Shamsi F A, Partal A, Sady C, Glomb M A, Nagaraj R H
Center for Vision Research, Department of Ophthalmology, Case Western Reserve University, Cleveland, Ohio 44106, USA.
J Biol Chem. 1998 Mar 20;273(12):6928-36. doi: 10.1074/jbc.273.12.6928.
The Maillard reaction, a non-enzymatic reaction of ketones and aldehydes with amino groups of proteins, contributes to the aging of proteins and to complications associated with diabetes. Methylglyoxal (MG) is a 2-oxoaldehyde derived from glycolytic intermediates and produced during the Maillard reaction. We reported previously the formation of a lysine-lysine protein cross-linking structure (imidazolysine) and a fluorescent arginine modification (argpyrimidine) from the Maillard reaction of MG. Here we show that rabbit antibodies to MG-modified ribonuclease A identify proteins modified by the Maillard reaction of glucose, fructose, ribose, glyceraldehyde, glyoxal, ascorbate, and ascorbate oxidation products (dehydroascorbate, 2,3-diketogulonate, L-xylosone, and L-threose) in addition to those modified by MG. The antibody recognized imidazolysine and argpyrimidine and a glyoxal-derived lysine-lysine cross-link. It did not react with Nepsilon-carboxymethyllysine. Incubations with amino acids revealed strongest reactivity with Nalpha-t-butoxycarbonylarginine and MG, and we identified argpyrimidine as one of the epitopes from this incubation mixture. Serum proteins from human diabetics reacted more strongly with the antibody than those from normal individuals, and the levels correlated with glycemic control. Collagen from human corneas contained MG-derived modifications, with those from older subjects containing higher levels of modified proteins than those from younger ones. An immunoaffinity-purified antibody showed higher reactivity with old corneas than with younger ones and localized the antigens primarily within the stromal region of the cornea. These results confirm reported MG-derived modifications in tissue proteins and show that dicarbonyl-mediated protein modification occurs during Maillard reactions in vivo.
美拉德反应是酮类和醛类与蛋白质氨基之间的非酶促反应，会导致蛋白质老化以及引发与糖尿病相关的并发症。甲基乙二醛（MG）是一种源自糖酵解中间产物并在美拉德反应过程中产生的2-氧代醛。我们之前报道过，MG的美拉德反应会形成赖氨酸-赖氨酸蛋白质交联结构（咪唑赖氨酸）和一种荧光精氨酸修饰物（精氨嘧啶）。在此我们表明，针对MG修饰的核糖核酸酶A的兔抗体，除了能识别被MG修饰的蛋白质外，还能识别被葡萄糖、果糖、核糖、甘油醛、乙二醛、抗坏血酸以及抗坏血酸氧化产物（脱氢抗坏血酸、2,3-二酮古洛糖酸、L-木酮糖和L-苏糖）的美拉德反应修饰的蛋白质。该抗体识别咪唑赖氨酸、精氨嘧啶以及一种源自乙二醛的赖氨酸-赖氨酸交联物。它不与Nε-羧甲基赖氨酸反应。与氨基酸的孵育实验表明，该抗体与Nα-叔丁氧羰基精氨酸和MG的反应性最强，并且我们从这个孵育混合物中鉴定出精氨嘧啶是其中一个表位。来自人类糖尿病患者的血清蛋白与该抗体的反应比正常个体的血清蛋白更强，且其水平与血糖控制相关。来自人类角膜的胶原蛋白含有MG衍生修饰物，老年受试者角膜中的修饰蛋白水平高于年轻受试者。一种免疫亲和纯化抗体对老年角膜的反应性高于年轻角膜，并将抗原主要定位在角膜的基质区域。这些结果证实了在组织蛋白中存在已报道的MG衍生修饰，并表明二羰基介导的蛋白质修饰在体内的美拉德反应过程中发生。
