title: Effects of spontaneous deamidation on the cytotoxic activity of the Bacillus anthracis protective antigen.
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.
authors.affiliation: Department of Biotechnology, Israel Institute for Biological Research, Ness-Ziona 74100, Israel.
authors.forename: Gil
authors.initials: G
authors.lastname: Zomber
authors.forename: Shaul
authors.initials: S
authors.lastname: Reuveny
authors.forename: Nissim
authors.initials: N
authors.lastname: Garti
authors.forename: Avigdor
authors.initials: A
authors.lastname: Shafferman
authors.forename: Eytan
authors.initials: E
authors.lastname: Elhanany
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D000942:Antigens, Bacterial / Q000737:chemistry* / Q000378:metabolism*; D001216:Asparagine / Q000737:chemistry; D001427:Bacterial Toxins / Q000737:chemistry* / Q000378:metabolism*; D001665:Binding Sites; D019921:Biotinylation; D016466:CHO Cells; D002470:Cell Survival; D006224:Cricetinae; D019281:Dimerization; D004591:Electrophoresis, Polyacrylamide Gel; D045683:Furin / Q000737:chemistry; D007525:Isoelectric Focusing; D007700:Kinetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010455:Peptides / Q000737:chemistry; D011485:Protein Binding; D020033:Protein Isoforms; D017434:Protein Structure, Tertiary; D021381:Protein Transport; D011506:Proteins / Q000737:chemistry; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013997:Time Factors; D014357:Trypsin / Q000737:chemistry
chemical_list: D000942:Antigens, Bacterial; D001427:Bacterial Toxins; D010455:Peptides; D020033:Protein Isoforms; D011506:Proteins; C030325:anthrax toxin; D001216:Asparagine; D014357:Trypsin; D045683:Furin