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Nitration of the Birch Pollen Allergen Bet v 1.0101: Efficiency and Site-Selectivity of Liquid and Gaseous Nitrating Agents

  • K. Reinmuth-Selzle
  • , C. Ackaert
  • , C.J. Kampf
  • , M. Samonig
  • , M. Shiraiwa
  • , S. Kofler
  • , H. Yang
  • , G. Gadermaier
  • , H. Brandstetter
  • , C.G. Huber
  • , A. Duschl
  • , G.J. Oostingh
  • , U. Pöschl
  • Multiphase Chemistry and Biogeochemistry Departments, Max Planck Institute for Chemistry
  • Department of Molecular Biology, University of Salzburg
  • Christian Doppler Laboratory for Allergy Diagnosis and Therapy, Department of Molecular Biology, University of Salzburg

Research output: Contribution to journalArticlepeer-review

Abstract

Nitration of the major birch pollen allergen Bet v 1 alters the immune responses toward this protein, but the underlying chemical mechanisms are not yet understood. Here we address the efficiency and site-selectivity of the nitration reaction of recombinant protein samples of Bet v 1.0101 with different nitrating agents relevant for laboratory investigations (tetranitromethane, TNM), for physiological processes (peroxynitrite, ONOO-), and for the health effects of environmental pollutants (nitrogen dioxide and ozone, O 3/NO2). We determined the total tyrosine nitration degrees (ND) and the NDs of individual tyrosine residues (NDY). High-performance liquid chromatography coupled to diode array detection and HPLC coupled to high-resolution mass spectrometry analysis of intact proteins, HPLC coupled to tandem mass spectrometry analysis of tryptic peptides, and amino acid analysis of hydrolyzed samples were performed. The preferred reaction sites were tyrosine residues at the following positions in the polypeptide chain: Y83 and Y81 for TNM, Y150 for ONOO-, and Y83 and Y158 for O 3/NO2. The tyrosine residues Y83 and Y81 are located in a hydrophobic cavity, while Y150 and Y158 are located in solvent-accessible and flexible structures of the C-terminal region. The heterogeneous reaction with O3/NO2 was found to be strongly dependent on the phase state of the protein. Nitration rates were about one order of magnitude higher for aqueous protein solutions (∼20% per day) than for protein filter samples (∼2% per day). Overall, our findings show that the kinetics and site-selectivity of nitration strongly depend on the nitrating agent and reaction conditions, which may also affect the biological function and adverse health effects of the nitrated protein. © 2014 American Chemical Society.
Original languageEnglish
Pages (from-to)1570-1577
Number of pages8
JournalJournal of Proteome Research
Volume13
Issue number3
DOIs
Publication statusPublished - 2014

Keywords

  • air pollution
  • Bet v 1.0101
  • HPLC-MS/MS
  • nitration sites
  • tyrosine nitration
  • pollen antigen
  • polypeptide
  • amino acid analysis
  • article
  • birch
  • high performance liquid chromatography
  • liquid chromatography
  • mass spectrometry
  • nitration
  • pollutant
  • priority journal
  • tandem mass spectrometry
  • Amino Acid Sequence
  • Antigens, Plant
  • Betula
  • Escherichia coli
  • Gene Expression
  • Kinetics
  • Models, Molecular
  • Molecular Sequence Data
  • Nitrogen Dioxide
  • Ozone
  • Peptides
  • Peroxynitrous Acid
  • Pollen
  • Protein Structure, Secondary
  • Recombinant Proteins
  • Tetranitromethane
  • Tyrosine

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