DEAMIDATION AND ISOASPARTIC ACID FORMATION IN PROTECTIVE ANTIGEN

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This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. The protective antigen (PA) protein of Bacillus anthracis plays an essential role for the pathology of anthrax by facilitating the translocation of toxic factors into targeted host cells. Since it is highly immunogenic and non-toxic by itself, recombinant PA (rPA) protein is also the proposed pharmaceutical substance for an improved human anthrax vaccine. PA has been known since discovery to comprise multiply charged isoforms, but the cause of heterogeneity in PA has eluded specific structural description. Two primary isoforms appear in vivo early during expression and represent the majority components after purification. Although both isoforms elicit equivalent protective immunity in animals as compared to the pharmaceutical product, structural heterogeneity of rPA requires further chemical and biological characterization since it is intended for eventual human use. Moreover, isoforms re-appear after improper handling in vitro; therefore, the source and nature of this degradation requires better definition. Using liquid chromatography-tandem mass spectrometry (LC-ESI-MS/MS) with verification of automated assignments for amino acid modification, we have recently demonstrated that pharmaceutical grade rPA contains measurable deamidation at 6 of 68 total asparagine (Asn) residues. A direct correlation between isoform complexity and percent deamidation was observed among various grades and treatments of rPA, as well as between isoforms purified by gel electrophoresis, such that both decreased with purity and increased with protein aging. With respect to biochemical and biological function, rPA with more isoforms and greater deamidation displayed lower in vitro activities for heptamerization, holotoxin formation, and macrophage toxicity. However, neither the overall complexity nor the identity of any given isoform was associated with percent deamidation at any observed site. Position N537 consistently showed the highest modification in all samples analyzed, even though it ranks 10th by a method of theoretical modeling (viewable at www.deamidation.org) which has otherwise been remarkably accurate in predicting protein and peptide deamidation from defined crystal structure. Since five other observed sites in rPA also did not align with predicted rank order for levels of deamidation, rPA appears to exhibit properties not yet incorporated into the popular modeling algorithm. A publication is being prepared.