Mass spectrometry identifies multiple organophosphorylated sites on tubulin

Abstract

Acute toxicity of organophosphorus poisons (OP) is explained by inhibition of acetylcholinesterase in nerve synapses. Low-dose effects are hypothesized to result from modification of other proteins, whose identity is not yet established. The goal of the present work was to obtain information that would make it possible to identify tubulin as a target of OP exposure. Tubulin was selected for study because live mice injected with a nontoxic dose of a biotinylated organophosphorus agent appeared to have OP-labeled tubulin in brain as determined by binding to avidin beads and mass spectrometry. The experiments with live mice were not conclusive because binding to avidin beads could be nonspecific. To be convincing, it is necessary to find and characterize the OP-labeled tubulin peptide. The search for OP-labeled tubulin peptides was begun by identifying residues capable of making a covalent bond with OP. Pure bovine tubulin (0.012 mM) was treated with 0.01-0.5 mM chlorpyrifos oxon for 24 h at 37 degrees C in pH 8.3 buffer. The identity of labeled amino acids and percent labeling was determined by mass spectrometry. Chlorpyrifos oxon bound covalently to tyrosines 83, 103, 108, 161, 224, 262, 272, 357, and 399 in bovine alpha tubulin, and to tyrosines 50, 51, 59, 106, 159, 281, 310, and 340 in bovine beta tubulin. The most reactive were tyrosine 83 in alpha and tyrosine 281 in beta tubulin. In the presence of 1 mM GTP, percent labeling increased 2-fold. Based on the crystal structure of the tubulin heterodimer (PDB 1jff) tyrosines 83 and 281 are well exposed to solvent. In conclusion seventeen tyrosines in tubulin have the potential to covalently bind chlorpyrifos oxon. These results will be useful when searching for OP-labeled tubulin in live animals.

Fig. 1

Structure of FP-biotin. The second order rate constant for the reaction of FP-biotin with human butyrylcholinesterase is 1.6 × 10⁸ M⁻¹ min⁻¹ and for the reaction with human acetylcholinesterase is 1.8 × 10⁷ M⁻¹ min⁻¹. These rates are comparable to the rates of reaction with chlorpyrifos oxon which are 1.7×10⁹ M⁻¹ min⁻¹ for human butyrylcholinesterase and 1.0 × 10⁷ M⁻¹ min⁻¹ for human acetylcholinesterase (Schopfer et al., 2005b).

Fig. 2

Preliminary detection of FP-biotinylated tubulin in mouse brain. Mouse brain supernatant was treated with 10 µM FP-biotin. Excess FP-biotin was separated by gel filtration. FP-biotinylated proteins were extracted by binding to avidin-agarose beads. The avidin-agarose beads were washed with 0.2% SDS, boiled in SDS gel loading buffer, and the extract and beads loaded on an SDS PAGE gradient gel (10–20%). Protein in Lanes A and B were transferred to PVDF membrane and stained with streptavidin Alexa-680. Lane A shows endogenously biotinylated proteins that appear in the absence of FP-biotinylation. Lane B shows 55 FP-biotinylated bands including an intense band for FP-biotinylated tubulin. Lanes C and D were stained with Coomassie blue. Lane C is the same sample as in lane B at a 20-fold higher concentration. Lane D contains molecular weight marker proteins. The molecular weight values for the marker proteins are shown to the right of the panel (MWM/kDa). The oval in lane C is an air bubble.

Fig. 3

MALDI-TOF MS spectrum of tryptic peptides from bovine tubulin treated with a 40 fold molar excess of chlorpyrifos oxon (0.5 mM). Singly-charged, unlabeled peptides with masses of 887.5, 1023.5, 1039.5, 1718.9, 1757.0, and 1959.0 amu are shown in regular font. Corresponding candidates for chlorpyrifos oxon labeled peptides (plus 136 amu) with masses of 1023.5, 1159.5, 1175.7, 1854.9, 1893.0, and 2095.1 amu are shown in bold and marked with an asterisk. The mass at 1023.5 m/z coincides with both the unlabeled peptide EDAANNYAR and the chlorpyrifos oxon labeled peptide FDLMYAK.

