Chem. Res. Toxicol. 1995, 8, 934-941
934
Alkylation of Escherichia coli Thioredoxin by S-(2-Chloroethy1)glutathioneand Identification of the Adduct on the Active Site Cysteine-32 by Mass Spectrometry John C. L. Erve,:s$ Elisabeth Barofsky,$ Douglas F. Barofsky,$ Max L. Deinzer,f and Donald J. Reed*>' Department of Biochemistry and Biophysics and Department of Agricultural Chemistry, Oregon State University, Corvallis, Oregon 97331-6502 Received February 16, 1995@ Alkylation of reduced Escherichia coli thioredoxin by the episulfonium ion derived from $42chloroethy1)glutathione (CEG) a t physiologic pH resulted in at least three different alkylation products. These adducts were separated by reverse phase chromatography, digested with trypsin, and peptide-mapped. The peptide containing the active site cysteines was collected and sequenced by tandem mass spectrometry. Results indicate that the site of alkylation was at Cys-32 exclusively with no alkylation at Cys-35. Raising the pH above the pKa of Cys-35 t o ionize the thiol before reacting with the episulfonium ion of CEG did not lead to alkylation at Cys-35, suggesting that a steric factor prevents the alkylating moiety of CEG from accessing this cysteine. A tryptic digest of a minor bis-adduct yielded an alkylated peptide which contained tyrosine, an amino acid known to be alkylated a t its hydroxyl group by CEG. Sequencing by tandem mass spectrometry, however, was unsuccessful due to fragmentation of the alkylating moiety from the peptide. Results of this study confirm that the episulfonium ion of CEG can adduct thioredoxin at the active site and may have important toxicologic significance regarding the mechanism of 172-dichloroethanetoxicity.
Introduction The cellular tripeptide glutathione (GSHY can protect cells from both endogenous and exogenous electrophiles that react with cellular constituents and cause cell damage (1). It is now clear that GSH conjugation can cause cytotoxicity by enhancing the reactivity of a number of xenobiotic chemicals, for example, some aliphatic halogenated alkenes (21,quinones (31, and isothiocyanates (4). GSH conjugation to form S42-chloroethyl)glutathione (CEG), as a consequence of 1,2-dichloroethane (DCE) exposure, is another example of a toxification reaction. Although DCE can be metabolized by an oxidative pathway via P4502E1 leading to the reactive metabolite 2-~hloroacetaldehyde,it is believed that mutagenicity is due to the GSH conjugate pathway (5). CEG can form an electrophilic episulfonium ion that can react with specific nucleophilic sites in DNA (6, 7). DCE metabolites also react with protein, and DCE-treated rats show signs of oxidative stress in the form of lipid peroxidation (8). Evidence for the cytotoxicity of CEG was demonstrated by the production of renal lesions (proximal tubule damage) in the Fisher rat treated with CEG (9). In the rat, GSH depletion as a consequence of DBE treatment may lead to oxidative stress conditions
* To whom correspondence should be addressed. Department of Biochemistry and Biophysics.
* Department of Agricultural Chemistry. +
Abstract published in Advance ACS Abstracts, July 15, 1995. Abbreviations: GSH, glutathione; CEG, S-(2-chloroethyl)glutathione; TCEP, tris(2-carboxyethy1)phosphine; CID, collisionally induced dissociation; FAB-MS, fast atom bombardment mass spectrometry; MALDI-MS, matrix-assisted laser desorption-ionization mass spectrometry; DCE, 1,2-dichloroethane;BCE, 1-bromo-2-chloroethane; PDS, 4,4'-dithiopyridine; TFA, trifluoroacetic acid; MA, iodoacetamide; HCCA, 4-hydroxy-a-cyanocinnamic acid; MSMS, tandem mass spectrometry.
