Conjugation of urate-derived electrophiles to proteins during normal metabolism and inflammation

Abstract

Urate is often viewed as an antioxidant. Here, we present an alternative perspective by showing that, when oxidized, urate propagates oxidative stress. Oxidation converts urate to the urate radical and the electrophilic products dehydrourate, 5-hydroxyisourate, and urate hydroperoxide, which eventually break down to allantoin. We investigated whether urate-derived electrophiles are intercepted by nucleophilic amino acid residues to form stable adducts on proteins. When urate was oxidized in the presence of various peptides and proteins, two adducts derived from urate (M_r 167 Da) were detected and had mass additions of 140 and 166 Da, occurring mainly on lysine residues and N-terminal amines. The adduct with a 140-Da mass addition was detected more frequently and was stable. Dehydrourate (M_r 166 Da) also formed transient adducts with cysteine residues. Urate-derived adducts were detected on human serum albumin in plasma of healthy donors. Basal adduct levels increased when neutrophils were added to plasma and stimulated, and relied on the NADPH oxidase, myeloperoxidase, hydrogen peroxide, and superoxide. Adducts of oxidized urate on serum albumin were elevated in plasma and synovial fluid from individuals with gout and rheumatoid arthritis. We propose that rather than acting as an antioxidant, urate's conversion to electrophiles contributes to oxidative stress. The addition of urate-derived electrophiles to nucleophilic amino acid residues, a process we call oxidative uratylation, will leave a footprint on proteins that could alter their function when critical sites are modified.

Keywords: NADPH oxidase; biomarker; lysine modification; myeloperoxidase; neutrophil; oxidative stress; post-translational modification (PTM); protein adduct; uratylation; uric acid.

Figure 1.

Oxidation of urate to electrophiles and their break down to allantoin. One-electron oxidation of urate produces the urate radical that, unless reduced by ascorbate (Asc), can either be further oxidized to dehydrourate or react with superoxide to produce urate hydroperoxide. Both of these electrophiles hydrolyze to 5-hydroxyisourate that, through a series of reactions (small arrows), breaks down to allantoin. Relative molecular masses (M_r) are given in parentheses.

Figure 2.

Oxidized urate forms stable covalent adducts with amino residues on peptides. Urate (380 μm) was oxidized by MPO (100 nm) and hydrogen peroxide (100 μm) in the presence of the peptide GGYR (100 μm) in 10 mm phosphate buffer, pH 7.4, for 30 min at 37 °C. The products were separated by LC-MS (A), and the fragmentation spectra for GGYR (B) and GGYR with mass additions of 140 Da (C) and 166 Da (D) were determined. The red residue G indicates the location of the urate-derived adduct.

Figure 3.

Superoxide enhances formation of urate-derived adducts with amino residues on peptides. The peptide MLTELEK (100 μm) or YGGFL (100 μm) was incubated with urate (380 μm), which was oxidized by MPO (100 nm) with xanthine oxidase (0.025 units/ml) and acetaldehyde (10 mm) to generate low fluxes of superoxide and hydrogen peroxide. Formation of the 140-Da adduct of oxidized urate on MLTELEK was confirmed by comparing fragmentation of the parent peptide (A) and the product identified by LC-MS (B). The red K residue indicates the location of the urate-derived adduct. C, urate-derived adducts on the peptides were generated in the presence and absence of SOD and measured using LC-MS/MS. Data are means ± S.D. (error bars) of triplicate experiments. Statistical differences were determined by Student's t test (**, p < 0.001; *, p = 0.02).

Figure 4.

Oxidized urate forms transient adducts with cysteine residues. A, urate (380 μm) was oxidized by MPO (100 nm) and hydrogen peroxide (100 μm) in the presence of the peptide PFVCG (100 μm) in 10 mm phosphate buffer, pH 7.4, at 37 °C for 1 min and kept at 4 °C until the products were identified by LC-MS. The product with an m/z of 688 is equivalent to PFVCG ([M + H]⁺ = 522 m/z) plus dehydrourate (166 Da). The mass spectra are shown for PFVCG (B) and PFVCG (C) with a mass addition of 166 Da. The red residue C indicates the location of the dehydrourate adduct.

Figure 5.

