The myeloperoxidase system of human phagocytes generates Nepsilon-(carboxymethyl)lysine on proteins: a mechanism for producing advanced glycation end products at sites of inflammation

Abstract

Reactive aldehydes derived from reducing sugars and peroxidation of lipids covalently modify proteins and may contribute to oxidative tissue damage. We recently described another mechanism for generating reactive aldehydes from free alpha-amino acids. The pathway begins with myeloperoxidase, a heme enzyme secreted by activated neutrophils. Conversion of alpha-amino acids to aldehydes requires hypochlorous acid (HOCl), formed from H2O2 and chloride by myeloperoxidase. When L-serine is the substrate, HOCl generates high yields of glycolaldehyde. We now demonstrate that a model protein, ribonuclease A (RNase A), exposed to free L-serine and HOCl exhibits the biochemical hallmarks of advanced glycation end (AGE) products -- browning, increased fluorescence, and cross-linking. Furthermore, Nepsilon-(carboxymethyl)lysine (CML), a chemically well-characterized AGE product, was generated on RNase A when it was exposed to reagent HOCl-serine, the myeloperoxidase-H2O2-chloride system plus L-serine, or activated human neutrophils plus L-serine. CML production by neutrophils was inhibited by the H2O2 scavenger catalase and the heme poison azide, implicating myeloperoxidase in the cell-mediated reaction. CML was also generated on RNase A by a myeloperoxidase-dependent pathway when neutrophils were activated in a mixture of amino acids. Under these conditions, we observed both L-serine-dependent and L-serine-independent pathways of CML formation. The in vivo production of glycolaldehyde and other reactive aldehydes by myeloperoxidase may thus play an important pathogenic role by generating AGE products and damaging tissues at sites of inflammation.

Figure 1

Progress curve of protein browning induced by the HOCl-serine system. HOCl and L-serine (0.9:1.0 mol/mol at the indicated final concentration) were incubated together in buffer A (50 mM sodium phosphate buffer [pH 7.0]) for 15 minutes at 37°C. RNase A (100 mg/mL in buffer A) was then added to a final concentration of 10 mg/mL, and the absorbance at 325 nm of the reaction mixture was monitored. When indicated, 20 mM NaCNBH₃ was present during the reaction of HOCl and L-serine.

Figure 2

Fluorescence excitation and emission spectra of RNase A modified by HOCl-serine. RNase A (10 mg/mL) was modified for 24 hours at 37°C in buffer A with the HOCl-serine system at 10 mM each (a), as described in the legend to Figure 1; with 10 mM glycolaldehyde (b); or with 9 mM HOCl alone (c). Reaction mixture was then diluted 1:10 with 50 mM sodium citrate-citric acid buffer (pH 5.0), 50 mM sodium phosphate buffer (pH 7.0), or 50 mM boric acid-borax buffer (pH 9.0). The excitation and emission spectra of each reaction mixture were then determined. (d) Excitation and emission spectra of 1 mg/mL RNase A in 50 mM sodium phosphate buffer (pH 7.0).

Figure 3

SDS-PAGE analysis of RNase A modified by HOCl-serine. RNase A (10 mg/mL) was incubated with the HOCl-serine system in buffer A, as indicated in the legend to Figure 1, for 24 hours at 37°C. RNase A (20 μg of protein) was then subjected to electrophoresis on a 10–20% gradient polyacrylamide gel under denaturing (0.1% SDS) and reducing (1% β-mercaptoethanol) conditions. Reaction conditions were as follows: lane 1, RNase A alone; lane 2, RNase A + 5 mM L-serine + 4.5 mM HOCl; lane 3, RNase A + 10 mM L-serine + 9 mM HOCl; lane 4, RNase A + 5 mM glycolaldehyde; lane 5, RNase A + 10 mM glycolaldehyde; lane 6, RNase A + 10 mM L-serine; lane 7, RNase A + 9 mM HOCl; lane 8, RNase A + 10 mM L-serine + 9 mM HOCl + 20 mM NaCNBH₃. NaCNBH₃ was present during the incubation of HOCl and L-serine (lane 8).

Figure 4

Progress curve and concentration dependence of CML formation on RNase A by HOCl-serine. (a) RNase A (1 mg/mL) was modified by the HOCl-serine system (0.9 mM and 1.0 mM, respectively) as described in the legend to Figure 1. At the indicated times, the reaction mixture was subjected to analysis for CML by electron ionization isotope dilution GC/MS with selected ion monitoring. (b) The indicated concentrations of HOCl and L-serine were incubated for 15 minutes at 37°C in buffer A, followed by addition of RNase A (final concentration 1 mg/mL) and incubation for 24 hours at 37°C.

