Inactivation of thiol-dependent enzymes by hypothiocyanous acid: role of sulfenyl thiocyanate and sulfenic acid intermediates

Abstract

Myeloperoxidase (MPO) forms reactive oxidants including hypochlorous and hypothiocyanous acids (HOCl and HOSCN) under inflammatory conditions. HOCl causes extensive tissue damage and plays a role in the progression of many inflammatory-based diseases. Although HOSCN is a major MPO oxidant, particularly in smokers, who have elevated plasma thiocyanate, the role of this oxidant in disease is poorly characterized. HOSCN induces cellular damage by targeting thiols. However, the specific targets and mechanisms involved in this process are not well defined. We show that exposure of macrophages to HOSCN results in the inactivation of intracellular enzymes, including creatine kinase (CK) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH). In each case, the active-site thiol residue is particularly sensitive to oxidation, with evidence for reversible inactivation and the formation of sulfenyl thiocyanate and sulfenic acid intermediates, on treatment with HOSCN (less than fivefold molar excess). Experiments with DAz-2, a cell-permeable chemical trap for sulfenic acids, demonstrate that these intermediates are formed on many cellular proteins, including GAPDH and CK, in macrophages exposed to HOSCN. This is the first direct evidence for the formation of protein sulfenic acids in HOSCN-treated cells and highlights the potential of this oxidant to perturb redox signaling processes.

Fig. 1

Inhibition of CK and GAPDH activity in J774A.1 lysates and intact cells after treatment with HOSCN. (a) CK and (c) GAPDH activity in J774A.1 cell lysates (1 × 10⁶ cells ml⁻¹) after HOSCN treatment (5–20 µM) for 15 min at 22 °C. (b) CK and (d) GAPDH activity in intact J774A.1 cells (1 × 10⁶ cells ml⁻¹) after HOSCN treatment (80–200 µM) for 15 min at 22 °C. *Significant decrease (p<0.05) in enzyme activity compared to control (untreated) lysates/cells by one-way ANOVA with Dunnett's post hoc test. Values are means ± SEM (n = 6).

Fig. 2

Inhibition of CK and GAPDH activity by HOSCN is reversible with DTT. (a) CK (5 µM) and (b) GAPDH (5 µM) were treated with HOSCN (2.5–25 µM) for 5 or 30 min, respectively, followed by further incubation in the absence (black bars) or presence (white bars) of DTT (40 µM) for 15 min. *Significant decrease (p<0.05) in enzyme activity of treated samples compared with controls by one-way ANOVA with Dunnett's post hoc testing; #significant increase (p<0.05) in enzyme activity on incubation in the presence of DTT compared to samples incubated in the absence of DTT by two-way ANOVA with Bonferroni post hoc testing. Values are means ± SEM (n = 6).

Fig. 3

¹⁴C incorporation after treatment of CK and GAPDH with HOS¹⁴CN. (a) CK (25 µM) and (b) GAPDH (25 µM) were treated with HOS¹⁴CN (12.5–50 µM) for 5 and 30 min, respectively, before incubation in the absence (white bars) and presence (gray bars) of DTT (1.25 mM). Controls were performed with oxidant-free, S¹⁴CN⁻ solutions with no DTT added (black bars) and after addition of DTT (1.25 mM; striped bars). A significant increase (p<0.05) in ¹⁴C incorporation was seen, compared with the control treated with oxidant-free S¹⁴CN⁻ solutions in the (*) absence and (#) presence of DTT, using two-way ANOVA with Bonferroni post hoc testing. Values are means ± SEM (n≥5).

Fig. 4

Effect of incubation time on the extent of ¹⁴C incorporation after treatment of CK and GAPDH with HOS¹⁴CN. (a) CK (25 µM) was treated with HOS¹⁴CN (25 µM) for various times before further incubation in the absence (triangle) and presence of DTT (1.25 mM; circle). (b) CK (25 µM) was treated with HOS¹⁴CN (25 µM) for 5 min before gel filtration to remove excess S¹⁴CN⁻ and potential decomposition products and further incubation in the absence (triangle) and presence of DTT (1.25 mM; circle). (c) GAPDH (25 µM) was treated with HOS¹⁴CN (25 µM) for various times before further incubation in the absence (triangle) and presence of DTT (1.25 mM; circle). (d) GAPDH (25 µM) was treated with HOS¹⁴CN (25 µM) for 30 min before gel filtration and analysis as in (b). *p<0.05; significant increase or decrease in ¹⁴C incorporation compared with initial incorporation by one-way ANOVA with Bonferroni post hoc testing. Values are means ± SEM (n≥5). Error bars have been plotted, but in some cases are smaller than the symbol used.

