Peroxynitrite Induces Formation of Nε-(Carboxymethyl)Lysine by the Cleavage of Amadori Product and Generation of Glucosone and Glyoxal From Glucose
Novel Pathways for Protein Modification by Peroxynitrite
- Ryoji Nagai1,
- Yuka Unno12,
- Miki Cristina Hayashi1,
- Shuichi Masuda2,
- Fumitaka Hayase3,
- Naohide Kinae2 and
- Seikoh Horiuchi1
- 1Department of Biochemistry, Kumamoto University School of Medicine, Kumamoto, Japan
- 2School of Food and Nutritional Sciences and Graduate School of Nutritional and Environmental Sciences, University of Shizuoka, Shizuoka, Japan
- 3Department of Agricultural Chemistry, Meiji University, Kawasaki, Japan
Abstract
Accumulation of advanced glycation end products (AGEs) on tissue proteins increases with pathogenesis of diabetic complications and atherosclerosis. Here we examined the effect of peroxynitrite (ONOO−) on the formation of Nε-(carboxymethyl)lysine (CML), a major AGE-structure. When glycated human serum albumin (HSA; Amadori-modified protein) was incubated with ONOO−, CML formation was detected by both enzyme-linked immunosorbent assay and high-performance liquid chromatography (HPLC) and increased with increasing ONOO− concentrations. CML was also formed when glucose, preincubated with ONOO−, was incubated with HSA but was completely inhibited by aminoguanidine, a trapping reagent for α-oxoaldehydes. For identifying the aldehydes that contributed to ONOO−-induced CML formation, glucose was incubated with ONOO− in the presence of 2,3-diaminonaphthalene. This experiment led to identification of glucosone and glyoxal by HPLC. Our results provide the first evidence that ONOO− can induce protein modification by oxidative cleavage of the Amadori product and also by generation of reactive α-oxoaldehydes from glucose.
Footnotes
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Address correspondence and reprint requests to Dr. Seikoh Horiuchi, Department of Biochemistry, Kumamoto University School of Medicine, Honjo, 2-2-1, Kumamoto 860-0811, Japan. E-mail: horiuchi{at}gpo.kumamoto-u.ac.jp.
Received for publication 15 January 2002 and accepted in revised form 3 June 2002.
R.N. and Y.U. contributed equally to this work.
AGE, advanced glycation end product; CML, Nε-(carboxymethyl)lysine; DAN, 2,3-diaminonaphthalene; ELISA, enzyme-linked immunosorbent assay; ESI-MS, electrospray ionization-mass spectrometry; HPLC, high-performance liquid chromatography; HRP, horseradish peroxidase; HSA, human serum albumin; iNOS, inducible nitric oxide synthase; KLH, keyhole limpet hemocyanin; NO, nitric oxide; O2−, superoxide anion radical; ONOO−, peroxynitrite; RAGE, receptor for AGE; TGF-β1, transforming growth factor-β1.
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