A capture method based on the VC1 domain reveals new binding properties of the human receptor for advanced glycation end products (RAGE)
A capture method based on the VC1 domain reveals new binding properties of the human receptor for advanced glycation end products (RAGE)
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DOI：
10.1016/j.redox.2016.12.017
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发表时间：
2017-04-01
期刊：
影响因子：
11.4
通讯作者：
Aldini, Giancarlo
中科院分区：
文献类型：
--
作者：
Degani, Genny;Altomare, Alessandra A.;Aldini, Giancarlo
高级糖化和脂氧化终产物（AGE 和 ALE）是一类异质化合物，源自脂氧化分解产物的非酶糖化或蛋白质加合。 AGE 受体 (RAGE) 参与基于持续炎症状态和氧化应激的慢性疾病的进展。 RAGE 是一种模式识别受体 (PRR)，抑制与其配体的相互作用或抑制配体的积累具有潜在的治疗作用。 RAGE 的 N 端结构域（V 结构域）是 AGE 结合的主要位点，并由相邻的 C1 结构域稳定。在这项研究中，我们建立了一种亲和力测定，依赖于 AGE 配体与 VC1 结构域的极其特异性的生物相互作用。将毕赤酵母中产生的 VC1 糖基化形式附着在磁珠上，用作不溶性亲和基质（VC1-树脂）。采用 VC1 相互作用测定从体外产生的 AGE-白蛋白中分离出特定的 VC1 结合伴侣，并通过质谱分析鉴定/定位修饰。有趣的是，这种方法还可以分离出丙二醛处理白蛋白产生的 ALE。计算研究为 V 结构域的特定修饰残基和氨基酸建立的接触提供了基于理性的解释。 VC1-树脂从血浆和牛奶等复杂生物混合物中捕获 AGE-白蛋白的验证可能会导致鉴定出可能参与促炎和促纤维化反应的新 RAGE 配体，与其结构或物理特性无关，并且无需使用任何共价衍生化过程。此外，该方法还可用于鉴定RAGE-配体相互作用的拮抗剂。
The Advanced Glycation and Lipoxidation End products (AGEs and ALEs) are a heterogeneous class of compounds derived from the non-enzymatic glycation or protein adduction by lipoxidation break-down products. The receptor for AGEs (RAGE) is involved in the progression of chronic diseases based on persistent inflammatory state and oxidative stress. RAGE is a pattern recognition receptor (PRR) and the inhibition of the interaction with its ligands or of the ligand accumulation have a potential therapeutic effect. The N-terminal domain of RAGE, the V domain, is the major site of AGEs binding and is stabilized by the adjacent C1 domain. In this study, we set up an affinity assay relying on the extremely specific biological interaction AGEs ligands have for the VC1 domain. A glycosylated form of VC1, produced in the yeast Pichia pastoris, was attached to magnetic beads and used as insoluble affinity matrix (VC1-resin). The VC1 interaction assay was employed to isolate specific VC1 binding partners from in vitro generated AGE-albumins and modifications were identified/ localized by mass spectrometry analysis. Interestingly, this method also led to the isolation of ALEs produced by malondialdehyde treatment of albumins. Computational studies provided a rational-based interpretation of the contacts established by specific modified residues and amino acids of the V domain. The validation of VC1-resin in capturing AGE-albumins from complex biological mixtures such as plasma and milk, may lead to the identification of new RAGE ligands potentially involved in pro-inflammatory and pro-fibrotic responses, independently of their structures or physical properties, and without the use of any covalent derivatization process. In addition, the method can be applied to the identification of antagonists of RAGE-ligand interaction.