Multiple Asparagine Deamidation of Bacillus anthracis Protective Antigen Causes Charge Isoforms Whose Complexity Correlates with Reduced Biological Activity
ARMY MEDICAL RESEARCH INST OF INFECTIOUS DISEASES FORT DETRICK MD BACTERIOLOGY DIV
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Protective antigen is essential to the pathology of Bacillus anthracis and is the proposed immunogen for an improved human anthrax vaccine. Known since discovery to comprise differentially charged isoforms, the cause of heterogeneity has eluded specific structural definition until now. Recombinant protective antigen PA contains similar isoforms which appear early in fermentation and are mostly removed through purification. By liquid chromatography-tandem mass spectrometry sequencing of the entire protein and inspection of spectral data for amino acid modifications, pharmaceutical grade rPA contained measurable deamidation at 7 of its 68 asparagine residues. A direct association between isoform complexity and percent deamidation was observed such that each decreased with purity and increased with protein aging. Position N537 consistently showed the highest level of modification, although its predicted rate of deamidation ranked 10th by theoretical calculation, and other asparagines of higher predicted rates were observed to be unmodifed. rPA with more isoforms and greater deamidation displayed lower activities for furin cleavage, heptamerization, and holotoxin formation. Lethal factor-mediated macrophage toxicity correlated inversely with rPA deamidation. The described method measures deamidation without comparison to theoretical isotopic distributions, algorithmic predictions of reactivity, or differentially treated samples, and is broadly applicable to the characterization of other deamidated proteins.
- Anatomy and Physiology