Abstract
Protective antigen (PA) is a central virulence factor of Bacillus anthracis and a key component in anthrax vaccines. PA binds to target cell receptors, is cleaved by the furin protease, self-aggregates to heptamers, and finally internalizes as a complex with either lethal or edema factors. Under mild room temperature storage conditions, PA cytotoxicity decreased (t(1/2) approximately 7 days) concomitant with the generation of new acidic isoforms, probably through deamidation of Asn residues. Ranking all 68 Asn residues in PA based on their predicted deamidation rates revealed five residues with half-lives of <60 days, and these residues were further analyzed: Asn10 in the 20-kDa region, Asn162 at P6 vicinal to the furin cleavage site, Asn306 in the pro-pore translocation loop, and both Asn713 and Asn719 in the receptor-binding domain. We found that PA underwent spontaneous deamidation at Asn162 upon storage concomitant with decreased susceptibility to furin. A panel of model synthetic furin substrates was used to demonstrate that Asn162 deamidation led to a 20-fold decrease in the bimolecular rate constant (k(cat)/Km) of proteolysis due to the new negatively charged residue at P6 in the furin recognition sequence. Furthermore, reduced PA cytotoxicity correlated with a decrease in PA cell binding and also with deamidation of Asn713 and Asn719. On the other hand, neither deamidation of Asn10 or Asn306 nor impairment of heptamerization could be observed upon prolonged PA storage. We suggest that PA inactivation during storage is associated with susceptible deamidation sites, which are intimately involved in both mechanisms of PA cleavage by furin and PA-receptor binding.
Highlights
The Gram-positive spore-forming bacterium Bacillus anthracis, the causative agent of anthrax, produces a bipartite A/B-type toxin
To determine putative deamidated residues, we first ranked all of the 68 Asn residues of protective antigen (PA) (Protein Data Bank code 1ACC) according to their theoretical deamidation half-lives calculated on the basis of the algorithm of Robinson and Robinson [39], which takes into account empirical studies as well as primary, secondary, and three-dimensional structures of the protein, under standard experimental conditions
PA has a complex mechanism of action, involving multiple forms of protein-protein interactions: as a substrate for furin, as a ligand to cell receptors, as a self-forming homooligomer, and as a complex with lethal factor (LF) and edema factor (EF) [6, 16, 68]
Summary
The Gram-positive spore-forming bacterium Bacillus anthracis, the causative agent of anthrax, produces a bipartite A/B-type toxin. Following PA binding to cell receptors [5,6,7,8], it is cleaved by a furin family protease [9] to a 20-kDa N-terminal fragment with no known further function and to a 63-kDa fragment that forms ring-shaped heptamers on the cell surface [10]. The deamidation process is of major concern in the production of pharmaceutical proteins mainly during their purification and under long-term storage, as reported for recombinant human growth hormone [51], growth hormone-releasing factor [52], recombinant plasminogen activator [53], and recombinant human interleukin-11 [54], and even in monoclonal antibody preparations following in vivo administration [55]. It was demonstrated recently that deamidation of tetanus vaccine under long-term storage results in the impairment of antigen processing and presentation [56]. We present evidence supporting the notion that inactivation of PA upon storage is caused by spontaneous deamidation of at least three vulnerable Asn sites, perturbing crucial steps in its mechanism of action
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