Protein oxidation by myeloperoxidase in vitro and in vivo.

摘要

Oxidative modification of proteins is implicated in the pathogenesis of disease. One potential pathway at sites of inflammation involves myeloperoxidase, an enzyme secreted by phagocytes. It is difficult to study oxidation chemistry in vivo since the intermediates are short-lived, so the analysis of stable markers has therefore become an important tool in elucidating the pathways responsible for tissue damage. Our studies began with the demonstration of a new pathway of potential modification, the generation of an alpha-hydroxy, and alpha,beta-unsaturated, aldehyde from free amino acids by myeloperoxidase. Next we demonstrated that one such aldehyde, glycolaldehyde, can react with protein lysine groups to form a stable adduct. This pathway occurs in vivo because adduct formation was impaired in mice deficient in oxidant generation, and was increased in human atherosclerotic tissue. To explore the role of myeloperoxidase in promoting protein oxidation in vivo, we collaborated with Dr. Aldons Lusis to generate a myeloperoxidase-deficient mouse. We used the mouse in an acute inflammatory model to look for markers suggested by in vitro studies to be generated by myeloperoxidase. Neutrophils isolated from knock-out mice were completely unable to generate hypochlorous acid, a characteristic product of myeloperoiddase. In the in vivo inflammatory model, wild-type mice generated 3-chlorotyrosine, a specific marker of myeloperoxidase in vitro. In contrast, the myeloperoxidase-deficient mice displayed a complete lack of tyrosine chlorination suggesting that myeloperoxidase is solely responsible for this modification in vivo. The final marker that we analyzed was 3-nitrotyrosine, a modification suggested to be formed through several pathways in vitro. In the inflammation model, the myeloperoxidase-deficient mouse had a 40% lower level of 3-nitrotyrosine compared to wild-type mice, suggesting that myeloperoxidase is playing a role in nitration of tyrosine in vivo. Collectively, our studies demonstrate that myeloperoxidase acts as a potent oxidant generator at sites of inflammation, and may play a role in tissue damage in inflammatory disease.