0893-228x/95/2708-0934$09.~0l0
that might play a role in the toxicity of the dihaloethanes such as DCE (10). Covalent binding of reactive intermediates to proteins is accepted to be important in the development of many chemical-induced toxicities, although the detailed mechanisms by which these covalent interactions produce cellular toxicity are not completely understood (11). Protein alkylation may cause toxicity by (a) altering the protein such that loss of function occurs or by (b) creating an immunogen that leads to a deleterious immune response (12). It is becoming clear that the amino acids alkylated in a protein are not randomly targeted, but are determined by the nature of the electrophile (hard, soft), available nucleophiles, and steric constraints imposed by the tertiary structure of the protein. Protein adducts are often the focus of m,olecular dosimetry studies aimed at quantifying exposure to carcinogens in the work place, such as measuring hemoglobin adducts of ethylene oxide (13, 14). Escherichia coli thioredoxin is a small, ubiquitous, redox-active protein that has been studied extensively (15-18). One of its many roles is as a hydrogen donor for ribonucleotide reductase in the enzymatic synthesis of deoxyribonucleotides. Due to its protein-disulfide reductase activity, the protective role of thioredoxin against the effects of oxidative stress has been investigated. For example, thioredoxin has been shown to enable lens epithelial cells exposed to hydrogen peroxide to recover from the resulting oxidative damage, based on regeneration of glyceraldehyde-3-phosphate dehydrogenase and leucine uptake (29). Recently, evidence has been obtained that the thioredoxidthioredoxin reductase system may regenerate enzymes inactivated by oxidative stress in endothelial cells (20). Previous work in this
0 1995 American Chemical Society
Alkylation of Thioredoxin by CEG laboratory has shown that, based on the insulin reduction assay (21), thioredoxin alkylated by CEG is inactivated. The results of this assay strongly implicate the active site as being targeted by CEG. Both eukaryote and prokaryote thioredoxin contains a conserved active site having the sequence Trp-Cys-Gly-Pro-Cys, which is present on a loop that protrudes from the threedimensional structure of the protein (22). The purpose of this investigation was to study the alkylation chemistry of CEG toward thioredoxin and, in particular, to identify which amino acids become alkylated following reaction with the episulfonium ion of CEG in vitro. To this end, we used a combination of peptide mapping and mass spectrometry to determine CEG alkylation sites in thioredoxin.
Materials and Methods Chemicals. Recombinant E. coli thioredoxin (MW = 11 669 Da) was purchased from Calbiochem-NovaBiochem (La Jolla, CA). Reduced GSH, 4,4'-dithiopyridine (PDS), and trifluoroacetic acid (TFA) were purchased from Sigma (St. Louis, MO). 1-Bromo-2-chloroethane (BCE) was purchased from Aldrich (Milwaukee, WI). Tris(2-carboxyethy1)phosphine (TCEP) was obtained from Pierce (Rockford, IL). Sequencing grade trypsin and Glu-C was from Boehringer-Mannheim (Indianapolis, IN). Labeled GSH was synthesized from glutamic acid a-tert-butyl ester, tritylcysteine and [15N]-glycine using a Wang resin (4alkoxybenzyl alcohol polystyrene), all purchased from Bachem (Torrance, CA). Bio-Gel P-2 gel (extra fine,
...., b,
244
Figure 6. (A) Ionspray mass spectrum of alkylated T3 showing doubly and triply charged molecular ions. This peptide was collected from a tryptic digest of the mono-adduct. (B)Mass spectrum of daughter ions formed by CID of the [M 2HI2+ parent ( m i z 1186) of the alkylated peptide T3. The ion denoted by an asterisk ( m / z 897) corresponds to Cys32-Gly-Pro-Cys35-L~~ alkylated by CEG at Cys~pand by IAA at Cys~s.This ion loses a glutamic acid residue from the adduct derived from CEG t o produce the ion signal at m / z 768. The insert indicates the nomenclature used to label the fragment ions in the mass spectrum using a tripeptide as a n example.
