ABSTRACT
Lethal factors are multifunctional oligomeric proteins found in the venomous apparatus of Scorpaeniformes fish. These toxins elicit not only an array of biological responses in vitro but also cardiovascular disorders and strong hemolytic, nociceptive and edematogenic activities in vivo. This work describes the cloning and molecular identification of two toxin subunits, denominated Sp-CTx-α and Sp-CTx-ß, from scorpionfish venom ( Scorpaena plumieri ). Methods: The primary structures were deduced after cDNA amplification by PCR with primers from conserved sequences described in Scorpaeniformes toxins. Following DNA sequencing and bioinformatic analysis, the tridimensional structures of both subunits were modeled. Results: The translated sequences (702 amino acids, each subunit) show homology with other lethal factors, while alignment between Sp-CTx-α and Sp-CTx-ß shows 54% identity. The subunits lack N-terminal signal sequences and display masses of approximately 80 kDa each. Both Sp-CTx subunits display a B30.2/SPRY domain at the C-terminal region with typically conserved motifs as described in these toxins. Secondary structure prediction identified six α-helices 18 residues long in both α and ß subunits, some of them amphiphilic with their N-terminal flanked by many basic residues, creating a cationic site associated with the cytolytic activity of these toxins. Antimicrobial potential sites were identified in Sp-CTx and share some features with other peptides presenting variable and broad-spectrum activity. A phylogenetic tree built to represent these toxins supports the proximity between scorpionfish, lionfish and stonefish. Conclusion: The study identified a putative toxin protein whose primary structure is similar to other fish toxins and with potential for production of antivenom against scorpionfish envenomation in Brazil. As a prelude to structure-function studies, we propose that the toxin is structurally related to pore-forming marine toxins.(AU)
Subject(s)
Animals , DNA, Complementary/analysis , Fish Venoms/toxicity , Peptides/analysis , Antivenins/classification , Polymerase Chain Reaction/methods , Amino Acid SequenceABSTRACT
Background: Lethal factors are multifunctional oligomeric proteins found in the venomous apparatus of Scorpaeniformes fish. These toxins elicit not only an array of biological responses in vitro but also cardiovascular disorders and strong hemolytic, nociceptive and edematogenic activities in vivo. This work describes the cloning and molecular identification of two toxin subunits, denominated Sp-CTx- and Sp-CTx-, from scorpionfish venom ( Scorpaena plumieri ). Methods: The primary structures were deduced after cDNA amplification by PCR with primers from conserved sequences described in Scorpaeniformes toxins. Following DNA sequencing and bioinformatic analysis, the tridimensional structures of both subunits were modeled. Results: The translated sequences (702 amino acids, each subunit) show homology with other lethal factors, while alignment between Sp-CTx- and Sp-CTx- shows 54% identity. The subunits lack N-terminal signal sequences and display masses of approximately 80 kDa each. Both Sp-CTx subunits display a B30.2/SPRY domain at the C-terminal region with typically conserved motifs as described in these toxins. Secondary structure prediction identified six -helices 18 residues long in both and subunits, some of them amphiphilic with their N-terminal flanked by many basic residues, creating a cationic site associated with the cytolytic activity of these toxins. Antimicrobial potential sites were identified in Sp-CTx and share some features with other peptides presenting variable and broad-spectrum activity...
Subject(s)
Animals , DNA, Complementary/analysis , Fishes, Poisonous , Fish Venoms/chemistryABSTRACT
Anthrax is a malignant infectious disease caused by Bacillus anthracis spores,after entering the host Bacillus an-thracis produces and releases anthrax toxin,which is the main cause leading to death of the host. The anthrax toxin is composed of two enzymatically active components:lethal factor(LF)and edema factor(EF),and one shared receptor binding and translocation com-ponent:protective antigen(PA). PA combined with LF is called lethal toxin(LeTx),while PA combined with EF called edema toxin (EdTx). Currently,the main drugs for treating anthrax are antibiotics,but antibiotics can only kill part of anthrax spores and bacte-ria,and cannot inhibit the activity of anthrax toxin. So it is necessary to develop novel drugs for inhibiting anthrax toxin. This review summarizes the evolution of small-molecule inhibitors of anthrax toxin respectively targeting PA,LF and EF.
ABSTRACT
Anthrax is a highly lethal infectious disease caused by the spore-forming bacterium Bacillus anthracis. The major virulence factor of B. anthracis consists of protective antigen (PA), lethal factor (LF) and edema factor (EF). PA binds with LF to form lethal toxin (LT), and PA binds with EF to form edema toxin (ET). Antibiotics is hard to work in advanced anthrax infections, because injuries and deaths of the infected are mainly caused by lethal toxin (LT). Thus, the therapeutic neutralizing antibody is the most effective treatment of anthrax. Currently most of the anthrax toxin antibodies are monoclonal antibodies (MAbs) for PA and US FDA has approved ABTHRAX humanized PA monoclonal antibody for the treatment of inhalational anthrax. Once B. anthracis was artificially reconstructed or PA had mutations within recognized neutralization epitopes, anti-PA MAbs would no longer be effective. Therefore, anti-LF MAbs is an important supplement for anthrax treatment. Most of the anti-LF antibodies are murine or chimeric antibodies. By contrast, fully human MAbs can avoid the high immunogenicity of murine antibodies. First, we used LF to immunize the transgenic mice and used fluorescent cell sorting to get antigen-specific memory B cells from transgenic mice spleen lymphocytes. By single cell PCR method, we quickly found two strains of anti-LF MAbs with binding activity, 1D7 and 2B9. Transiently transfected Expi 293F cells to obtain MAbs protein after purification. Both 1D7 and 2B9 efficiently neutralized LT in vitro, and had good synergistic effect when mixed with anti-PA MAbs. In summary, combining the advantages of transgenic mice, fluorescent cell sorting and single-cell PCR methods, this study shows new ideas and methods for the rapid screening of fully human monoclonal antibodies.
ABSTRACT
Objective To study the role of protective antigen(PA)and N-terminal segment of lethal factor (LFn)in the entrance of EGFP(enhanced green fluorescent protein)into HeLa cells. Methods The DNA fragments encoding LFn and EGFP were amplified,respectively,and cloned into the plasmid pET-21 a(+)one after another to construct a recombinant plasmid pET-LFn-EGFP. The plasmid was txansformed into BL21 cells to express LFn-EGFP protein under the induction of IPTG. The protein was purified by Ni chelating chromatography. After incubation with LFn-EGFP in the presence of PA or not, the HeLa cells were analyzed by flow cytometry or laser confocal microscopy. Results The fusion protein LFn-EGFP was purified by over 90% homogeneity and retained the ability of LF to bind with PA when incubated with J774A.1 macrophage cells,and could get into HeLa cells. Conclusion The LFn-EGFP could enter the HeLa cells in a PA independent pathway. But PA could help more LFn-EGFP molecules enter into HeLa cells.
ABSTRACT
For transferable and immunologieal studies on shock toxin (lethal faetor), a hind limbs ischemia tourniquet shock in rats has been examined. The blood of rats subjectd to severe shock transfused into mild shock rats has not significantly increased the mortality of the latter. Both the recruited plasma from shock rats and the plasma from the rats immunized with severe shork plasma had no therapeutic effects on tourniquet shock. It therefore appears that there is no specific and immunogenic shock toxin produced in the tourniquet shock in rats