RESUMO
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.
Assuntos
Antígenos de Bactérias/química , Antígenos de Bactérias/metabolismo , Toxinas Bacterianas/química , Toxinas Bacterianas/metabolismo , Sequência de Aminoácidos , Animais , Asparagina/química , Sítios de Ligação , Biotinilação , Células CHO , Sobrevivência Celular , Cricetinae , Dimerização , Eletroforese em Gel de Poliacrilamida , Furina/química , Focalização Isoelétrica , Cinética , Modelos Moleculares , Dados de Sequência Molecular , Peptídeos/química , Ligação Proteica , Isoformas de Proteínas , Estrutura Terciária de Proteína , Transporte Proteico , Proteínas/química , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Fatores de Tempo , Tripsina/químicaRESUMO
Vaccination with vaccinia virus is carried out in order to induce protection against variola virus, the causative agent of smallpox. Serum titer of vaccinia virus-neutralizing antibodies is considered to be well-correlated with in vivo protection. Plaque reduction neutralization test (PRNT) is the gold standard for detecting and quantifying vaccinia virus-neutralizing antibodies in sera of vaccinees. However, PRNT is time and labor consuming, which does not allow large-scale screening needed for a population survey. A simplified, sensitive, standardized, reproducible and rapid method, neutralization tissue-culture enzyme immunoassay (NTC-EIA) was developed for quantitation of neutralizing antibodies against vaccinia virus. The assay consists of the following steps: neutralization of the virus with serially diluted sera, infection of cells in culture and measurement of residual virus replication using an enzyme immunoassay. The assay can be used for animal (rabbit) or human sera. Titer averages obtained using NTC-EIA were highly correlated (R2=0.9994) to those obtained using PRNT. The assay is carried out in 96-well plates and takes only 2 days to complete. With the appropriate setup, it can be automated fully to allow screening of a large number of sera.
