Cross-linking of the human DNA repair protein O6-alkylguanine DNA alkyltransferase to DNA in the presence of 1,2,3,4-diepoxybutane

Abstract

1,2,3,4-Diepoxybutane (DEB) is a key carcinogenic metabolite of the important industrial chemical 1,3-butadiene. DEB is a bifunctional alkylating agent capable of reacting with DNA and proteins. Initial DNA alkylation by DEB produces N7-(2'-hydroxy-3',4'-epoxybut-1'-yl)-guanine monoadducts, which can react with another nucleophilic site to form cross-linked adducts. A recent report revealed a strong correlation between cellular expression of the DNA repair protein O6-alkylguanine DNA alkyltransferase (AGT) and the cytotoxic and mutagenic activity of DEB, suggesting that DEB induces AGT-DNA cross-links (Valadez, J. G., et al. (2004) Activation of bis-electrophiles to mutagenic conjugates by human O6-alkylguanine-DNA alkyltransferase. Chem. Res. Toxicol. 17, 972-982). The purpose of our study was to analyze the formation and structures of DEB-induced AGT-DNA conjugates and to identify specific amino acid residues within the protein involved in cross-linking. DNA-protein cross-link formation was detected by SDS-PAGE when 32P-labeled double-stranded oligodeoxynucleotides were exposed to DEB in the presence of either wild-type hAGT or a C145A hAGT mutant. Capillary HPLC-electrospray ionization mass spectrometry (ESI-MS) analysis of hAGT that had been treated with N7-(2'-hydroxy-3',4'-epoxybut-1'-yl)-deoxyguanosine (dG monoepoxide) revealed the ability of the protein to form either one or two butanediol-dG cross-links, corresponding to mass shifts of +353 and +706 Da, respectively. HPLC-ESI+ -MS/MS sequencing of the tryptic peptides obtained from dG monoepoxide-treated protein indicated that the two cross-linking sites were the alkyl acceptor site, Cys145, and a neighboring active site residue, Cys150. The same two amino acid residues of hAGT became covalently cross-linked to DNA following DEB treatment. Modification of Cys145 was further confirmed by HPLC-ESI+ -MS/MS analysis of dG monoepoxide-treated synthetic peptide GNPVPILIPCHR which represents the active site tryptic fragment of hAGT (C = Cys145). The replacement of the catalytic cysteine residue with alanine in the C145A hAGT mutant abolished DEB-induced cross-linking at this site, while the formation of conjugates via neighboring Cys150 was retained. The exact chemical structure of the cross-linked lesion was established as 1-(S-cysteinyl)-4-(guan-7-yl)-2,3-butanediol by HPLC-ESI+ -MS/MS analysis of the amino acids resulting from the total digestion of modified proteins analyzed in parallel with an authentic standard. AGT-DNA cross-linking is a likely mechanism of DEB-mediated cytotoxicity in cells expressing this important repair protein.

Figure 1

12% SDS-PAGE analysis of ³²P-end labeled DNA duplexes (5′-GGA GCT GGT GGC GTA GGC-3′, (+) strand) following incubation with DEB and hAGT (lanes 4–6) or C145A hAGT (lane 7). AGT-DNA cross-links are observed as slowly moving bands on the gel. Lanes 1–3 serve as negative controls.

Figure 2

HPLC-ESI⁺-MS analysis of dG monoepoxide-induced butanediol cross-links to hAGT. (A) HPLC-ESI⁺-MS of untreated hAGT. Top: Total ion chromatogram; Bottom: ESI⁺ mass spectrum of the 16.6 min protein peak; Inset: Deconvoluted mass spectrum of the 16.6 min peak (observed M = 21 880 Da, calculated M = 21 876 Da). (B) HPLC-ESI⁺-MS of hAGT following incubation with 50 molar equivalents dG monoepoxide. Top: Total ion chromatogram; Bottom: ESI⁺ mass spectrum of the 16.3 min protein peak; Inset: Deconvoluted mass spectrum of the 16.3 min peak: A = hAGT containing a single cross-link to dG (observed M = 22 230 Da, calculated M = 22 229 Da); B = double cross-link to dG (observed M = 22 586 Da, calculated M = 22 582 Da); C = unmodified hAGT (observed M = 21 877 Da, calculated M = 21 876 Da).

Figure 3

HPLC-ESI⁺-MS analysis of C145A hAGT protein following treatment with dG monoepoxide. (A) HPLC-ESI⁺-MS of untreated C145A hAGT. Top: Total ion chromatogram; Bottom: ESI⁺ mass spectrum of the 16.2 min protein peak; Inset: Deconvoluted mass spectrum of the 16.2 min peak (observed M = 23 015 Da, calculated M = 23 012 Da). (B) HPLC-ESI⁺-MS of C145A hAGT mutant following incubation dG monoepoxide. Top: Total ion chromatogram; Bottom: ESI⁺ mass spectrum of the 16.1 min protein peak; Inset: Deconvoluted mass spectrum of the 16.1 min peak: A = C145A hAGT containing a single cross-link to dG (observed M = 23 367 Da, calculated M = 23 365 Da); B = unmodified protein (observed M = 23 014 Da, calculated M = 23 012 Da).

