Internalization of a Bacillus anthracis Protective Antigen-c-Myc Fusion Protein Mediated by Cell Surface Anti-c-Myc Antibodies

Varughese, Mini; Chi, Angela; Teixeira, Avelino; Nicholls, Peter J.; Keith, Jerry M.; Leppla, Stephen H.

doi:10.1007/bf03401732

Cited by 55 publications

(52 citation statements)

References 37 publications

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“…Thioglycolate-induced peritoneal macrophages were harvested from BALB/cJ mice and *Lethal toxin (LeTx) dose of 0ϫ ϭ diluent only; 1ϫ ϭ 20 g/kg protective antigen (PA) ϩ 10 g/kg lethal factor (LF); 2.5ϫ ϭ 50 g/kg PA ϩ 25 g/kg LF; 5ϫ ϭ 100 g/kg PA ϩ 50 g/kg LF; 7.5ϫ ϭ 150 g/kg PA ϩ 75 g/kg LF; 10ϫ ϭ 200 g/kg PA ϩ 100 g/kg LF; 50ϫ ϭ 1,000 g/kg PA ϩ 500 g/kg LF; 100ϫ ϭ 2,000 g/kg PA ϩ 1,000 g/kg LF; 500ϫ ϭ 10,000 g/kg PA ϩ 5,000 g/kg LF noninduced resident cells from Sprague-Dawley and Fischer rats by saline lavage. These macrophages or RAW 264.7 cells were plated in 96-well plates in DMEM supplemented with 10% fetal bovine serum, 10 mM HEPES, 2 mM Glutamax I, and 50 g/ml gentamicin for 4 h. LeTx (PA ϩ LF) was added at a range of concentrations (0-2,000 ng/ml) in duplicate, and cells were incubated at 37°C for 2 h. Cell viability was assessed by the 3-(4,5-dimethyl-2-thiazyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) as previously described (37 …”

Section: Methodsmentioning

confidence: 99%

Lethality during continuous anthrax lethal toxin infusion is associated with circulatory shock but not inflammatory cytokine or nitric oxide release in rats

Cui¹,

Moayeri²,

Li³

et al. 2004

American Journal of Physiology-Regulatory, Integrative and Comp

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114

172

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. Lethality during continuous anthrax lethal toxin infusion is associated with circulatory shock but not inflammatory cytokine or nitric oxide release in rats. Am J Physiol Regul Integr Comp Physiol 286: R699-R709, 2004. First published January 8, 2004 10.1152/ajpregu.00593.2003.-Although circulatory shock related to lethal toxin (LeTx) may play a primary role in lethality due to Bacillus anthracis infection, its mechanisms are unclear. We investigated whether LeTx-induced shock is associated with inflammatory cytokine and nitric oxide (NO) release. SpragueDawley rats with central venous and arterial catheters received 24-h infusions of LeTx (lethal factor 100 g/kg; protective antigen 200 g/kg) that produced death beginning at 9 h and a 7-day mortality rate of 53%. By 9 h, mean arterial blood pressure, heart rate, pH, and base excess were decreased and lactate and hemoglobin levels were increased in LeTx nonsurvivors compared with LeTx survivors and controls (diluent only) (P Յ 0.05 for each comparing the 3 groups). Despite these changes, arterial oxygen and circulating leukocytes and platelets were not decreased and TNF-␣, IL-1␤, IL-6, and IL-10 levels were not increased comparing either LeTx nonsurvivors or survivors to controls. Nitrate/nitrite levels and tissue histology also did not differ comparing LeTx animals and controls. In additional experiments, although 24-h infusions of LeTx and Esherichia coli LPS produced similar mortality rates (54 and 56%, respectively) and times to death (13.2 Ϯ 0.8 vs. 11.0 Ϯ 1.7 h, respectively) compared with controls, only LPS reduced circulating leukocytes, platelets, and IL-2 levels and increased TNF-␣, IL-1␣ and -1␤, IL-6, IL-10, interferon-␥, granulocyte macrophage-colony stimulating factor, RANTES, migratory inhibitory protein-1␣, -2, and -3, and monocyte chemotactic protein-1, as well as nitrate/nitrite levels (all P Յ 0.05 for the effects of LPS). Thus, in contrast to LPS, excessive inflammatory cytokine and NO release does not appear to contribute to the circulatory shock and lethality occurring with LeTx in this rat model. Although therapies to modulate these host mediators may be applicable for shock caused by LPS or other bacterial toxins, they may not with LeTx.

