Multiple Asparagine Deamidation of Bacillus anthracis Protective Antigen Causes Charge Isoforms Whose Complexity Correlates with Reduced Biological Activity

Abstract

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.

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Document Details

Document Type
Technical Report
Publication Date
Jan 01, 2007
Accession Number
ADA470145

Entities

People

  • Bradford S. Powell
  • Gerard P. Andrews
  • Jeffrey J. Adamovicz
  • Jeffrey T. Enama
  • Stephen Little
  • Timothy Hoover
  • Wendy Webster
  • Wilson J. Ribot

Organizations

  • United States Army Medical Research Institute of Infectious Diseases

Tags

Communities of Interest

  • Biomedical

DTIC Thesaurus Topics

  • Amino Acids
  • Biomedical Information Systems
  • Biomedical Research
  • Capillary Electrophoresis
  • Chromatography
  • Computational Biology
  • Gel Electrophoresis
  • Infectious Diseases
  • Information Operations
  • Ion Traps
  • Liquid Chromatography
  • Mass Spectrometry
  • Spectrometry
  • Two Dimensional
  • United States
  • Vaccines

Readers

  • Analytical Chemistry
  • Electrochemical Surface Science
  • Microbial Pathology

Technology Areas

  • Biotechnology