Figure 4

MS/MS spectra of chlorpyrifos oxon-labeled peptides from bovine tubulin obtained by LC/MS/MS on the QTRAP 2000 mass spectrometer. Panel (A) peptide Y₃₁₀LTVAAVFR from beta tubulin; (B) peptide EDAANNY₁₀₃AR from alpha tubulin; (C) peptide, FDLMY₃₉₉AK from alpha tubulin; (D) peptide IHFPLATY₂₇₂APVISAEK from alpha tubulin. In panels A-C, singlycharged y ion series derived from doubly-charged parent ions at 588.43 (A), 580.23 (B) and 512.32 (C) m/z are indicated. For IHFPLATY₂₇₂APVISAEK, in panel D, singly-charged y and b series ions derived from a triply-charged parent ion at 632.01 m/z are marked. In all cases the masses of the y and b ions are consistent with O-diethylphosphate attached to tyrosine (marked by an asterisk in the peptide sequence). Masses enclosed in a box are the immonium ion of phosphotyrosine (216), the immonium ion of O-monoethylphosphotyrosine (244), and the immonium ion of O-diethylphosphotyrosine (272). Peaks that are not labeled include internal fragments and masses that have lost a molecule of water or ammonia.

Figure 5

Reaction of chlorpyrifos oxon with tyrosine. Covalent binding of chlorpyrifos oxon to tyrosine increases the mass of tyrosine by 136 amu due to the addition of diethoxyphosphate.

Figure 6

Reactivity of chlorpyrifos oxon with various tyrosines from tubulin. Bars represent the percentage of chlorpyrifos oxon-modified tyrosine in 3 peptides from alpha tubulin and 5 peptides from beta tubulin. Twelve μM tubulin was incubated with various concentrations of chlorpyrifos oxon at 37 °C for 24 h (pH 8.3).

Figure 7

Progress curves for chlorpyrifos oxon binding to tyrosines from bovine tubulin. The percentage of labeled peptides was calculated after 3, 6.5, 10 and 24 hr of incubation at 37 °C. Experimental data for alpha tubulin (tyrosines 83 and 224) are presented with empty symbols for TGTY₈₃R and NLDIERPTY₂₂₄TNLNR. Data for beta tubulin (tyrosines 281, 59, 159, 310 and 106) are shown with filled symbols for GSQQY₂₈₁R, Y₅₉VPR, EY₁₅₉PDR, Y₃₁₀LTVAAVFR and GHY₁₀₆TEGAELVDSVLDVVR. The values of the initial velocities are presented next to each curve. Each point is the average of 3 trials, with an error of approximately 10%.

Figure 8

Effect of GTP on extent of chlorpyrifos oxon labeling. MS spectra were acquired on the MALDI TOF mass spectrometer. (A) labeled in the absence of GTP; (B) labeled in the presence of 1 mM GTP. Unlabeled peptides are indicated by regular font and labeled peptides (+136 amu added mass from chlorpyrifos oxon) are indicated in bold and marked by asterisks. Seven pairs of peptides are represented. Their masses and amino acid sequences are listed in Table 2. The peak at 1023.5 m/z corresponds to the unlabeled EDAANNYAR and the chlorpyrifos oxon labeled FDLMY*AK peptides. The peak height of each labeled peptide compared to the unlabeled peptide is higher in panel B (with GTP) than in panel A (without GTP).

Fig. 9

Crystal structure of the bovine brain tubulin heterodimer with subunit alpha in red and beta in blue (Protein Data Bank number, 1jff). Eight chlorpyrifos oxon labeled tyrosines (3 in alpha and 5 in beta tubulin) are shown as solid green sticks. GTP in the GTP non-exchangeable binding site in alpha tubulin and GDP in the exchangeable binding site in beta tubulin are shown as balls surrounded by amino acid residues that directly interact with these nucleotides. Amino acid numbers correspond to the numbers in pdb 1jff. These numbers are high compared to the numbers in the protein sequence because a gap of two residues was introduced in the numbers of the beta tubulin crystal structure (Nogales et al., 1999). The structure was drawn with PyMol software www.pymol.sourceforge.net