Preliminary detection of FP-biotinylated tubulin
Preliminary screening for OP-targets other than AChE was conducted with the biotinylated organophosphorus agent FP-biotin, whose structure is shown in . FP-biotin was employed because the biotin moiety provided a convenient way for enriching and extracting OP-reactive proteins from crude mixtures (Kidd et al., 2001
; Nomura et al., 2005
; Peeples et al., 2005
; Schopfer et al., 2005a
; Schopfer et al., 2005b
; Ding et al., 2008
; Grigoryan et al., 2008
; Nomura et al., 2008
; Tuin et al., 2009
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 (more ...)
In vitro labeling of mouse brain supernatant (100,000 × g) with 10 µM FP-biotin, was followed by binding to avidin beads, SDS gel electrophoresis, transfer of proteins to PVDF membrane, and staining with Streptavidin Alexa-680. This sensitive, fluorescent dye revealed as many as 55 separate bands from a single SDS PAGE gel lane for FP-biotinylated mouse brain supernatant (, lane B). In the absence of FP-biotin, no more than four bands were normally detected.
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. (more ...)
Coomassie staining is less sensitive than staining with Streptavidin Alexa-680; twenty-two bands were detected in the Coomassie-stained lane shown in . Proteins identified by mass spectrometry from tryptic, in-gel digests of these bands included serine hydrolases, dehydrogenases, a phosphatase, a monooxygenase, peroxiredoxin, heat shock proteins, albumin, and tubulin. See . In addition, two endogenously biotinylated enzymes (pyruvate carboxylase and propionyl CoA-carboxylase) were identified.
Proteins in mouse brain homogenates that bind FP-biotin.
An intensely stained band at 55 kDa from the SDS PAGE gel contained tubulin as the major component. Both the alpha and beta subunits of tubulin were found through probability-based searching of the NCBI database using the Mascot algorithm (Perkins et al., 1999
). The MOWSE scores showed high confidence in the identification. The band at 55 kDa did not appear in brain preparations that had not been treated with FP-biotin ().
Of the 36 proteins in , only 11 have an active site serine as indicated by Yes in the column headed by GXSXG. Proteins that have an active site serine are expected to covalently bind organophosphorus agents on serine. However, the 25 proteins without the consensus sequence GXSXG bind organophosphorus agent at unknown sites.
Mice treated with FP-biotin
Similar analyses were conducted on brain supernatant from mice that had been injected intraperitoneally with 5.5 mg/kg FP-biotin. These animals showed no signs of cholinergic toxicity and no AChE inhibition (Peeples et al., 2005
). Proteins that bound to avidin beads were separated by SDS gel electrophoresis, digested with trypsin, and analyzed by mass spectrometry. A gel band at about 55 kDa contained tubulin as the primary component. Alpha tubulin gave a MOWSE score of 322, which was based on 9 peptides, 6 of which scored in the identity range and 3 in the homology range. Beta tubulin gave a MOWSE score of 217, which was based on 4 peptides, 3 of which scored in the identity range and 1 in the homology range.
Mascot clearly identified tubulin as the major component in the avidin-bound material extracted from the brain of a mouse that had been treated with FP-biotin. However, the FP-biotin-labeled peptides for neither alpha nor beta tubulin were found. Identification of the labeled peptide is essential to confirm that tubulin truly reacts with OP. This question is particularly important in the case of tubulin, because this protein does not carry the serine hydrolase active site, which is the traditionally accepted target for OP.
Chlorpyrifos oxon labeled tryptic peptides from bovine tubulin identified by mass spectrometry
In an effort to find an OP-labeled peptide from tubulin, the reaction of pure bovine tubulin with a different OP was studied. The OP chosen for these more focused studies was chlorpyrifos oxon. Chlorpyrifos oxon was chosen because it is the active metabolite of chlorpyrifos, a commercially used pesticide that is more relevant than FP-biotin in terms of human exposure. The chlorpyrifos oxon studies complement those from the more exotic FP-biotin and address the question of whether the FP-biotin results are relevant to real-world exposure situations. Though FP-biotin is very convenient for finding unknown proteins that react with OP, it is subject to the criticism that it is an atypical OP and that its reactions may not reflect the selectivity that can be expected for commonly used OP.