Figure 4

HPLC-ESI⁺-MS/MS analysis of hAGT tryptic peptide G¹³⁶NPVPILIPCHR¹⁴⁷ containing a dG monoepoxide-induced butanediol cross-link between Cys¹⁴⁵ and guanine. Top: Extracted ion chromatogram of m/z 777.0 [M + 2H]²⁺; Bottom: MS/MS spectrum. Modified fragment ions are indicated by “*”.

Figure 5

HPLC-ESI⁺-MS/MS analysis of hAGT tryptic peptide V¹⁴⁸VCSSGGAVGNYSG GLAVK¹⁶⁵ containing dG monoepoxide-induced butanediol cross-link between Cys¹⁵⁰ and guanine. Top: Extracted ion chromatogram of m/z 953.6 [M + 2H]²⁺; Bottom: MS/MS spectrum. Modified fragment ions are indicated by “*”.

Figure 6

HPLC-ESI⁺-MS/MS analysis of hAGT tryptic peptides containing DEB-induced butanediol cross-links to guanine. (A) Top: Extracted ion chromatogram of hAGT tryptic peptide G¹³⁶NPVPILIPCHR¹⁴⁷ cross-linked to guanine (m/z 776.9 [M + 2H]²⁺); Bottom: MS/MS spectrum of tryptic peptide G¹³⁶NPVPILIPCHR¹⁴⁷ mapping the cross-link to Cys¹⁴⁵. (B) Top: Extracted ion chromatogram of hAGT tryptic peptide V¹⁴⁸VCSSGGAVGNYSGGLAVK¹⁶⁵ cross-linked to guanine (m/z 952.9 [M + 2H]²⁺); Bottom: MS/MS spectrum of tryptic peptide V¹⁴⁸VCSSGGAVGNYSGGLAVK¹⁶⁵ mapping the cross-link to Cys¹⁵⁰. Modified fragment ions are indicated by “*”.

Figure 6

HPLC-ESI⁺-MS/MS analysis of hAGT tryptic peptides containing DEB-induced butanediol cross-links to guanine. (A) Top: Extracted ion chromatogram of hAGT tryptic peptide G¹³⁶NPVPILIPCHR¹⁴⁷ cross-linked to guanine (m/z 776.9 [M + 2H]²⁺); Bottom: MS/MS spectrum of tryptic peptide G¹³⁶NPVPILIPCHR¹⁴⁷ mapping the cross-link to Cys¹⁴⁵. (B) Top: Extracted ion chromatogram of hAGT tryptic peptide V¹⁴⁸VCSSGGAVGNYSGGLAVK¹⁶⁵ cross-linked to guanine (m/z 952.9 [M + 2H]²⁺); Bottom: MS/MS spectrum of tryptic peptide V¹⁴⁸VCSSGGAVGNYSGGLAVK¹⁶⁵ mapping the cross-link to Cys¹⁵⁰. Modified fragment ions are indicated by “*”.

Figure 7

HPLC-ESI⁺-MS/MS analysis of hAGT tryptic peptides G¹³⁶NPVPILIPCHR¹⁴⁷ and V¹⁴⁸VCSSGGAVGNYSGGLAVK¹⁶⁵containing DEB-induced 2′,3′,4′-trihydroxybut-1′-yl adducts to active site cysteine residues Cys¹⁴⁵ (A) and Cys¹⁵⁰ (B). Top: Extracted ion chromatograms; Bottom: MS/MS spectra. Modified fragment ions are indicated by “*”.

Figure 7

HPLC-ESI⁺-MS/MS analysis of hAGT tryptic peptides G¹³⁶NPVPILIPCHR¹⁴⁷ and V¹⁴⁸VCSSGGAVGNYSGGLAVK¹⁶⁵containing DEB-induced 2′,3′,4′-trihydroxybut-1′-yl adducts to active site cysteine residues Cys¹⁴⁵ (A) and Cys¹⁵⁰ (B). Top: Extracted ion chromatograms; Bottom: MS/MS spectra. Modified fragment ions are indicated by “*”.

Figure 8

HPLC-ESI⁺-MS/MS analysis of 1-(S-cysteinyl)-4-(guan-7-yl)-2,3-butanediol (Cys-Gua-BD) in total digests of AGT protein treated with dG monoepoxide. (A) Extracted ion chromatogram of Cys-Gua-BD (m/z 359.7 [M + H]⁺) resulting from the total digestion of dG monoepoxide-treated hAGT; Inset: MS/MS fragmentation of Cys-Gua-BD. (B) Extracted ion chromatogram of Cys-Gua-BD (m/z 359.4 [M + H]⁺) resulting from the total digestion of dG monoepoxide-treated C145A hAGT; Inset: MS/MS fragmentation of Cys-Gua-BD.

Scheme 1

DNA-AGT cross-linking by dihaloethanes and DEB.

Scheme 2

Mass spectrometric analysis of DEB-induced AGT-DNA conjugates.