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Section: Methodsmentioning

confidence: 99%

Lethality during continuous anthrax lethal toxin infusion is associated with circulatory shock but not inflammatory cytokine or nitric oxide release in rats

Cui¹,

Moayeri²,

Li³

et al. 2004

American Journal of Physiology-Regulatory, Integrative and Comp

Self Cite

114

172

View full text Add to dashboard Cite

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“…Cells were incubated at 37°C for 4 h. Cell viability was determined by the addition of 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT). Viable cells converted the MTT to an insoluble blue pigment, which was measured as previously described (34). Results were plotted and analyzed with Prism software (GraphPad Software Inc., San Diego) as the percentage viability of control wells containing LF without PA. EC 50 values were determined by nonlinear regression sigmoidal dose-response analysis with variable slopes.…”

Section: Methodsmentioning

confidence: 99%

Alanine-scanning Mutations in Domain 4 of Anthrax Toxin Protective Antigen Reveal Residues Important for Binding to the Cellular Receptor and to a Neutralizing Monoclonal Antibody

Rosovitz

Schuck

Varughese

et al. 2003

Journal of Biological Chemistry

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131

136

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A panel of variants with alanine substitutions in the small loop of anthrax toxin protective antigen domain 4 was created to determine individual amino acid residues critical for interactions with the cellular receptor and with a neutralizing monoclonal antibody, 14B7. Substituted protective antigen proteins were analyzed by cellular cytotoxicity assays, and their interactions with antibody were measured by plasmon surface resonance and analytical ultracentrifugation. Residue Asp 683 was the most critical for cell binding and toxicity, causing an ϳ1000-fold reduction in toxicity, but was not a large factor for interactions with 14B7. Substitutions in residues Tyr 681 , Asn 682 , and Pro 686 also reduced toxicity significantly, by 10 -100-fold. Of these, only Asn 682 and Pro 686 were also critical for interactions with 14B7. However, residues Lys 684 , Leu 685 , Leu 687 , and Tyr 688 were critical for 14B7 binding without greatly affecting toxicity. The K684A and L685A variants exhibited wild type levels of toxicity in cell culture assays; the L687A and Y688A variants were reduced only 1.5-and 5-fold, respectively.Bacillus anthracis secretes two toxins: edema toxin and lethal toxin. Each is composed of a common binding component, protective antigen (PA), 1 together with an enzymatic component, edema factor (EF), in the case of edema toxin and lethal factor (LF) in the case of lethal toxin (1-3). The current model for toxin entry into the cell illustrates the centrality of PA for toxin action. PA binds to cellular receptors, recently identified as splice variants of either tumor endothelial marker 8 (TEM8) (4 -6) or the closely related capillary morphogenesis protein 2 (CMG2) (7). Furin cleaves PA, releasing a 20-kDa fragment and leaving behind a 63-kDa portion (PA 63 ) capable of forming a heptamer, which has a newly exposed surface that binds . Heptamer complexes enter the endocytic pathway by receptor-mediated endocytosis (13), and upon acidification of the vesicle, the PA 63 heptamer undergoes a conformational change to form a pore through which EF and LF translocate into the cytoplasm (10, 11, 14 -16). Once in the cytoplasm, EF and LF exert their toxic effects.The PA protein can be divided into four domains based on its crystal structure, and functions can be attributed to the different domains based on mutational and biochemical analyses (16). Domain 1 (residues 1-258) contains the furin cleavage site as well as the hydrophobic portion of PA, which is exposed upon furin cleavage to allow EF and LF to bind (16,17). Several lines of evidence indicate that domain 2 (residues 259 -487) is involved in oligomerization and contains the loop that inserts into the membrane to form the channel through which the LF and EF enter the cytosol (16, 18 -20). Various amino acids in domain 3 (residues 488 -595) are necessary for oligomerization, and this has been the only function attributed to domain 3 to date (21,22). Domain 4 (residues 596 -735) is essential for binding to cellular receptor as indicated by several lines of...