+
Table 1. Predicted and Experimental Masses of Tryptic Peptides of Thioredoxin Treated with CEG peptide
sequence
theoretical mlz
observed mlz
T1 T11 T7 T5 T10 T9 T12 T8 T6 T11,12 T2 T8,9 T4 T4e
1-3 97-100 70-73 53-57 91-96 83-90 101-018 74-82 58-69 97-108 4-18 74-90 37-52 37-52
349.2 445.3 508.3 531.4 574.4 789.4 892.4 1001.6 1267.7 1318.7 1731.9 1772.0 1805.9 2140.0
T1,2 T3 f T3'
1-18 19-36 19-36
2062.0 2095.9 2370.9
nda nd 508.0b 531.4c 574.4c 790 .4c 892.4' 1001.6c 1267.6c 1318.5' 1731& 1772.ad 18O6.Oc 2139.9; 2139.2,d 2139.4b 2O62.Oc 2095.0d 2371.6,' 2371.0; 2371.2b
nd, not detected. Ionspray MS. FAB-MS. MALDI-MS. Peptide with adduct derived from CEG.f Cysteines are carboxamidomethylated.
e
bz through bg fragment ions (Figure 6B). The y5-y14 ions were shifted 334 Da above their expected mass values in the unalkylated peptide, whereas y3 and y4 were only shifted by the 58 Da mass increment associated with alkylation by IAA. In addition, y5 lost a glutamic acid residue from the adduct derived from CEG to produce the observed ion a t m l z 768. These observations are consistent with an adduct derived from CEG on Cys-32
and a carboxamidomethyl moiety on Cys-35. No fragment ions could be identified that would indicate alkylation by CEGs episulfonium ion at Cys-35. The peptide, which produced a MALDI signal a t m l z 2139, displayed a doubly charged ion at mlz 1070 and a triply charged ion at m l z 714 in the ionspray mass spectrum. The triply charged ion was subject to CID because the doubly charged ion was too weak for this type of experiment. The product ion spectrum for alkylated T4 was not as informative as that obtained for alkylated T3. Fragment ions were formed, but no peaks shifted by the mass of the alkylating species could be observed. An ion signal observed at m l z 334 is consistent with extensive loss of the alkylating species from the peptide during CID; this probably accounts for our inability to ascertain the site of alkylation on T4. Alkylation with 15N-LabeledCEG. This study as well as a previous one (21)produced evidence that trisand tetra- adducts of thioredoxin can be formed, albeit, in low amounts. The tetra-alkylated thioredoxin was formed with a much larger excess of CEG than used to produce mono- and bis-adducts. In an attempt to identify sites of alkylation other than at Cys-32, oxidized thioredoxin was alkylated with a mixture of CEG that contained 50% I5N-labeledglycine. It was expected that the isotopic pattern of the alkylated peptides would aid in identifying them. Oxidized thioredoxin, which has no free thiols, was used to assure that alkylation could not occur at the cysteines. The reaction products of oxidized thioredoxin alkylated with 35-fold excess CEG were chromatographed by HPLC; the resulting chromatogram
Alkylation of Thioredoxin by CEG
showed an adduct peak representing 11%of oxidized thioredoxin and two smaller adduct peaks that account for 2.2% and 1.4% of the area of oxidized thioredoxin, respectively. The largest adduct peak in the chromatogram was produced primarily of bis-adduct, but ionspray mass analysis (data not shown) indicated a small contribution from tris-adducted thioredoxin. Mass analysis of the minor chromatographic peaks also showed evidence for coelution of mono- and bis-adducts. Another batch of alkylated oxidized thioredoxin was prepared in order to carry out peptide mapping on a tryptic digest. Reaction of 300-fold excess CEG over oxidized thioredoxin increased the amount of bis-adduct (HPLC chromatographic peak area) to approximately 45% of that of the unreacted oxidized thioredoxin. Mass analysis of the tryptic digest of the bis-adduct by MALDI showed the presence of the alkylated form of a tryptic peptide designated as T4. This peptide was examined a t medium resolution by FAB-MS. The isotopic pattern observed for this peptide matches the theoretically predicted pattern well (data not shown) and, thus, gives additional support for alkylation of this peptide by the episulfonium ion of CEG. No other alkylated peptides were observed, possibly because sensitivity for the CEGalkylated peptides was not very high in FAB-MS. It is also possible, however, that CEG alkylation at amino acids other than cysteine is not stable, and consequently, the alkylating moiety is lost during digestion andlor peptide mapping. Reaction