Acid induced unfolding of anthrax protective antigen

Acidic pH plays an important role in the membrane insertion of protective antigen (PA) of anthrax toxin leading to the translocation of the catalytic moieties. The structural transitions occurring in PA as a consequence of change in pH were investigated by fluorescence and circular dichroism measurements. Our studies revealed the presence of two intermediates on-pathway of acid induced unfolding; one at pH 2.0 and other at pH 4-5. Intrinsic fluorescence measurements of these intermediates showed a red shift in the wavelength of emission maximum with a concomitant decrease in fluorescence intensity, indicative of the exposure of tryptophan residues to the bulk solvent. Furthermore, no significant change was seen in the secondary structure of PA at a pH of 2.0, as indicated by far UV-CD spectra. The low pH intermediate of PA was characterized using the hydrophobic dye, 8-anilino-1-naphthalenesulfonate, and was found to have properties similar to those of a molten globule state.     

Unfolding transitions of Bacillus anthracis protective antigen

Protective antigen (PA) is an 83kDa protein which, although essential for toxicity of Bacillus anthracis, is harmless and an effective vaccine component. In vivo it undergoes receptor binding, proteolysis, heptamerisation and membrane insertion. Here we probe the response of PA to denaturants, temperature and pH. We present analyses (including barycentric mean) of the unfolding and refolding behavior of PA and reveal the origin of two critical steps in the denaturant unfolding pathway in which the first step is a calcium and pH dependent rearrangement of domain 1. Thermal unfolding fits a single transition near 50 degrees C. We show for the first time circular dichroism (CD) spectra of the heptameric, furin-cleaved PA63 and the low-pH forms of both PA83 and PA63. Although only PA63 should reach the acidic endosome, both PA83 and PA63 undergo similar acidic transitions and an unusual change from a beta II to a beta I CD spectrum.     

Asporogenic recombinant producer of anthrax protective antigen

An asporogenic recombinant strain Bacillus anthracis 55ΔTPA-1(Spo⁻) producing anthrax protective antigen (PA) was obtained. The strain contains structural gene pag as a part of a hybrid replicon pUB110PA-1 and lacks determinants encoding the synthesis of main factors of anthrax pathogenicity. The level of PA production by asporogenic genetically engineered strain is approximately 80 μg/ml that is 4–5 times more than the values determined for vaccine strains B. anthracis STI-1 and B. anthracis 55. The strain preserves asporogenicity and ability to replicate the hybrid plasmid after in vitro passages. Biologically active PA was isolated from the constructed strain B. anthracis 55ΔTPA-1(Spo⁻). Double immunization of rabbits with 50 μg of the purified recombinant product provides their 100% protection from infection with 50 LD₅₀ of a highly virulent anthrax strain.     

Production and purification of recombinant protective antigen and protective efficacy against Bacillusanthracis

Recombinant protective antigen (rPA), expressed by Bacillus subtilis WB600 (pPA101), has been purified to homogeneity and the protective efficacy against a Bacillus anthracis challenge has been investigated. rPA was fractionated from culture supernatant fluid by ammonium sulphate, followed by anion exchange chromatography using DEAE Streamline™, anion-exchange chromatography on FPLC MonoQ HR 10/10 and finally, gel filtration chromatography on FPLC Superose 12 HR 10/30, to yield 7 mg rPA per litre of culture. The protective efficacy of rPA against an airborne challenge with the AMES strain of B. anthracis was determined in the presence of the adjuvants, alhydrogel and Ribi, and compared to that achieved by the current UK human vaccine in guinea pigs. rPA combined with the Ribi adjuvant was found to provide 100% protection against challenge.     

Plant-derived EpCAM antigen induces protective anti-cancer response

Immunotherapy holds great promise for treatment of infectious and malignant diseases and might help to prevent the occurrence and recurrence of cancer. We produced a plant-derived tumor-associated colorectal cancer antigen EpCAM (pGA733) at high yields using two modern plant expression systems. The full antigenic domain of EpCAM was efficiently purified to confirm its antigenic and immunogenic properties as compared to those of the antigen expressed in the baculovirus system (bGA733). Recombinant plant-derived antigen induced a humoral immune response in BALB/c mice. Sera from those mice efficiently inhibited the growth of SW948 colorectal carcinoma cells xenografted in nude mice, as compared to the EpCAM-specific mAb CO17-1A. Our results support the feasibility of producing anti-cancer recombinant vaccines using plant expression systems.     

Anthrax protective antigen: prepore-to-pore conversion.

PA(63), the active 63 kDa form of anthrax protective antigen, forms a heptameric ring-shaped oligomer that is believed to represent a precursor of the membrane pore formed by this protein. When maintained at pH >/=8.0, this "prepore" dissociated to monomeric subunits upon treatment with SDS at room temperature, but treatment at pH 相似文献   

Characterization of dominant-negative forms of anthrax protective antigen

Certain mutations within the protective antigen (PA) moiety of anthrax toxin endow the protein with a dominant-negative (DN) phenotype, converting it into a potent antitoxin. Proteolytically activated PA oligomerizes to form ring-shaped heptameric complexes that insert into the membrane of an acidic intracellular compartment and promote translocation of bound edema factor and/or lethal factor to the cytosol. DN forms of PA co-oligomerize with the wild-type protein and block the translocation process. We prepared and characterized 4 DN forms: a single, a double, a triple, and a quadruple mutant. The mutants were made by site-directed mutation of the cloned form of PA in Escherichia coli and tested by various assays conducted on CHO cells or in solution. All 4 mutant PAs were competent for heptamerization and ligand binding but were defective in the pH-dependent functions: pore formation, ability to convert to the SDS-resistant heptamer, and ability to translocate bound ligand. The single mutant (F427K) showed less attenuation than the others in the pH-dependent functions and lower DN activity in a CHO cell assay. The quadruple (K397D + D425K + F427A + 2beta2-2beta3) deletion showed the most potent DN activity at low concentrations but also gave indications of low stability in a urea-mediated unfolding assay. The double mutant (K397D + D425K) and the triple (K397D + D425K + F427A) showed strong DN activity and slight reduction in stability relative to the wild-type protein. The properties of the double and the triple mutants make these forms worthy of testing in vivo as a new type of antitoxic agent for treatment of anthrax.     