Initial analysis of the tryptic digest of chlorpyrifos oxon treated bovine tubulin was made on the MALDI-TOF/TOF 4800 mass spectrometer. The MS peaks from the labeled preparation were compared with theoretical mass lists of tryptic peptides from unlabeled tubulin (alpha1, gi 73586894 and beta2 gi 75773583 NCBI database). Alpha1 and beta2 are the most abundant tubulin isotypes in the brain. Therefore we took these isotypes for generation of the theoretical mass lists by Protein Prospector. The list of theoretical masses was generated with Protein Prospector software (UCSF, www.prospector.ucsf.edu
). Peaks from the labeled preparation whose mass was equal to that of an unlabeled peptide plus the added mass from chlorpyrifos oxon (136 amu) were considered to be candidates for chlorpyrifos oxon modified peptides.
Labeling did not reach 100 % under the experimental conditions employed. Therefore, masses for both labeled and unlabeled peptides appeared in the same mass spectrum. A MALDI-TOF MS spectrum showing some unlabeled peptides and their corresponding chlorpyrifos oxon-labeled candidates is shown in .
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 (more ...)
Not all labeled peptides could be detected in the MALDI mass spectrometer and MS/MS fragmentation in the MALDI mass spectrometer was not always sufficient to fully characterize the labeled peptides. Complementary MS/MS spectra for the candidate peptides were obtained via LC/MS/MS in the tandem, quadrupole, linear-ion trap, QTRAP 2000 mass spectrometer, using electrospray ionization to introduce the sample.
MS/MS data from the QTRAP 2000 were submitted to the Mascot search engine for identification of chlorpyrifos oxon labeled peptides. The Mascot results were checked manually to confirm the sequences of putative, chlorpyrifos oxon-labeled peptides and to identify the covalently modified amino acid residue. In total, 16 labeled peptides and 17 amino acid residues were identified (one peptide carried two labels). Each labeled peptide was covalently attached to an O-diethyl phosphate fragment. Nine tyrosines were from alpha tubulin and 8 tyrosines were from beta tubulin. Four of the tyrosines (59, 83, 159 and 281) had been identified and reported previously (Grigoryan et al., 2008
). Representative MS/MS spectra of labeled peptides are shown in .
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 (more ...)
Theoretical fragment ion masses for each labeled peptide were calculated using the MS/MS Fragment Ion Calculator from Systems Biology (http://db.systemsbiology.net
) and compared to the observed data. Extensive y-ion series were identified for each peptide. A delta mass corresponding to the labeled tyrosine fit into the sequence ions for the y-series of each peptide.
Characteristic, non-sequence fragments at 216, 244, and/or 272 m/z were found for each peptide. The mass at 216 m/z is consistent with the immonium ion of phosphotyrosine, the mass at 244 m/z is consistent with the immonium ion of O-monoethylphosphotyrosine, and the mass at 272 m/z is consistent with the immonium ion of O-diethylphosphotyrosine. These characteristicions provide additional evidence that the chlorpyrifos oxon is bound to tyrosine in these peptides. In , the characteristic ion masses are enclosed in boxes (Schopfer et al., 2009
The scheme for the reaction of chlorpyrifos oxon with tyrosine is presented in .
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.
Aging is a secondary, enzymatically catalyzed reaction that occurs with OP-labeled acetyl- and butyrylcholinesterase. It results in the loss of one alkoxy side-chain from the phosphorus atom. For example, aging of chlorpyrifos oxon modified acetylcholinesterase would result in loss of ethylene from one ethoxy group (28 amu). Chlorpyrifos oxon-labeled tyrosine did not lose an ethylene group from the phosphate. If aging had occurred the added mass from chlorpyrifos oxon modification would have been +108 rather than +136. No peptides with an added mass of +108 were found.
OP-modified and phosphate-modified tyrosines
The complete list of organophosphorylated peptides from bovine alpha and beta tubulin is presented in . Four of the peptides (TGTYR, YVPR, GSQQYR and EEYPDR) were previously reported to have been labeled with a variety of OP (Grigoryan et al., 2008
Chlorpyrifos oxon-labeled tryptic peptides from bovine alpha1 and beta2 tubulin identified by LC/MS/MS.