of Reduced Thioredoxin at High pH. In order to investigate the reactivity of Cys-35 toward the episulfonium ion of CEG, reduced thioredoxin was first treated with IAA at pH 7.0. Only Cys-32 is present as the thiolate anion and will react with IAA (33). Mass analysis of the resulting product by MALDI revealed a peak at m l z 11 728 that corresponds to the expected product, namely, thioredoxin with carboxamidomethylation at Cys-32 but no alkylation at Cys-35. After removal of IAA by centrifugal size-exclusion chromatography and readjustment of pH to 8.5, CEG was added in excess. The higher pH would result in at least 65% of the Cys-35 thiol ionized, assuming a pKa of 7.9 (34). Following this reaction and addition of more IAA, HPLC showed the formation of one major product. This thioredoxin product gave a MALDI peak at m l z of 11786, which corresponds to thioredoxin with two molecules of IAA but no adduct derived from CEG.
Discussion The results of this study confirm prior findings that the episulfonium ion of CEG is a chemically reactive species capable of alkylating nucleophilic sites in the protein thioredoxin at physiologic pH. The focus of the present investigation was to identify the specific amino acids that undergo alkylation when adducts are formed between the episulfonium ion of CEG and thioredoxin. Reaction of reduced thioredoxin resulted in the formation of a t least three adducts, the major of which is a monoadduct that readily forms with only a 10-fold molar excess of CEG over thioredoxin. The mono-adduct was digested with trypsin, and the peptide containing the alkylating moiety was sequenced by MSMS. The sequencing analysis showed the site of alkylation to be exclusively at Cys-32, which is one of the two active site cysteines. The preference for this alkylation site was anticipated because Cys-32 has a lower pKa than Cys-35. The pKa
Chem. Res. Toxicol., Vol. 8, No. 7, 1995 939
values of Cys-32 and Cys-35 have been measured by Raman spectroscopy t o be 7.1 f 0.2 and 7.9 f 0.2 (34). The y5 peak present in the CID spectrum (Figure 6B) contains the alkylating moiety and also fragments by loss of a glutamic acid residue from the adduct derived from CEG. A similar observation was observed for the adducts of oxytocin alkylated by CEG at its two cysteines (24). Cys-35's lack of reactivity a t pH 8.5 was unexpected because at this pH it is presumed to be at least 65% ionized. It was found in this study, as in a previous one (331, that the alkylating agent IAA,which is smaller relative to CEG, is able to alkylate Cys-35 a t this pH. The active site of reduced thioredoxin has been determined by NMR (35) and is known to have the cysteines on a protruding loop with Cys-32 more exposed than Cys35 (36). Given this fact and the results of this study, it seems reasonable to conclude that the relatively large episulfonium ion of CEG has limited access to Cys-35. This conclusion is in accord with the idea that tertiary structure is important in directing alkylation toward specific nucleophiles and away from others. A bis-adduct of reduced thioredoxin produced a peptide digest that contained alkylated T3 and T4 tryptic peptides. Like the mono-adduct, the bis-adduct was also alkylated at Cys-32. Attempts to determine the site of alkylation in the peptide T4 by MSMS failed, ostensibly because the adduct derived from CEG fragmented during CID. The most likely amino acid in this peptide to be alkylated by the episulfonium ion of CEG is Tyr-49, because after thiol groups the hydroxyl functional group on tyrosine is the next most reactive nucleophile toward the alkylating species derived from CEG (37). Tyrosine in the peptide angiotensin was shown to be alkylated following treatment with styrene oxide (38). A molecular model (Brookhaven Protein Data Base) of thioredoxin shows that Tyr-49 is on the surface of the protein, further supporting the plausibility of this target. Tyrosine in the peptide oxytocin was also suspected of being alkylated by the episulfonium ion of CEG; however, this could not be confirmed by MSMS because the complete adduct fragmented off the peptide (24). Regardless of where the bond is formed in this second alkylation of thioredoxin, it is definitely more labile under CID conditions (with the collision parameters used) than is the sulfur-adduct bond. The