Multiple asparagine deamidation of Bacillus anthracis protective antigen causes charge isoforms whose complexity correlates with reduced biological activity.
Protective antigen is essential for 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 (rPA) contains similar isoforms that 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 rPA contained measurable deamidation at seven 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 unmodified. 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 deamidation at residues N466 and N408. The described method measures deamidation without employing theoretical isotopic distributions, comparison between differentially treated samples or computational predictions of reactivity rates, and is broadly applicable to the characterization of other deamidated proteins.
Powell
Bradford S
BS
Bacteriology Division, United States Army Medical Research Institute of Infectious Diseases, Fort Detrick, Frederick, Maryland 21702-5011, USA. bradford.powell@det.amedd.army.mil
Enama
Jeffrey T
JT
Ribot
Wilson J
WJ
Webster
Wendy
W
Little
Stephen
S
Hoover
Timothy
T
Adamovicz
Jeffrey J
JJ
Andrews
Gerard P
GP
D000595:Amino Acid Sequence; D000942:Antigens, Bacterial / Q000378:metabolism*; D001216:Asparagine / Q000737:chemistry / Q000378:metabolism*; D001224:Aspartic Acid / Q000737:chemistry / Q000378:metabolism*; D001408:Bacillus anthracis / Q000378:metabolism*; D001426:Bacterial Proteins / Q000737:chemistry; D013058:Mass Spectrometry; D008958:Models, Molecular; D008969:Molecular Sequence Data; D020033:Protein Isoforms / Q000737:chemistry* / Q000378:metabolism*; D011499:Protein Processing, Post-Translational; D011994:Recombinant Proteins / Q000737:chemistry
D000942:Antigens, Bacterial; D001426:Bacterial Proteins; D020033:Protein Isoforms; D011994:Recombinant Proteins; D001224:Aspartic Acid; D001216:Asparagine