Crystallization of the protective antigen protein of Bacillus anthracis

The protective antigen protein, one of the three separate proteins constituting the exotoxin system of Bacillus anthracis, has been crystallized in a form suitable for structural studies. The crystal form which is most amenable to x-ray analysis is orthorhombic, space group P2(1)2(1)2(1), a = 101.1 A, b = 95.4 A, c = 87.3 A, with one protective antigen monomer/asymmetric unit. The crystals diffract to approximately 3.0-A resolution.     

Cloning of the protective antigen gene of Bacillus anthracis

The tripartite protein toxin of Bacillus anthracis consists of protective antigen (PA), edema factor (EF), and lethal factor (LF). As a first step in developing a more efficacious anthrax vaccine, recombinant plasmids containing the PA gene have been isolated. A library was constructed in the E. coli vector pBR322 from Bam HI-generated fragments of the anthrax plasmid, pBA1. Two clones producing PA were identified by screening lysates with ELISA (enzyme-linked immunosorbent assay). Western blots revealed a full-size PA protein in the recombinant E. coli, and a cell elongation assay demonstrated biological activity. Both positive clones had a 6 kb insert of DNA, which mapped in the Bam HI site of the vector. The two inserts are the same except that they lie in opposite orientations with respect to the vector. Thus PA is encoded by the plasmid pBA1.     

Immunochemical properties of a protective cross-reacting antigen of meningococci

The study of protective cross-reacting antigenic preparations isolated from meningococci of groups A and C in the blot immunoassay has shown the presence of a group of proteins with a molecular weight ranging from 23 to 31 KD and common for 8 tested serological groups of meningococci, gonococci and 4 nonpathogenic Neisseria species. The possible role of these structures as common Neisseria antigen in the formation of natural resistance to meningococcal infection is discussed.     

Interactions between anthrax toxin receptors and protective antigen

Since the anthrax mail attacks of 2001, much has been learned about the interactions between anthrax toxin and its receptors. Two distinct cellular receptors for anthrax toxin have been identified and are designated capillary morphogenesis protein 2 (CMG2) and anthrax toxin receptor/tumor endothelial marker 8 (ATR/TEM8). The molecular details of the toxin-receptor interactions have been revealed through crystallographic, biochemical and genetic studies. In addition, a novel pathway by which anthrax toxin enters cells is starting to be uncovered.     

Functional characterization of protease-treated Bacillus anthracis protective antigen.

Characterization of the functional domains of Bacillus anthracis protective antigen (PA, 83-kDa), the common cellular binding molecule for both anthrax edema toxin and anthrax lethal toxin, is important for understanding the mechanism of entry and action of the anthrax toxins. In this study, we generated both biologically active (facilitates killing of J774A.1 cells in combination with lethal factor, LF) and inactive preparations of PA by protease treatment. Limited proteolytic digestion of PA in vitro with trypsin generated a 20-kDa fragment and a biologically active 63-kDa fragment. In contrast, limited digestion of PA with chymotrypsin yielded a preparation containing 37- and 47-kDa fragments defective for biological activity. Treatment with both chymotrypsin and trypsin generated three major fragments, 20, "17," and 47 kDa as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. This PA preparation was also biologically inactive. To investigate the nature of the defect resulting from chymotrypsin treatment, we assayed PA preparations for the ability to bind to the cellular receptor and to bind and internalize 125I-LF. All radiolabeled PA preparations bound with specificity to J774A.1 cells and exhibited affinities similar to native 83-kDa PA. Once bound to the cell surface receptor, both trypsin-treated PA and chymotrypsin/trypsin-treated PA specifically bound 125I-LF with high affinity. Finally, these PA preparations delivered 125I-LF to a Pronase-resistant cellular compartment in a time- and temperature-dependent fashion. Thus, the biological defect exhibited by chymotrypsin-treated PA is not at the level of cell binding or internalization but at a step later, such as toxin routing or processing by J774A.1 cells. These protease-treated preparations of PA should prove useful in both elucidating the intracellular processing of anthrax lethal toxin and determining the structure-function relationship of PA and LF.     

Serum protease cleavage of Bacillus anthracis protective antigen.

The protective antigen component of anthrax lethal toxin, produced in vitro, has a molecular mass of 83 kDa. Cell-culture studies by others have demonstrated that upon binding of the 83 kDa protective antigen to cell-surface receptors, the protein is cleaved by an unidentified cell-associated protease activity. The resultant 63 kDa protein then binds lethal factor to form lethal toxin, which has been proposed to be internalized by endocytosis. We found that, in the blood of infected animals, the protective antigen exists primarily as a 63 kDa protein and appears to be complexed with the lethal factor component of the toxin. Conversion of protective antigen from 83 to 63 kDa was catalysed by a calcium-dependent, heat-labile serum protease. Except for being complexed to protective antigen, there was no apparent alteration of lethal factor during the course of anthrax infection. The protective antigen-cleaving protease appeared to be ubiquitous among a wide range of animal species, including primates, horses, goats, sheep, dogs, cats and rodents.