significance of this putative alkylated Tyr-49 on thioredoxin is not known. The tris-adduct, being formed in much smaller amounts, could be neither purified nor digested so that the third site of alkylation could not be identified. When oxidized thioredoxin was alkylated by the episulfonium ion of CEG and then chromatographed, several smaller adduct peaks eluted before oxidized thioredoxin. These adducts were formed in much smaller amounts than the major mono-adduct formed with reduced thioredoxin, indicating that nucleophilic sites other than the cysteine thiol are much less reactive toward CEGs episulfonium ion. MALDI mass analysis shows that the largest chromatographic peak is actually the result of two coeluting peaks. The tryptic digest was mass analyzed by MALDI, and an alkylated T4 was identified. There is not much difference between the tertiary structure of reduced and oxidized thioredoxin (35),and it is probable, therefore, that this T4 peptide adduct of oxidized thioredoxin is alkylated at the same site (putatively at Tyr49) as that on reduced thioredoxin. We were not able to identify other alkylated peptides in the tryptic digests of these adducts by either FAB- or MALDI-MS. It is
940 Chem. Res. Toxicol., Vol. 8, No. 7, 1995
possible that the adduct formed is unstable and is cleaved off the amino acid during proteolytic digestion. This possibility is supported by our attempt to further digest the T4 peptide adduct with endoproteinase Glu-C. A 12 h incubation of the peptide with enzyme at 50 "C produced no observable digestion of the peptide, but it did result in loss of the adduct derived from CEG. That is, we no longer observed alkylated T4 but instead only unalkylated T4, suggesting that the adduct was unstable at this incubation temperature. Protein alkylation is known to play a role in the mechanism of toxicity of a number of drugs, such as acetaminophen (391,and xenobiotics, such as bromobenzene (40). It is possible, however, that a given protein adduct has no toxicologic consequence, and therefore, it is not sufficient to simply show that a protein has been adducted to infer a toxicologic mechanism. For example, alkylation of alcohol dehydrogenase by the active metabolite of NJV-dimethyl-4-aminoazobenzene causes only minor alterations in enzyme activity and has been regarded as a detoxification pathway for this compound (411. Therefore, the capability to identify the particular amino acid that has undergone alkylation by CEGs episulfonium ion in a protein might point to a toxicological role for protein alkylation as a mechanism of toxicity, especially if the amino acid is vital to the functioning of the protein. In this work, we have used a combination of MALDI and ionspray MS/MS to show that one of the active site cysteines of the protein thioredoxin, Cys-32, is alkylated by CEGs episulfonium ion at physiologic pH. This finding supports previous work in this laboratory that showed the partial loss of thioredoxin insulin reduction activity following in vitro treatment with CEG (21). Such loss of activity suggests that, if levels of CEG in target cells were high enough, an important enzyme required for a number of functions in vivo might be inactivated. To ascertain the consequences of our findings, an antibody against CEG-alkylated thioredoxin would aid in determining if thioredoxin is alkylated in isolated hepatocytes or in vivo following the administration of a toxic dose of DCE. Demonstration of alkylated thioredoxin as a critical target in vivo would strengthen the implication that CEG plays a role in the toxicity of DCE. Microsomal metabolites of DCE are known to be reactive toward protein, e.g., lung microsomal proteins (42). Hence, it would be useful to compare the reactivity of 2-~hloroacetaldehyde,a P450 metabolite of DCE, toward thioredoxin in order to better understand the relative importance of the GSH conjugation pathway and the microsomal oxidation pathway in the toxicity of DCE. It would also be of interest to investigate the fate of thioredoxin alkylated by CEG in cells. Is it possible, for instance, that alkylated thioredoxin will be degraded more quickly than unalkylated thioredoxin? Eventually, it will be important to know whether human thioredoxin is alkylated by the episulfonium ion of CEG, because certain workers are exposed to DCE (43).
