title: V-type nerve agents phosphonylate ubiquitin at biologically relevant lysine residues and induce intramolecular cyclization by an isopeptide bond.
abstract: Toxic organophosphorus compounds (e.g., pesticides and nerve agents) are known to react with nucleophilic side chains of different amino acids (phosphylation), thus forming adducts with endogenous proteins. Most often binding to serine, tyrosine, or threonine residues is described as being of relevance for toxicological effects (e.g., acetylcholinesterase and neuropathy target esterase) or as biomarkers for post-exposure analysis (verification, e.g., albumin and butyrylcholinesterase). Accordingly, identification of novel protein targets might be beneficial for a better understanding of the toxicology of these compounds, revealing new bioanalytical verification tools, and improving knowledge on chemical reactivity. In the present study, we investigated the reaction of ubiquitin (Ub) with the V-type nerve agents Chinese VX, Russian VX, and VX in vitro. Ub is a ubiquitous protein with a mass of 8564.8 Da present in the extra- and intracellular space that plays an important physiological role in several essential processes (e.g., proteasomal degradation, DNA repair, protein turnover, and endocytosis). Reaction products were analyzed by matrix-assisted laser desorption/ionization-time-of-flight- mass spectrometry (MALDI-TOF MS) and μ-high-performance liquid chromatography online coupled to UV-detection and electrospray ionization MS (μHPLC-UV/ESI MS). Our results originally document that a complex mixture of at least mono-, di, and triphosphonylated Ub adducts was produced. Surprisingly, peptide mass fingerprint analysis in combination with MALDI and ESI MS/MS revealed that phosphonylation occurred with high selectivity in at least 6 of 7 surface-exposed lysine residues that are essential for the biological function of Ub. These reaction products were found not to age. In addition, we herein report for the first time that phosphonylation induced intramolecular cyclization by formation of an isopeptide bond between the ε-amino group of a formerly phosphonylated lysine and the side chain of an adjacent acidic glutamic acid residue.
authors.affiliation: Bundeswehr Institute of Pharmacology and Toxicology, Neuherbergstrasse 11, 80937, Munich, Germany.
authors.forename: Christian
authors.initials: C
authors.lastname: Schmidt
authors.forename: Felicitas
authors.initials: F
authors.lastname: Breyer
authors.forename: Marc-Michael
authors.initials: MM
authors.lastname: Blum
authors.forename: Horst
authors.initials: H
authors.lastname: Thiermann
authors.forename: Franz
authors.initials: F
authors.lastname: Worek
authors.forename: Harald
authors.initials: H
authors.lastname: John
mesh_terms: D000595:Amino Acid Sequence; D002619:Chemical Warfare Agents / Q000737:chemistry*; D002851:Chromatography, High Pressure Liquid; D003500:Cyclization; D018698:Glutamic Acid / Q000737:chemistry*; D006801:Humans; D008239:Lysine / Q000737:chemistry*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009946:Organothiophosphorus Compounds / Q000737:chemistry*; D010757:Phosphorous Acids / Q000737:chemistry*; D012996:Solutions; D021241:Spectrometry, Mass, Electrospray Ionization; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D025801:Ubiquitin / Q000737:chemistry*
chemical_list: D002619:Chemical Warfare Agents; D018698:Glutamic Acid; D008239:Lysine; D009946:Organothiophosphorus Compounds; D010757:Phosphorous Acids; D012996:Solutions; D025801:Ubiquitin; C009680:VX-agent