Figure 5

Progress curve and reaction requirements for generation of CML on RNase A by the myeloperoxidase-H₂O₂-chloride-serine system. (a) The complete myeloperoxidase system (60 nM myeloperoxidase, 1 mM H₂O₂, 1 mM L-serine, and 150 mM NaCl in buffer A) was incubated at 37°C for 90 minutes. RNase A (1 mg/mL final concentration) was added, and the reaction mixture was incubated for the indicated period at 37°C. The reaction mixture was then subjected to analysis for CML by electron ionization isotope dilution GC/MS with selected ion monitoring. (b) The complete myeloperoxidase system was incubated at 37°C as described for a. After the addition of 1 mg/mL RNase A, the reaction mixture was incubated for 24 hours. Reaction mixture was then subjected to analysis for CML by GC/MS. When indicated, chloride ion (Cl^–), myeloperoxidase (MPO), L-serine, or H₂O₂ (peroxide) was omitted from the complete myeloperoxidase system.

Figure 6

Influence of plasma amino acids on CML formation by myeloperoxidase. The complete myeloperoxidase system (Complete; 30 nM myeloperoxidase, 300 ng/mL glucose oxidase, 100 μg/mL glucose) was incubated for 16 hours at 37°C in buffer A supplemented with 1 mg/ml RNase A, 260 μM L-glutamate, 210 μM L-alanine, 200 μM L-serine, 175 μM glycine, 165 μM L-valine, 100 μM L-proline, and 100 μM L-lysine. The reaction mixture was then subjected to analysis for CML by negative-ion electron capture GC/MS with selected ion monitoring. Sodium azide (Azide; 5 mM) or catalase (Cat; 400 nM) was added when indicated. The bottom 3 bars represent control reactions containing RNase A alone or supplemented with catalase or glucose oxidase. G, glucose; GO, glucose oxidase; MPO, myeloperoxidase.

Figure 7

Detection of the trifluoroacetyl methyl ester derivative of CML generated by activation human neutrophils by electron ionization GC/MS with selected ion monitoring. Freshly harvested human neutrophils (10⁶/mL) were incubated at 37°C in medium B supplemented with 1 mM L-serine. Cells were stimulated with 250 nM PMA and maintained in suspension by intermittent inversion. After a 45-minute incubation, cells were removed by centrifugation, and RNase A (1 mg/mL final concentration) was added. After a 96-hour incubation at 37°C, protein-bound CML in the incubation medium was detected by electron ionization GC/MS with selected ion monitoring. The ions at m/z 392 (M^•+ – CH₃OH) and m/z 305 (M^•+ – 2 COOCH₃ –H) represent the most abundant ions in the full-scan mass spectrum of CML. Note that the ions derived from d₄-labeled CML at m/z 309 and m/z 396 elute slightly earlier than the corresponding ions of CML, as has been shown for many other deuterated compounds (45).

Figure 8

Progress curve and reaction requirements for generation of CML on RNase A by activated human neutrophils. (a) Freshly harvested human neutrophils (10⁶/mL) were incubated at 37°C in medium B supplemented with 1 mM L-serine as described in the legend to Figure 6. After incubation for the indicated period at 37°C, the reaction was stopped by freezing at –20°C. The incubation medium was then subjected to analysis for CML by electron ionization isotope dilution GC/MS with selected ion monitoring. (b) Freshly harvested human neutrophils (10⁶/mL) were incubated at 37°C in medium B supplemented with 1 mM L-serine (Complete) as in a. After the addition of 1 mg/mL RNase A, the reaction mixture was incubated for 96 hours. Reactions were then subjected to analysis for CML by electron ionization selected ion monitoring GC/MS. When indicated, cells, L-serine,or PMA was omitted, or azide (5 mM) or catalase (500 nM) was included in the complete cellular system. Values are corrected for the endogenous CML content (12.2 μmol/mol lysine) of RNase A.

Figure 9

Reaction requirements for generation of CML on RNase A by activated human neutrophils in medium supplemented with plasma concentrations of free amino acids. Isolated neutrophils (10⁶/mL) were activated with 200 nM PMA in medium B supplemented with 1 mg/mL RNase A, 260 μM L-glutamate, 210 μM L-alanine, 200μm L-serine, 175 μM glycine, 165 μM L-valine, 100 μM L-proline, and 100 μM L-lysine. When indicated, L-serine was omitted (–Ser) from the medium, and sodium azide (Azide; 5 mM) or 3-aminotriazole (ATA; 10 mM) was included. After a 1-hour incubation at 37°C, cells were removed by centrifugation, and the medium was incubated for another 16 hours at 37°C. Reaction mixtures were then subjected to analysis for CML by negative-ion electron capture isotope dilution GC/MS with selected ion monitoring. Values are corrected for the endogenous CML content (21.0 μmol/mol lysine) of RNase A and represent the mean ± SEM of 6 determinations.