Multiple asparagine deamidation of Bacillus anthracis protective antigen causes charge isoforms whose complexity correlates with reduced biological activity

Protective antigen is essential for the pathology of Bacillus anthracis and is the proposed immunogen for an improved human anthrax vaccine. Known since discovery to comprise differentially charged isoforms, the cause of heterogeneity has eluded specific structural definition until now. Recombinant protective antigen (rPA) contains similar isoforms that appear early in fermentation and are mostly removed through purification. By liquid chromatography-tandem mass spectrometry sequencing of the entire protein and inspection of spectral data for amino acid modifications, pharmaceutical rPA contained measurable deamidation at seven of its 68 asparagine residues. A direct association between isoform complexity and percent deamidation was observed such that each decreased with purity and increased with protein aging. Position N537 consistently showed the highest level of modification, although its predicted rate of deamidation ranked 10th by theoretical calculation, and other asparagines of higher predicted rates were observed to be unmodified. rPA with more isoforms and greater deamidation displayed lower activities for furin cleavage, heptamerization, and holotoxin formation. Lethal factor-mediated macrophage toxicity correlated inversely with deamidation at residues N466 and N408. The described method measures deamidation without employing theoretical isotopic distributions, comparison between differentially treated samples or computational predictions of reactivity rates, and is broadly applicable to the characterization of other deamidated proteins.     

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.     

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.     

Genetic diversity in the protective antigen gene of Bacillus anthracis

Bacillus anthracis is a gram-positive spore-forming bacterium that causes the disease anthrax. The anthrax toxin contains three components, including the protective antigen (PA), which binds to eucaryotic cell surface receptors and mediates the transport of toxins into the cell. In this study, the entire 2,294-nucleotide protective antigen gene (pag) was sequenced from 26 of the most diverse B. anthracis strains to identify potential variation in the toxin and to further our understanding of B. anthracis evolution. Five point mutations, three synonymous and two missense, were identified. These differences correspond to six different haploid types, which translate into three different amino acid sequences. The two amino acid changes were shown to be located in an area near a highly antigenic region critical to lethal factor binding. Nested primers were used to amplify and sequence this same region of pag from necropsy samples taken from victims of the 1979 Sverdlovsk incident. This investigation uncovered five different alleles among the strains present in the tissues, including two not seen in the 26-sample survey. One of these two alleles included a novel missense mutation, again located just adjacent to the highly antigenic region. Phylogenetic (cladistic) analysis of the pag corresponded with previous strain grouping based on chromosomal variation, suggesting that plasmid evolution in B. anthracis has occurred with little or no horizontal transfer between the different strains.     

炭疽杆菌保护性抗原基因的克隆与序列测定

采用聚合酶链反应从炭疽芽孢杆菌减毒株ＹＢ１中扩增其保护性抗原（ＰＡ）的编码区基因,将其克隆至ｐＧＥＭ－Ｔ载体中,并分步测定其序列。序列测定表明,该基因长２２０５ｂｐ,编码７３５个氨基酸残基,与献报道的标准菌株Ｓｔｅｒｎｅ株的ＰＡ序列只有４个碱基的差异。     

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.     

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.     

Expression and secretion of the protective antigen of Bacillus anthracis in Bacillus brevis

We used the Bacillus brevis-pNU212 system to develop a mass production system for the protective antigen (PA) of Bacillus anthracis. A moderately efficient expression-secretion system for PA was constructed by fusing the PA gene from B. anthracis with the B. brevis cell-wall protein signal-peptide encoding region of pNU212, and by introducing the recombinant plasmid, pNU212-mPA, into B. brevis 47-5Q. The clone producing PA secreted about 300 microg of recombinant PA (rPA) per ml of 5PY-erythromycin medium after 4 days incubation at 30 degrees C. The rPA was fractionated from the culture supernatant of B. brevis 47-5Q carrying pNU212-mPA using ammonium sulfate at 70% saturation followed by anion exchange chromatography on a Hitrap Q, a Hiload 16/60 Superdex 200 gel filtration column and a phenyl sepharose hydrophobic interaction column, yielding 70 mg rPA per liter of culture. The N-terminal sequence of the purified rPA was identical to that of native PA from B. anthracis. The purified rPA exhibited cytotoxicity towards J774A.1 cells when combined with lethal factor. The rPA formulated in either Rehydragel HPA or MPL-TDM-CWS adjuvant (Ribi-Trimix) elicited the expression of a large amount of anti-PA and neutralizing antibodies in guinea pigs and completely protected them against a 100 LD50 challenge with fully virulent B. anthracis spores.     