No serine in tubulin was found to be organophosphorylated by chlorpyrifos oxon.
Phosphoproteomic data for a variety of proteins from different sources are available from www.phosphosite.org
. In vivo and in vitro experiments showed that in cancer cells alpha and beta tubulin can be phosphorylated on tyrosine using GTP and different kinases (Rush et al., 2005
; Zheng et al., 2005
; Rikova et al., 2007
; Guo et al., 2008
). Some of the tyrosines that have been reported to be phosphorylated are organophosphorylated by chlorpyrifos oxon as well. Both phosphorylation and organophosphorylation occur at positions 103, 224, 272 and 357 on alpha and 50, 51, and 340 on beta tubulin (). Hyper phosphorylation can interfere with the polymerization of tubulin (Wandosell et al., 1987
; Ley et al., 1994
). It follows that multiple organophosphorylation of tubulin might also interfere with tubulin polymerization and consequently with its functions.
Dose dependent organophosphorylation of tyrosines in tubulin by chlorpyrifos oxon
With so many chlorpyrifos oxon binding sites on tubulin, it was of interest to determine which was more reactive and to what extent. To characterize the dynamics of chlorpyrifos oxon-binding, both the reactivity (i.e. the fraction of each tyrosine that became organophosphorylated) and the kinetics of organophosphorylation were determined.
Eight peptides were studied: TGTY83R, NLDIERPTY224TNLNR, IHFPLATY272APVISAEK, Y59VPR, GHY106TEGAELVDSVLDVVR, EEY159PDR, GSQQY281R and Y310LTVAAVFR. The reactivity of the remaining peptides could not be determined for one of the following reasons: i) the mass of the unlabeled peptide coincided with the m/z value for another peptide labeled with chlorpyrifos oxon. For example, 1023.5 amu is the mass of both unlabeled EDAANNYAR and chlorpyrifos oxon-labeled FDLMY*AK, ii) some peptides were labeled on more than one tyrosine. For example, the peptide INVYYNEATGGK was labeled on either tyrosine, iii) the isotope clusters for some labeled peptides overlapped isotope clusters of other peptides. For example, the isotope cluster for VGINY*QPPTVVPGGDLAK at 1961.0 m/z overlapped the isotope cluster of a peak at 1959.0 m/z, and the isotope cluster for FDGALNVDLTEFQTNLVPY*PR at 2545.2 m/z overlapped the isotope cluster of a peak at 2544.2 m/z and iv) labeled peptides were not detectable by MALDI -TOF/TOF though they could be identified in the QTRAP. For example, masses 911.4, 917.4, 1832.8 amu for labeled-peptides GHY*TIGK, LSVDY*GK and NSSY*FVEWIPNNVK could not be detected in the MALDI.
For those peptides that were studied, the percentage of each tyrosine that was labeled is presented in .
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 (more ...)
As shown in , Tyr 83 in alpha tubulin was the most reactive tyrosine. It was the only tyrosine labeled (12%) by an equimolar concentration of chlorpyrifos oxon. Tyr 83 was the most extensively labeled residue (61 %) in the presence of 0.5 mM chlorpyrifos oxon. For beta tubulin, tyrosine 281 exhibited the largest amount of labeling, with 34% labeling at 0.5 mM chlorpyrifos oxon. Residues 224, 272, 59, 106, 159 and 310 were substantially less reactive than 83 and 281. Reaction with 0.5 mM chlorpyrifos oxon resulted in 17%, 10%, 19%, 5%, 22% and 8.3% labeling of these tyrosines, respectively. Tyr 83 showed 3 – 12-fold higher reactivity than all other tyrosines. Tyrosines 106 and 310 from beta tubulin were the least reactive. The difference in the reactivity between tyrosines can be rationalized by their location in the crystal structure of the tubulin heterodimer and by the effect of neighboring amino acids.
Kinetics of the chlorpyrifos oxon reaction with tubulin tyrosines
Progress curves for chlorpyrifos oxon binding to tyrosine were obtained by plotting the percentage of labeled peptide against the corresponding incubation time. shows the progress curves are linear (R2 = 0.7 – 1).
