Unfolding transitions of Bacillus anthracis protective antigen

Chalton, David A.; Kelly, Ian F.; McGregor, Alistair; Ridley, Helen; Watkinson, Allan; Miller, Julie Ann; Lakey, Jeremy H.

doi:10.1016/j.abb.2007.04.030

Cited by 18 publications

(24 citation statements)

References 33 publications

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“…The solid-state (31, 34) far-UV CD spectra of rPA-AlOH closely resembled those of soluble rPA (35) (Fig. 5A), indicating minimal secondary structure perturbation upon binding to Alhydrogel.…”

Section: Resultsmentioning

confidence: 81%

“…The phosphate-subsaturated 0.25 mM phosphate rPA-AlOH formulation was significantly less potent than the 4 mM phosphate rPA-AlOH formulation, and one contrasting feature of the two formulations was the pH of the bulk solution, with the 0.25 mM phosphate rPA-AlOH formulation having a pH of 5.9 and the 4 mM phosphate rPA-AlOH formulation a value of around 7.1. Consequently, the differing bulk solution pH values might explain the differences in ED 50 values, particularly since rPA is acid labile (35,42). However, the biophysical analysis of the protein structure of rPA-AlOH under low-phosphate conditions does not support any major acid-induced transformation of the protein structure (31,35).…”

Section: Discussionmentioning

confidence: 92%

“…The near-UV solidstate CD spectra of rPA-AlOH at 0.25 mM phosphate, 4 mM phosphate, and 10 mM phosphate were also similar, with each showing the two prominent positive CD bands for tryptophan at 284 nm and 291 nm. These two bands, which are characteristic of rPA (31,35), were weaker than for soluble rPA in solution (Fig. 5B); in the 10 mM phosphate sample, two bands (272 nm and 276 nm) that were not present for soluble rPA were observed.…”

Section: Resultsmentioning

confidence: 99%

“…Consequently, the differing bulk solution pH values might explain the differences in ED 50 values, particularly since rPA is acid labile (35,42). However, the biophysical analysis of the protein structure of rPA-AlOH under low-phosphate conditions does not support any major acid-induced transformation of the protein structure (31,35). Furthermore, with 0.25 mM phosphate rPA-AlOH, the Alhydrogel surface and bound antigen would have a local pH approximately 2 units higher, due to the positive surface charge attracting hydroxyl anions (27).…”

Section: Discussionmentioning

confidence: 99%

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Increasing the Potency of an Alhydrogel-Formulated Anthrax Vaccine by Minimizing Antigen-Adjuvant Interactions

Watkinson

Soliakov

Ganesan³

et al. 2013

Clin Vaccine Immunol

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f Aluminum salts are the most widely used vaccine adjuvants, and phosphate is known to modulate antigen-adjuvant interactions. Here we report an unexpected role for phosphate buffer in an anthrax vaccine (SparVax) containing recombinant protective antigen (rPA) and aluminum oxyhydroxide (AlOH) adjuvant (Alhydrogel). Phosphate ions bind to AlOH to produce an aluminum phosphate surface with a reduced rPA adsorption coefficient and binding capacity. However, these effects continued to increase as the free phosphate concentration increased, and the binding of rPA changed from endothermic to exothermic. Crucially, phosphate restored the thermostability of bound rPA so that it resembled the soluble form, even though it remained tightly bound to the surface. Batches of vaccine with either 0.25 mM (subsaturated) or 4 mM (saturated) phosphate were tested in a disease model at batch release, which showed that the latter was significantly more potent. Both formulations retained their potency for 3 years. The strongest aluminum adjuvant effects are thus likely to be via weakly attached or easily released native-state antigen proteins.

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Section: Resultsmentioning

confidence: 81%

Section: Discussionmentioning

confidence: 92%

Section: Resultsmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

See 2 more Smart Citations

Increasing the Potency of an Alhydrogel-Formulated Anthrax Vaccine by Minimizing Antigen-Adjuvant Interactions

Watkinson

Soliakov

Ganesan³

et al. 2013

Clin Vaccine Immunol

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“…The molecular events that lead to such destabilization remain to be elucidated. Among possible causes for the observed destabilization are direct interaction of previously buried or inaccessible regions of rPA with Alhydrogel, accelerated deamidation of asparagine residues important for structural integrity of the protein due to the Alhydrogel microenvironment (38), and loss of one or both of the calcium ions that are important in the stabilization of PA structure in solution (5,12,14,15,28). Another possibility is that minute quantities of contaminating proteases copurifying with rPA could lead to proteolysis of the adsorbed protein upon prolonged storage; although SDS-PAGE analysis of rPA stored in solution for up to 3 weeks at 25°C did not show significant proteolysis (data not shown).…”

