimensional structure of Sol g 4.1 venom protein are described. The recombinant Sol g 4.1 protein (rSol g 4.1) was produced in E. coli , and its possible function as a hydrophobic binding protein was characterized by paralyzing crickets using the 50% piperidine dose (PD50). Moreover, an antiserum was produced in mice to determine the allergenic properties of Sol g 4.1, and the antiserum was capable of binding to Sol g 4.1, as determined by Western blotting. Results: The molecular weight of Sol g 4.1 protein is 16 kDa, as determined by SDS-PAGE. The complete cDNA is 414 bp in length and contains a leader sequence of 19 amino acids. The protein consists of six cysteines that presumably form three disulfide bonds, based on a predicted three-dimensional model, creating the interior hydrophobic pocket and stabilizing the structure. The rSol g 4.1 protein was expressed in inclusion bodies, as determined by SDS-PAGE. Dialysis techniques were used to refold the recombinant protein into the native form. Its secondary structure, which primarily consists of α-helices, was confirmed by circular dichroism analysis, and the three-dimensional model was also verified. The results of allergenic analysis performed on mice showed that the obtained protein was predicted to be allergenically active. Moreover, we report on the possible role of the Sol g 4.1 venom protein, which significantly reduced the PD50 from 0.027 to 0.013% in paralyzed crickets via synergistic effects after interactions with piperidine alkaloids. Conclusions: The primary structure of Sol g 4.1 showed high similarity to that of venom proteins in the Solenopsis 2 and 4 family. Those proteins are life-threatening and produce IgE-mediated anaphylactic reactions in allergic individuals. The possible function of this protein is the binding of the interior hydrophobic pockets with piperidine alkaloids, as determined by the analysis of the structural model and PD50 test.(AU)

Identification, expression and characterization of the recombinant Sol g 4.1 protein from the venom of the tropical fire ant Solenopsis geminata

Background: Fire ant venom is a complex mixture consisting of basic piperidine alkaloids, various biologically active peptides and protein components, including a variety of major allergenic proteins. Tropical fire ant Solenopsis geminata is an important stinging ant species that causes anaphylaxis and serious medical problems. Although the biological activities of allergenic venom proteins that are unique to ant venom, particularly Solenopsis 2 and 4, are still unknown, these proteins are believed to play important roles in mediating the effects of the piperidine derivatives in the venom. Methods: In the present study, the cDNA cloning, sequencing and three-dimensional structure of Sol g 4.1 venom protein are described. The recombinant Sol g 4.1 protein (rSol g 4.1) was produced in E. coli , and its possible function as a hydrophobic binding protein was characterized by paralyzing crickets using the 50% piperidine dose (PD50). Moreover, an antiserum was produced in mice to determine the allergenic properties of Sol g 4.1, and the antiserum was capable of binding to Sol g 4.1, as determined by Western blotting. Results: The molecular weight of Sol g 4.1 protein is 16 kDa, as determined by SDS-PAGE. The complete cDNA is 414 bp in length and contains a leader sequence of 19 amino acids. The protein consists of six cysteines that presumably form three disulfide bonds, based on a predicted three-dimensional model, creating the interior hydrophobic pocket and stabilizing the structure. The rSol g 4.1 protein was expressed in inclusion bodies, as determined by SDS-PAGE. Dialysis techniques were used to refold the recombinant protein into the native form. Its secondary structure, which primarily consists of -helices, was confirmed by circular dichroism analysis, and the three-dimensional model was also verified. The results of allergenic analysis performed on mice showed that the...(AU)

Identification, expression and characterization of the recombinant Sol g 4.1 protein from the venom of the tropical fire ant Solenopsis geminata

Background: Fire ant venom is a complex mixture consisting of basic piperidine alkaloids, various biologically active peptides and protein components, including a variety of major allergenic proteins. Tropical fire ant Solenopsis geminata is an important stinging ant species that causes anaphylaxis and serious medical problems. Although the biological activities of allergenic venom proteins that are unique to ant venom, particularly Solenopsis 2 and 4, are still unknown, these proteins are believed to play important roles in mediating the effects of the piperidine derivatives in the venom. Methods: In the present study, the cDNA cloning, sequencing and three-dimensional structure of Sol g 4.1 venom protein are described. The recombinant Sol g 4.1 protein (rSol g 4.1) was produced in E. coli , and its possible function as a hydrophobic binding protein was characterized by paralyzing crickets using the 50% piperidine dose (PD50). Moreover, an antiserum was produced in mice to determine the allergenic properties of Sol g 4.1, and the antiserum was capable of binding to Sol g 4.1, as determined by Western blotting. Results: The molecular weight of Sol g 4.1 protein is 16 kDa, as determined by SDS-PAGE. The complete cDNA is 414 bp in length and contains a leader sequence of 19 amino acids. The protein consists of six cysteines that presumably form three disulfide bonds, based on a predicted three-dimensional model, creating the interior hydrophobic pocket and stabilizing the structure. The rSol g 4.1 protein was expressed in inclusion bodies, as determined by SDS-PAGE. Dialysis techniques were used to refold the recombinant protein into the native form. Its secondary structure, which primarily consists of -helices, was confirmed by circular dichroism analysis, and the three-dimensional model was also verified. The results of allergenic analysis performed on mice showed that the...

Characterization of the allergen Sol gem 2 from the fire ant venom, Solenopsis geminata

Sol i 2 is a potent allergen in Solenopsis invicta venom, and most humans exhibit reactivity to it. The Sol gem 2 allergen found in the venom of the Thai tropical fire ant Solenopsis geminata was analysed in the present study. The protein was present in higher amounts than other proteins, as determined by SDS-PAGE, and presumably has allergenic properties similar to those of Sol i 2. Sol gem 2 molecular weight is 28 and 15 kDa, respectively, under non-reducing and reducing conditions, indicating that its native form is a dimer. LC-MS/MS analysis confirmed its similarity to Sol i 2. The mono/dimeric form of Sol gem 2 was determined to be relevant by proteomic approach and immunoblotting. An anti-Sol gem 2 antibody was produced in mice, with a titer greater than 1:800 according to the Western blotting analysis. The Sol gem 2-neutralising activity of this antibody was determined in crickets. The paralytic dose 50 (PD50) of crude S. geminata venom was elevated from 0.18 mg/g of body weight to more than 0.90 mg/g of body weight after preincubation with antibody at a ratio of 1:1. These results suggest that Sol gem 2 plays an important role in mediating the effects of the piperidine derivatives in the venom.(AU)