Expression and purification of the Bacillus anthracis protective antigen domain 4

The protective antigen (PA) of Bacillus anthracis plays a crucial role in the pathogenesis of the anthrax disease. The fourth domain of PA (PA-D4) is responsible for initial binding of the anthrax toxin to the cellular receptor, and thus, is an attractive target for structure-based drug therapies. A synthetic gene for PA-D4 has been prepared by recursive PCR. PA-D4 has been expressed as a fusion protein in Escherichia coli. PA-D4 has been purified to near homogeneity and its identity has been verified by mass spectrometry. The recombinant PA-D4 exhibits CD and NMR spectra that suggest that it is folded and amenable for biophysical studies. Moreover, recombinant PA-D4 binds to HeLa cells, which suggests that recombinant PA-D4 is functional to bind to its cellular receptor.     

Location of receptor-binding region of protective antigen from Bacillus anthracis

The pag gene, which codes for protective antigen (PA), a common component of the lethal and edema toxins of Bacillus anthracis, was cloned and expressed in Escherichia coli. Nested deletions of pag were generated into the C-terminus coding region. Recombinant proteins were analyzed by Western blot with either an anti-PA polyclonal antisera or two monoclonal antibodies that neutralized lethal toxin and edema toxin activities by inhibiting the binding of PA to cell receptors. Localization of the receptor binding domain within the C-terminal region of PA was suggested by the inability of the monoclonal antibodies 3B6 and 14B7 to recognize the recombinant proteins expressed by C-terminal deletions of the pag gene.     

Scalable purification of Bacillus anthracis protective antigen from Escherichia coli

The anthrax toxin consists of three proteins, protective antigen (PA), lethal factor, and edema factor that are produced by the Gram-positive bacterium, Bacillus anthracis. Current vaccines against anthrax use PA as their primary component. In this study, we developed a scalable process to produce and purify multi-gram quantities of highly pure, recombinant PA (rPA) from Escherichia coli. The rPA protein was produced in a 50-L fermentor and purified to >99% purity using anion-exchange, hydrophobic interaction, and hydroxyapatite chromatography. The final yield of purified rPA from medium cell density fermentations resulted in approximately 2.7 g of rPA per kg of cell paste (approximately 270 mg/L) of highly pure, biologically active rPA protein. The results presented here exhibit the ability to generate multi-gram quantities of rPA from E. coli that may be used for the development of new anthrax vaccines and anthrax therapeutics.     

Monoclonal antibodies directed against protective antigen of Bacillus anthracis enhance lethal toxin activity in vivo

Protective antigen (PA) from Bacillus anthracis binds to cellular receptors, combines with lethal factor (LF) forming lethal toxin (LeTx), and facilitates the translocation of LF into the cytosol. LeTx is cytotoxic for J774A.1 cells, a murine macrophage cell line, and causes death of Fisher 344 rats when injected intravenously. PA is also the major protective component in anthrax vaccines. Antibody-dependent enhancement has been reported for several viral diseases, a bacterial infection, and for B. anthracis LeTx in vitro cytotoxicity. Further screening of our 73 PA monoclonal antibodies (mAbs) identified a total of 17 PA mAbs that enhanced in vitro cytotoxicity at suboptimal concentrations of LeTx. A competitive binding enzyme-linked immunosorbent assay showed that these 17 PA mAbs identified eight different antigenic regions on PA. Eight of the 17 PA mAbs that enhanced LeTx in vitro cytoxicity were examined for their activity in vivo. Of the eight mAbs that were injected intravenously with a sublethal concentration of LeTx into male Fisher 344 rats, four mAbs enhanced the lethality of LeTx and resulted in the death of animals, whereas control animals did not succumb to intoxication. This is the first demonstration that PA mAbs can enhance LeTx intoxication in vivo.     