Section: Figmentioning

confidence: 99%

Structural and Immunological Analysis of Anthrax Recombinant Protective Antigen Adsorbed to Aluminum Hydroxide Adjuvant

Wagner

Verma

Meade

et al. 2012

Clin Vaccine Immunol

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ABSTRACTNew anthrax vaccines currently under development are based on recombinant protective antigen (rPA) and formulated with aluminum adjuvant. Because long-term stability is a desired characteristic of these vaccines, an understanding of the effects of adsorption to aluminum adjuvants on the structure of rPA is important. Using both biophysical and immunological techniques, we compared the structure and immunogenicity of freshly prepared rPA-Alhydrogel formulations to that of formulations stored for 3 weeks at either room temperature or 37°C in order to assess the changes in rPA structure that might occur upon long-term storage on aluminum adjuvant. Intrinsic fluorescence emission spectra of tryptophan residues indicated that some tertiary structure alterations of rPA occurred during storage on Alhydrogel. Using anti-PA monoclonal antibodies to probe specific regions of the adsorbed rPA molecule, we found that two monoclonal antibodies that recognize epitopes located in domain 1 of PA exhibited greater reactivity to the stored formulations than to freshly prepared formulations. Immunogenicity of rPA-Alhydrogel formulations in mice was assessed by measuring the induction of toxin-neutralizing antibodies, as well as antibodies reactive to 12-mer peptides spanning the length of PA. Mice immunized with freshly prepared formulations developed significantly higher toxin-neutralizing antibody titers than mice immunized with the stored preparations. In contrast, sera from mice immunized with stored preparations exhibited increased reactivity to nine 12-mer peptides corresponding to sequences located throughout the rPA molecule. These results demonstrate that storage of rPA-Alhydrogel formulations can lead to structural alteration of the protein and loss of the ability to elicit toxin-neutralizing antibodies.

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Effect of metal ion substitutions in anticoagulation factor I from the venom of Agkistrodon acutus on the binding of activated coagulation factor X and on structural stability

Zhang

Shen

et al. 2009

J Biol Inorg Chem

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Anticoagulation factor I (ACF I) isolated from the venom of Agkistrodon acutus is an activated coagulation factor X (FXa)-binding protein that binds in a Ca(2+)-dependent fashion with marked anticoagulant activity. The thermodynamics of the binding of alkaline earth metal ions to ACF I and the effects of alkaline earth metal ions on the guanidine hydrochloride (GdnHCl)-induced unfolding of ACF I and the binding of ACF I to FXa were studied by isothermal titration calorimetry, fluorescence, circular dichroism, and surface plasmon resonance, respectively. The results indicate that the ionic radii of the cations occupying Ca(2+)-binding sites in ACF I crucially affect the binding affinity of ACF I for alkaline earth metal ions as well as the structural stability of ACF I against GdnHCl denaturation. Sr(2+) and Ba(2+), with ionic radii larger than the ionic radius of Ca(2+), can bind to Ca(2+)-free ACF I (apo-ACF I), while Mg(2+), with an ionic radius smaller than that of Ca(2+), shows significantly low affinity for the binding to apo-ACF I. All bindings of Ca(2+), Sr(2+), and Ba(2+) ions in two sites of ACF I are mainly enthalpy-driven and the entropy is unfavorable for them. Sr(2+)-stabilized ACF I exhibits slightly lower resistance to GdnHCl denaturation than Ca(2+)-ACF I, while Ba(2+)-stabilized ACF I exhibits much lower resistance to GdnHCl denaturation than Ca(2+)-ACF I. Mg(2+) and Sr(2+), with ionic radii close to that of Ca(2+), can bind to FXa and therefore also induce the binding of ACF I to FXa, whereas Ba(2+), with a much larger ionic radius than Ca(2+), cannot support the binding of ACF I with FXa. Our observations suggest that bindings of Ca(2+), Sr(2+), and Ba(2+) ions in two sites of ACF I increase the structural stability of ACF I, but these bindings are not essential for the binding of ACF I with FXa, and that the binding of Mg(2+), Ca(2+), and Sr(2+) ions to FXa may be essential for the recognition between FXa and ACF I.

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Unfolding transitions of Bacillus anthracis protective antigen

Cited by 18 publications

References 33 publications

Increasing the Potency of an Alhydrogel-Formulated Anthrax Vaccine by Minimizing Antigen-Adjuvant Interactions

Increasing the Potency of an Alhydrogel-Formulated Anthrax Vaccine by Minimizing Antigen-Adjuvant Interactions

Structural and Immunological Analysis of Anthrax Recombinant Protective Antigen Adsorbed to Aluminum Hydroxide Adjuvant

Effect of metal ion substitutions in anticoagulation factor I from the venom of Agkistrodon acutus on the binding of activated coagulation factor X and on structural stability

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