The myeloperoxidase system of human phagocytes generates Nε-(carboxymethyl)lysine on proteins: a mechanism for producing advanced glycation end products at sites of inflammation
Melissa M. Anderson, … , Suzanne R. Thorpe, Jay W. Heinecke
Melissa M. Anderson, … , Suzanne R. Thorpe, Jay W. Heinecke
Published July 1, 1999
Citation Information: J Clin Invest. 1999;104(1):103-113. https://doi.org/10.1172/JCI3042.
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Article

The myeloperoxidase system of human phagocytes generates Nε-(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 α-amino acids. The pathway begins with myeloperoxidase, a heme enzyme secreted by activated neutrophils. Conversion of α-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, Nε-(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.

Authors

Melissa M. Anderson, Jesús R. Requena, Jan R. Crowley, Suzanne R. Thorpe, Jay W. Heinecke

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Fluorescence excitation and emission spectra of RNase A modified by HOCl...
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).