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 (more ...)
The initial velocity for each tyrosine was taken from the slope of the plot. No significant labeling was observed for any peptide during the first hour of incubation. Tyr 272 is not depicted in because it showed labeling only after 24 hr of incubation. Tyr 83 yielded the fastest rate at 1.87 % labeling/min, but no labeling was detected until the 6.5 h time point. At that time it was found to be 25 % labeled. Thereafter, the rate of labeling was rapid. This observation suggests that there might have been a reorientation of tyrosine 83 within the structure of tubulin. Tyr 83 reacted with chlorpyrifos oxon 1.5 – 9 times faster than other tyrosines. Tyrosine 281 showed the second fastest rate at 1.36 % labeling/min. These findings are consistent with the reactivity measurements described above.
Bovine alpha tubulin (accession # 73586894) has 19 tyrosines, of which 9 can be labeled by chlorpyrifos oxon. Bovine beta tubulin (accession # 75773583) has 16 tyrosines of which 8 can be labeled. About half of the tyrosines are reactive and half are unreactive.
In summary, Tyr 83 from alpha and Tyr 281 from beta bovine tubulin were more reactive toward chlorpyrifos oxon than the other tyrosines that became labeled. They both showed a higher percentage of labeling: Tyr 83 and Tyr 281 were labeled 61 % and 34 %, respectively, after 24 h of incubation at 37°C (). And, they reacted with chlorpyrifos oxon more rapidly than the other tyrosines, being labeled at a rate of 1.87 and 1.36 % labeled peptide/min, respectively ().
Effect of GTP on the reaction of chlorpyrifos oxon with tubulin
Tubulin dimers are incorporated into microtubules only after they have bound GTP to the exchangeable GTP binding site on beta tubulin. The binding of GTP alters the structure of the tubulin dimer. It was of interest to determine whether the structural change induced by GTP binding was reflected in the amount of chlorpyrifos oxon covalently bound to tubulin. The effect of GTP on chlorpyrifos oxon binding to tubulin was determined by incubation of 0.012 mM bovine tubulin heterodimer with 0.5 mM chlorpyrifos oxon at 37 °C for 24 hr in the presence and absence of 1 mM GTP. Tryptic peptides were analyzed in the MALDI-TOF/TOF 4800 mass spectrometer. presents the MS spectra of pairs of unlabeled and chlorpyrifos oxon-labeled tryptic peptides without (panel A) and with GTP (panel B). The presence of 1 mM GTP increased the extent of tubulin organophosphorylation. The peak intensities of chlorpyrifos oxon-labeled peptides were increased about 2 fold in the presence of GTP relative to the intensities of the unlabeled peaks.
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 (more ...)
Location of chlorpyrifos oxon labeled tyrosines in the crystal structure of the tubulin heterodimer
Tyrosines of bovine tubulin differed in their sensitivity toward chlorpyrifos oxon. To explain these differences, the location of modified tyrosines was of interest. The crystal structure of bovine brain tubulin heterodimer, reported by Nogales et al (Protein Data Bank number, 1jff) is shown in (Nogales et al., 1999
). The 8 tyrosines for which reactivity data were obtained are indicated. Nogales et al introduced gaps into the numbers of the beta tubulin amino acid sequence, which explains why the numbers in the crystal structure are higher than the numbers in the protein database.
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 (more ...)
The phenolic oxygen of Tyr 272 in alpha tubulin and Tyr 108 (Y106) and Tyr 312 (Y310) in beta tubulin face the inside of the protein. As a consequence, it can be assumed that access of chlorpyrifos oxon to these tyrosines was limited, which is consistent with the relatively low percentage of chlorpyrifos oxon-labeling observed for these tyrosines (see ). Tyrosines 61 (59), 83, 161 (159), 224 and 283 (281) are exposed on the surface of tubulin. Tyr 224, in addition to being exposed to solvent, makes a π-πinteraction with the guanine moiety of the GTP molecule. Tyrosine 83 and Tyr 283 (281) are exposed on the surface of the tubulin dimer making them readily available for reaction with chlorpyrifos oxon, which would contribute to their high reactivity.