Nω -(Carboxymethyl)arginine Is One of the Dominant Advanced Glycation End Products in Glycated Collagens and Mouse Tissues.

Sho Kinoshita,Hiroko Ichikawa,Katsumi Mera,Katsumasa Iijima,Yuki Taga,Jun-Ichi Shirakawa,Shunji Hattori,Satoko Shimasaki,Ryoji Nagai,Yukio Fujiwara,Mime Nagai

doi:10.1155/2019/9073451

Abstract

Advanced glycation end products (AGEs) accumulate in proteins during aging in humans. In particular, the AGE structure Nω-(carboxymethyl)arginine (CMA) is produced by oxidation in glycated collagen, accounting for one of the major proteins detected in biological samples. In this study, we investigated the mechanism by which CMA is generated in collagen and detected CMA in collagen-rich tissues. When various protein samples were incubated with glucose, the CMA content, detected using a monoclonal antibody, increased in a time-dependent manner only in glycated collagen, whereas the formation of Nε-(carboxymethyl)lysine (CML), a major antigenic AGE, was detected in all glycated proteins. Dominant CMA formation in glycated collagen was also observed by electrospray ionization-liquid chromatography-tandem mass spectrometry (LC-MS/MS). During incubation of glucose with collagen, CMA formation was enhanced with increasing glucose concentration, whereas it was inhibited in the presence of dicarbonyl-trapping reagents and a metal chelator. CMA formation was also observed upon incubating collagen with glyoxal, and CMA was generated in a time-dependent manner when glyoxal was incubated with type I–IV collagens. To identify hotspots of CMA formation, tryptic digests of glycated collagen were applied to an affinity column conjugated with anti-CMA. Several CMA peptides that are important for recognition by integrins were detected by LC-MS/MS and amino acid sequence analyses. CMA formation on each sequence was confirmed by incubation of the synthesized peptides with glyoxal and ribose. LC-MS detected CMA in the mouse skin at a higher level than other AGEs. Furthermore, CMA accumulation was greater in the human aorta of older individuals. Overall, our study provides evidence that CMA is a representative AGE structure that serves as a useful index to reflect the oxidation and glycation of collagen.

Highlights

Incubation of proteins with glucose leads to the generation of advanced glycation end products (AGEs) from the Maillard reaction through the formation of Schiff bases and Amadori products [1]
Immunoreaction of 3F5 to CMA-conjugated Human serum albumin (HSA) was significantly inhibited by the free form of CMA, whereas the reactivities of free CML and S-(carboxymethyl)cysteine (CMC) were negligible (Figure 2(b))
0 01247 01247 01247 01247 01247 Time. These results suggested that intermediate aldehydes may play a role in CMA formation during the incubation of collagen with glucose

Summary

Introduction

Incubation of proteins with glucose leads to the generation of advanced glycation end products (AGEs) from the Maillard reaction through the formation of Schiff bases and Amadori products [1]. The levels of AGE-modified proteins increase during the normal aging process [2]; this increase is markedly accelerated in a diabetic condition with sustained hyperglycemia. Nε-(Carboxymethyl)lysine (CML), one of the major antigenic AGE structures, accumulates in several tissue proteins, including the kidneys of patients with diabetic nephropathy [3] and chronic renal failure [4], atherosclerotic lesions of arterial walls [5], amyloid fibrils in hemodialysisrelated amyloidosis [6], and actinic elastosis of photoaged skin [7]. Methylglyoxal is generated through the Embden-Meyerhof and polyol pathways and reacts with proteins to form Nε-(carboxyethyl)lysine (CEL) [9] and Nδ-(5-hydro-5-methyl-4-imidazolon-2-yl)-ornithine (MG-H1) [10]. Detection of CEL and MG-H1 in biological samples is used as evidence for the modification of proteins by methylglyoxal

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Conclusion