ToxSci Advance Access published online on October 16, 2008
Toxicological Sciences, doi:10.1093/toxsci/kfn211
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Tyrosines of human and mouse transferrin covalently labeled by organophosphorus agents: a new motif for binding to proteins that have no active site serine
* Eppley Institute, University of Nebraska Medical Center, Omaha, NE 68198-6805, binli{at}unmc.edu, lmschopf{at}unmc.edu, hgrigoryan{at}unmc.edu, olockrid{at}unmc.edu
Department of Biomedical and Pharmaceutical Sciences, University of Montana, Missoula, MT 59812 cmthomp{at}spahs.umt.edu
Department of Pathology and Microbiology, University of Nebraska Medical Center, Omaha, NE 68198 shinrich{at}unmc.edu
Centre de Recherches d Service de Santé des Armées, Unité d'Enzymologie, BP87, 38702 La Tronche Cedex, France pmasson{at}unmc.edu
Corresponding author: Oksana Lockridge, Eppley Institute, University of Nebraska Medical Center, Omaha, NE 68198-6805, olockrid{at}unmc.edu, phone 402 559-6032, FAX 402 559-4651.
Received August 5, 2008; revision received September 25, 2008; accepted September 26, 2008
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Abstract |
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The expectation from the literature is that organophosphorus agents (OP) bind to proteins that have an active site serine. However, transferrin, a protein with no active site serine, was covalently modified in vitro by 0.5 mM 10-fluoroethoxyphosphinyl-N-biotinamido pentyldecanamide (FP-biotin), chlorpyrifos oxon, diisopropylfluorophosphate, dichlorvos, sarin, and soman. The site of covalent attachment was identified by analyzing tryptic peptides in the mass spectrometer. Tyr 238 and Tyr 574 in human transferrin and Tyr 238, Tyr 319, Tyr 429, Tyr 491, and Tyr 518 in mouse transferrin were labeled by OP. Tyrosine in the small synthetic peptide ArgTyrThrArg made a covalent bond with diisopropylfluorophosphate, chlorpyrifos oxon, and dichlorvos at pH 8.3. These results, together with our previous demonstration that albumin and tubulin bind OP on tyrosine, lead to the conclusion that OP bind covalently to tyrosine, and that OP-binding to tyrosine is a new OP binding residue. The OP-reactive tyrosines are activated by interaction with Arg or Lys. It is suggested that many proteins in addition to those already identified may be modified by OP on tyrosine. The extent to which tyrosine modification by OP can occur in vivo and the toxicological implications of such modifications require further investigation.
Key Words: plasma; soman; sarin; mass spectrometry; tyrosine residue; transferrin.