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“…An exciting variation on stimulating immunity involves the anticancer capabilities of lethal toxin (129,216). Like previous studies with a diphtheria toxin chimera containing the C fragment of C. tetani tetanus toxin for specific targeting of neurons (128), it is also possible to direct PA toward unique surface receptors, as evidenced by fusing a c-Myc epitope consisting of 10 amino acids to the C terminus of PA, which subsequently binds to (and kills with LF) a hybridoma line expressing c-Myc antibody (233,448).…”

Section: Protein Shuttlesmentioning

confidence: 99%

Binary Bacterial Toxins: Biochemistry, Biology, and Applications of CommonClostridiumandBacillusProteins

Barth

Aktories

Popoff

et al. 2004

Microbiol Mol Biol Rev

375

388

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Certain pathogenic species of Bacillus and Clostridium have developed unique methods for intoxicating cells that employ the classic enzymatic “A-B” paradigm for protein toxins. The binary toxins produced by B. anthracis, B. cereus, C. botulinum, C. difficile, C. perfringens, and C. spiroforme consist of components not physically associated in solution that are linked to various diseases in humans, animals, or insects. The “B” components are synthesized as precursors that are subsequently activated by serine-type proteases on the targeted cell surface and/or in solution. Following release of a 20-kDa N-terminal peptide, the activated “B” components form homoheptameric rings that subsequently dock with an “A” component(s) on the cell surface. By following an acidified endosomal route and translocation into the cytosol, “A” molecules disable a cell (and host organism) via disruption of the actin cytoskeleton, increasing intracellular levels of cyclic AMP, or inactivation of signaling pathways linked to mitogen-activated protein kinase kinases. Recently, B. anthracis has gleaned much notoriety as a biowarfare/bioterrorism agent, and of primary interest has been the edema and lethal toxins, their role in anthrax, as well as the development of efficacious vaccines and therapeutics targeting these virulence factors and ultimately B. anthracis. This review comprehensively surveys the literature and discusses the similarities, as well as distinct differences, between each Clostridium and Bacillus binary toxin in terms of their biochemistry, biology, genetics, structure, and applications in science and medicine. The information may foster future studies that aid novel vaccine and drug development, as well as a better understanding of a conserved intoxication process utilized by various gram-positive, spore-forming bacteria

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Internalization of a Bacillus anthracis Protective Antigen-c-Myc Fusion Protein Mediated by Cell Surface Anti-c-Myc Antibodies

Abstract: This study shows that PA can be redirected to alternate receptors by adding novel epitopes to the C-terminus of PA, enabling the creation of cell-directed toxins for therapeutic purposes.

Cited by 55 publications

References 37 publications

Lethality during continuous anthrax lethal toxin infusion is associated with circulatory shock but not inflammatory cytokine or nitric oxide release in rats

Lethality during continuous anthrax lethal toxin infusion is associated with circulatory shock but not inflammatory cytokine or nitric oxide release in rats

Alanine-scanning Mutations in Domain 4 of Anthrax Toxin Protective Antigen Reveal Residues Important for Binding to the Cellular Receptor and to a Neutralizing Monoclonal Antibody

Binary Bacterial Toxins: Biochemistry, Biology, and Applications of CommonClostridiumandBacillusProteins

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