Acknowledgment. This work was supported in part by grants from the NIH (ES-00040 and ES-00210). J.C.L.E. was supported by a NIEHS predoctoral training grant (ES-07060). The Sciex I11 plus ionspray mass spectrometer was funded in part through a grant from the NSF (BIR-9214371) and from the Anheuser-Busch Companies. We thank Jane Aldrich for designing the synthesis of labeled GSH and her students for help in
Erve et al. carrying out the reactions. We also thank Brian Prbogast for performing the FAB mass analyses and Marian Meyer for helpful discussions and her critical review of manuscript.
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Chem. Res. Toxicol., Vol. 8, No. 7, 1995 941 (34) Huimin, L., Fuchs, J. A., Woodward, C., and Thomas, G. J. (1993) Determination of the pK, values of active-center cysteines, cysteine-32 and cysteine-35, in Escherichia coli thioredoxin by raman spectroscopy. Biochemistry 32, 5800-5808. (35) Dyson, H. J., Gippert, G. P., Case, D. A., Holmgren, A., and Wright, P. E. (1990) Three-dimensional solution structure of the reduced form of Escherichia coli thioredoxin determined by nuclear magnetic resonance spectroscopy. Biochemistry 29,41294136. (36) Eklund, H., Gleason, F. K., and Holmgren, A. (1991) Structural and functional relations among thioredoxins of different species. Proteins: Struct., Funct., Genet. 11, 13-28. (37) Jean, P. A,, and Reed, D. J. (1989) In vitro dipeptide, nucleoside, and glutathione alkylation by S-(2-~hloroethyl)-~-cysteine. Chem. Res. Toxicol. 2, 455-460. (38) Ferranti, P., Carbone, V., Sannolo, N., Fiume, I., Milone, A., Ruoppolo, M., Gallo, M., and Malorini, A. (1992) Study of interaction of styrene oxide with angiotensin by mass spectrometry. Carcinogenesis 13, 1397-1401. (39) Mitchell, J. R., Jollow, D. J., Potter, W. Z., Davis, D. C., Gillette, J. R., and Brodie, B. B. (1973) Acetaminophen induced hepatic necrosis. I. Role of drug metabolism. J . Pharmacol. Exp. Ther. 187, 185-194. (40) Jollow, D. J., Mitchel, J . R., Zampaglione, N., and Gilette, J. R. (1974) Bromobenzene-induced liver necrosis. Protective role of glutathione and evidence for 3,4-bromobenzene oxide as the hepatotoxic metabolite. Pharmacology 11, 151-169. (41) Coles, B., Beale, D., Miller, D., Lay, J., and Kadlubar, F. (1987) The binding of an aminoazo dye carcinogen to a specific methionine residue in rat liver alcohol dehydrogenase in vivo. Chem. Biol. Interact. 64, 181-192. (42) Banerjee, S., Van Duuren, B. L., and Oruambo, F. I. (1980) Microsome-mediated covalent binding of 1,2-dichloroethane to lung microsomal protein and salmon sperm DNA. Cancer Res. 40, 2170-2173. (43) Williams, M., and Diwan, S. (1994) Toxicological Profile for 1,2Dichloroethane (Update), pp 3-4, US Department of Health and Human Services, Agency for toxic substances and disease registry, Atlanta.
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