Determination of the structure of the 140-Da adduct of oxidized urate with amino groups. Labeled (¹⁵N) (M_r 169 Da) or unlabeled urate (M_r 167 Da) (380 μm) was oxidized by MPO (100 nm) and hydrogen peroxide (400 μm) in the presence of phenethylamine (PEA) (M_r 121 Da; 100 μm) in 10 mm phosphate buffer, pH 7.4, for 30 min at 20–22 °C, and products (A) with an addition of 140 Da ([M + 140 + H]⁺ m/z 262 (blue line) or 264 Da (red line)) were detected by selected ion-monitoring LC-MS. The fragmentation spectra of the unlabeled ([M + H]⁺ 262 m/z) (B) and labeled ([M + H]⁺ 264 m/z) (C) products were then determined. D, the proposed reaction sequence for breakdown of 5-hydroxyisourate to the bicylic imidazolone that adds to an amino group (NH₂R) to form the stable 140-Da adduct.

Figure 6.

Urate electrophiles inactivate GAPDH. A, residual enzyme activity (μm/min) was determined after GAPDH (2 μm) was treated for 10 min at pH 7.4 with a solution of urate electrophiles that contained urate hydroperoxide (5 μm). Urate was oxidized using XO, hypoxanthine (HX; 100 μm), urate (400 μm), and lactoperoxidase (LPO; 160 nm) in 50 mm phosphate buffer, pH 7.4, for 20 min. Other additions included SOD (20 μg/ml) or CAT (20 μg/ml). Data are representative of two independent experiments. Error bars, S.D. of triplicate readings for one experiment. A one-way ANOVA followed by Dunnett's multiple-comparison test on triplicate readings was used to identify samples that were significantly different from the full system. All treatments were significantly different from the control (p < 0.001) except catalase. The enzyme GAPDH was treated with oxidized urate containing increasing concentrations of urate hydroperoxide, and its residual activity (B) was determined either before (●) or after (▴) reduction with DTT. C, the content of reduced thiols after treatment with increasing concentrations of urate hydroperoxide.

Figure 7.

Oxidized urate forms stable adducts on amino residues of proteins. Urate (380 μm) was oxidized by MPO (100 nm) and hydrogen peroxide (100 μm) in the presence of either ubiquitin (A and B; M_r 8,565 Da; 0.1 mg/ml) or β-lactoglobulin (C and D; M_r 18,278 Da; 0.1 mg/ml) in 10 mm phosphate buffer, pH 7.4, for 30 min at 37 °C. The molecular masses of the modified proteins were determined by MS (A and C), and tryptic peptides (B and D) containing the 140-Da adducts from oxidized urate were identified by LC-MS/MS. The red residues M and K indicate the locations of the urate-derived adduct.

Figure 8.

Oxidized urate forms stable adducts with human serum albumin, which are present in normal plasma and elevated by oxidative stress. A, urate was oxidized by MPO and hydrogen peroxide in the presence of human serum albumin under the conditions listed in Fig. 7. Adducts of urate (140 Da) were detected on the protein's lysine residues indicated in blue on the crystal structure of albumin (yellow) drawn using PyMOl (Protein Data Bank code 1AO6). The red residue is Lys-432. B, the fragmentation spectrum of NLGK⁴³²VGSK from albumin containing the urate-derived adduct (140 Da) on lysine 432 (red residue in A). C, using multiple-reaction-monitoring MS, NLGK⁴³²VGSK was measured in plasma from healthy donors without (black) or with neutrophils (5 × 10⁶/ml) that had been stimulated with CytB (10 μg/ml) and PMA (100 ng/ml) for 45 min at 37 °C (red). D, urate-derived adducts on serum albumin were measured in plasma from healthy donors alone or with neutrophils. When stimulated, neutrophils were incubated without or with inhibitors of the NADPH oxidase (DPI; 20 μm), myeloperoxidase (TX1; 10 μm), CAT (100 μg/ml), or SOD (20 μg/ml). Data are means ± S.D. (error bars) of three independent experiments with blood from three donors. Significant difference (*, p < 0.05; ***, p < 0.0001) from control plasma (blue line) was determined by ANOVA.

Figure 9.

Urate-derived adducts on albumin are elevated during inflammation. A, the LC-MS signal for NLGK⁴³²VGSK from albumin was higher in plasma and synovial fluid from a patient with rheumatoid arthritis compared with that from a healthy control. B, the levels of urate-derived adducts on NLGK⁴³²VGSK from albumin relative to the normal tryptic peptide NLGK were significantly (p < 0.014) elevated in plasma and synovial fluid from individuals with rheumatoid arthritis (n = 6) and gout (n = 6) compared with healthy controls (n = 8). Plots show individual points, with error bars indicating means and standard deviations. Significant differences were determined by ANOVA after Dunnett's multiple-comparison test.