Fig. 5

Treatment of CK with HOSCN results in oxidation of the active-site Cys-282. CK (25 µM) was treated with HOSCN (12.5–250 µM) for 5 min, before reductive alkylation, tryptic digestion, and LC–MS/MS analysis. (a) Loss of the alkylated active-site Cys-containing peptide of CK (Cys-282, [M + 2H]²⁺, 1465.6). (b) Formation of the +138 product peptide corresponding to the addition of dimedone at the Cys-282 active-site of CK ([M + 2H]²⁺, 1505.6). (c) Formation of the +32 product peptide corresponding to sulfinic acid generation at Cys-282 ([M + 2H]²⁺, 1452.6). (d) Formation of the +48 product peptide corresponding to sulfonic acid generation at Cys-282 ([M + 2H]²⁺, 1460.6). Peak areas were obtained from extracted ion chromatograms (selected characteristic ions presented in Supplementary Table 3) and were standardized to the area of the CK oxidant-insensitive peptide SEEEYPDLSK ([M + 2H]²⁺, 598.5). *p<0.05; significant decrease or increase in the concentration of each peptide compared to control by one-way ANOVA with Dunnett's post hoc test. Values are means ± SEM (n = 6).

Fig. 6

Fragmentation ion spectra of the [M + 2H]²⁺, [M + 138 + 2H]²⁺, [M + 32 + 2H]²⁺, and [M + 48 + 2H]²⁺ for the modified active-site (Cys-282) peptides from tryptic digests of HOSCN-treated CK. (a) MS/MS spectrum of the doubly charged species m/z 1465.6 (carboxymethyl peptide), with the fragment ions specific for the Cys-282 + 58 product. Fragment ions are singly charged, except where specified. Equivalent data are shown in (b), (c), and (d), obtained by monitoring the doubly charged peptide species m/z 1505.6, 1452.6, and 1460.6, respectively. Data are representative of at least five independent experiments.

Fig. 7

Treatment of GAPDH with HOSCN results in oxidation of Cys-149 and Cys-153. GAPDH (25 µM) was treated with HOSCN (12.5–250 µM) for 30 min, before reductive alkylation, tryptic digestion, and LC–MS/MS analysis. (a) Loss of the active-site Cys-containing peptide of GAPDH (Cys-149, [M + 2H]²⁺, 912.0). (b) Formation of the +138 product peptide corresponding to the addition of dimedone at the Cys-149 active site of GAPDH (black bars, [M + 2H]²⁺, 952.0) and addition of dimedone at the Cys-153 site of GAPDH (white bars, [M + 2H]²⁺, 952.0). (c) Formation of the +32 product peptide corresponding to sulfinic acid generation on Cys-149 ([M + 2H]²⁺, 899.0). (d) Formation of the +48 product peptide corresponding to sulfonic acid generation on Cys-149 ([M + 2H]²⁺, 907.0). Peak areas were obtained from extracted ion chromatograms (selected characteristic ions presented in Supplementary Table 4) and were standardized to the area of the oxidant-insensitive GAPDH peptide GAAQNIIPASTGAAK ([M + 2H]²⁺, 685.6). *p<0.05; significant decrease or increase in the tryptic peptide of interest compared to control conditions by one-way ANOVA with Dunnett's post hoc test. Values are means ± SEM (n = 6).

Fig. 8

Fragmentation ion spectra of [M + 2H]²⁺, [M + 138 + 2H]²⁺ (Cys-149 and Cys-153), [M + 32 + 2H]²⁺ (Cys-149), and [M + 48 + 2H]²⁺ (Cys-149) peptides from tryptic digests of HOSCN-treated GAPDH. (a) MS/MS spectrum of the doubly charged species m/z 912.0 (carboxymethyl peptide), with the fragment ions specific for the Cys-149 + 58 and Cys-153 + 58 product. Fragment ions are singly charged. Equivalent data are shown in (b), (c), (d), and (e) via monitoring of the doubly charged species m/z 952.0, 899.0, and 907.0, respectively. Data are representative of at least five independent experiments.

Fig. 9

Exposure of J774A.1 cells to HOSCN results in the formation of protein-derived sulfenic acid species. J774A.1 cells (1 × 10⁶ cells ml⁻¹) were pretreated with DAz-2 (5 mM in 1% v/v DMSO) or DMSO (1% v/v) for 30 min at 37 °C before the addition of PBS or HOSCN (50–100 µM) and further incubation for 15 min at 37 °C. The cells were then lysed, and Staudinger ligation was carried out with p-biotin (200 µM) and DTT (5 mM) for 2 h at 37 °C. The reaction was quenched and proteins were separated by 1D SDS–PAGE (4–12%). Proteins (50 µg per lane) were then transferred to PVDF membranes and biotinylated proteins detected by HRP–streptavidin. Asterisks denote endogenously biotinylated proteins.