Sequence and analysis of the DNA encoding protective antigen of Bacillus anthracis

The nucleotide sequence of the protective antigen (PA) gene from Bacillus anthracis and the 5' and 3' flanking sequences were determined. PA is one of three proteins comprising anthrax toxin; and its nucleotide sequence is the first to be reported from B. anthracis. The open reading frame (ORF) is 2319 bp long, of which 2205 bp encode the 735 amino acids of the secreted protein. This region is preceded by 29 codons, which appear to encode a signal peptide having characteristics in common with those of other secreted proteins. A consensus TATAAT sequence was located at the putative -10 promoter site. A Shine-Dalgarno site similar to that found in genes of other Bacillus sp. was located 7 bp upstream from the ATG start codon. The codon usage for the PA gene reflected its high A + T (69%) base composition and differed from those of genes for bacterial proteins from most other sequences examined. The TAA translation stop codon was followed by an inverted repeat forming a potential termination signal. In addition, a 192-codon ORF of unknown significance, theoretically encoding a 21.6-kDa protein, preceded the 5' end of the PA gene.     

Efficacy of non-toxic deletion mutants of protective antigen from Bacillus anthracis

Current human anthrax vaccines available in the United States and Europe consist of alum-precipitated supernatant material from cultures of a toxigenic, nonencapsulated strain of Bacillus anthracis. The major component of human anthrax vaccine that confers protection is protective antigen (PA). A second-generation human vaccine using the recombinant PA (rPA) is being developed. In this study, to prevent the toxicity and the degradation of the native rPA by proteases, we constructed two PA variants, delPA (163-168) and delPA (313-314), that lack trypsin (S(163)-R(164)-K(165)-K(166)-R(167)-S(168)) or chymotrypsin cleavage sequence (F(313)-F(314)), respectively. These proteins were expressed in Bacillus brevis 47-5Q. The delPAs were fractionated from the culture supernatant of B. brevis by ammonium sulfate at 70% saturation, followed by anion exchange chromatography on a Hitrap Q, Hiload 16/60 superdex 200 gel filtration column and phenyl sepharose hydrophobic interaction column. In accordance with previous reports, both delPA proteins combined with lethal factor protein did not show any cytotoxicity on J774A.1 cells. The delPA (163-168) and delPA (313-314) formulated either in Rehydragel HPA or MPL-TDM-CWS (Ribi-Trimix), elicited a comparable amount of anti-PA and neutralizing antibodies to those of native rPA in guinea pigs, and confers full protection of guinea pigs from 50xLD50 of fully virulent B. anthracis spore challenges. Ribi-Trimix was significantly more effective in inducing anti-PA and neutralizing antibodies than Rehydragel HPA. These results indicate the possibility of delPA (163-168) and delPA (313-314) proteins being developed into nontoxic, effective and stable recombinant vaccine candidates.     

Evaluation of Bacillus subtilis strain IS53 for the production of Bacillus anthracis protective antigen

An asporogenous strain of Bacillus subtilis , IS53, transformed with plasmid pPA102, produces the protective antigen (PA) component of the tripartite toxin of B. anthracis . Addition of yeast extract was required to support growth and PA production in all the media examined. Protective antigen expression was down-regulated during exponential growth and extracellular proteases caused marked degradation of the mature protein.     

Preparation and characterization of monoclonal antibodies to Bacillus anthracis protective antigen

Anthrax is the widespread acute infection disease, affecting animals and humans, refers to the bioterrorist threat agents of category A, because of the high resistance of Bacillus anthracis spores to adverse environmental factors and the ease of receiving them. We obtain a representative panel of 20 monoclonal antibodies against the key component of pathogenic exotoxins, anthrax protective antigen. Quantitative sandwich-ELISA for protective antigen with antibody obtained was developed. Six pairs of monoclonal antibodies showed the detection limit up to 1 ng/ml concentration of the protective antigen in blood serum.