Nω -(羧甲基)精氨酸是糖化胶原和小鼠组织中主要的高级糖化终产物之一。,Oxidative Medicine and Cellular Longevity

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Nω -(羧甲基)精氨酸是糖化胶原和小鼠组织中主要的高级糖化终产物之一。
Oxidative Medicine and Cellular Longevity Pub Date : 2019-09-10 , DOI: 10.1155/2019/9073451
Sho Kinoshita ₁ , Katsumi Mera ₂ , Hiroko Ichikawa ₁ , Satoko Shimasaki ₃ , Mime Nagai ₁ , Yuki Taga ₄ , Katsumasa Iijima ₄ , Shunji Hattori ₄ , Yukio Fujiwara ₅ , Jun-Ichi Shirakawa ₁ , Ryoji Nagai ₁

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人类衰老过程中，晚期糖基化终末产物 (AGE) 在蛋白质中积累。特别是，AGE 结构N ^ω -(羧甲基)精氨酸 (CMA) 是通过糖化胶原中的氧化产生的，是生物样品中检测到的主要蛋白质之一。在本研究中，我们研究了胶原蛋白中生成 CMA 的机制，并在富含胶原蛋白的组织中检测到了 CMA。当各种蛋白质样品与葡萄糖一起孵育时，使用单克隆抗体检测到的 CMA 含量仅在糖化胶原蛋白中以时间依赖性方式增加，而N ^ε -(羧甲基)赖氨酸 (CML)（一种主要抗原性 AGE）的形成，在所有糖化蛋白中均检测到。还通过电喷雾电离-液相色谱-串联质谱 (LC-MS/MS) 观察到糖化胶原蛋白中主要的 CMA 形成。在葡萄糖与胶原蛋白的孵育过程中，CMA 的形成随着葡萄糖浓度的增加而增强，而在二羰基捕获剂和金属螯合剂的存在下则受到抑制。在将胶原蛋白与乙二醛一起孵育时也观察到了 CMA 的形成，并且当乙二醛与 I-IV 型胶原蛋白一起孵育时，CMA 以时间依赖性方式生成。为了识别 CMA 形成的热点，将糖化胶原蛋白的胰蛋白酶消化物应用于与抗 CMA 缀合的亲和柱。通过 LC-MS/MS 和氨基酸序列分析检测到了对整合素识别很重要的几种 CMA 肽。通过将合成的肽与乙二醛和核糖一起温育来确认每个序列上的 CMA 形成。LC-MS 检测到小鼠皮肤中的 CMA 水平高于其他 AGE。此外，老年人主动脉中的 CMA 积累量更大。总体而言，我们的研究提供了证据表明CMA是具有代表性的AGE结构，可作为反映胶原蛋白氧化和糖化的有用指标。

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

Advanced glycation end products (AGEs) accumulate in proteins during aging in humans. In particular, the AGE structure N^ω-(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 N^ε-(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.

更新日期：2019-09-10

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