Mass Spectrometric Characterization of Modifications to Angiotensin II by Lipid Peroxidation Products, 4-Oxo-2(E)-nonenal and 4-Hydroxy-2(E)-nonenal
Mass Spectrometric Characterization of Modifications to Angiotensin II by Lipid Peroxidation Products, 4-Oxo-2(E)-nonenal and 4-Hydroxy-2(E)-nonenal
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DOI：
10.1021/tx100228q
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发表时间：
2010-11-01
影响因子：
4.1
通讯作者：
Oe, Tomoyuki
中科院分区：
文献类型：
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作者：
Lee, Seon Hwa;Takahashi, Ryo;Oe, Tomoyuki
八肽血管紧张素 II (Ang Asp(1)-Arg(2)-Val(3)-Tyr(4)-Ile(5)-His(6)-Pro(7)-Phe(8)) 是肾素/血管紧张素系统 (RAS) 的主要活性激素，与多种心血管疾病有关。大量结构活性关系研究已确定 Ang II 的 Asp(1)、Arg(2) 和 His(6) 对于其生物活性和受体结合至关重要。从Ang II与脂质过氧化衍生的醛类4-oxo-2(E)-nonenal (ONE)或4-羟基-2(E)-nonenal (HNE)的反应中，我们通过液相色谱/质谱(LC/MS)和基质辅助激光解吸鉴定了Ang II的N末端、Asp(1)、Arg(2)和His(6)的主要修饰电离飞行时间/MS (MALDI-TOF/MS)。在与硼氢化钠反应之前和之后，通过串联质谱 (MS/MS) 和源后衰变 (PSD)-TOF/MS 确认了 ONE 和 HNE 修饰的 Ang II 的身份。在与 ONE 的反应中，通过 N 端 Asp 氧化脱羧形成的丙酮酰胺-Ang If 在孵育 48 小时后被检测为最丰富的产物。随后是 Arg 修饰的 [Arg(2)(ONE H(2)O)]-Ang II 和 [N-ONE]-Ang II 的 N 末端修饰的 4-酮酰胺形式。 [His(6)(HNE)]-Ang II 的迈克尔加成产物是与 HNE 反应开始时最丰富的产物，其次是[His(6)(HNE H(2)O)]Ang II 的脱水迈克尔加成产物。 [His(6)(HNE)]-Ang II 在长时间孵育过程中脱水为[His(6)(HNE H(2)O)]-Ang H，7天后[His(6)(HNE H(2)O)]-Ang II成为主要产物。进行了N(α)-叔丁氧羰基(tBoc)-Arg与ONE和tBoc-His与HNE的模型反应，并与Ang II反应进行了比较。 tBoc-Arg 很容易与 ONE 反应生成一种类似于 [Arg(2)(ONE H(2)O)]Ang II 的化合物，这证实了 Arg 是 ONE 的重要目标亲核试剂之一。然而，tBoc-His 在与 HNE 反应时仅形成迈克尔加成产物。 [His(6)(HNE H(2)O)]-Ang II 的意外形成可以通过 Ang II 的特定构象中 His(6) 与 C 端羧酸盐的接近来解释，这有助于迈克尔加成产物的脱水。因此，我们的结果表明，模型氨基酸和内源生物活性肽的 ONE 和 HNE 加合化学可能存在差异，这是由肽的微环境（例如特定的氨基酸序列和构象）控制的。这种稳定的 ONE 和 HNE 衍生的 Ang H 修饰可能通过破坏与 Ang H 1 型 (AT(1)) 受体的相互作用和/或抑制氨肽酶 A (APA) 的酶活性来潜在地调节其体内功能，氨肽酶 A (APA) 会裂解 Ang II 的 N 端天冬氨酸残基以产生 Ang III。
The octapeptide angiotensin II (Ang Asp(1)-Arg(2)-Val(3)-Tyr(4)-Ile(5)-His(6)-Pro(7)-Phe(8)) is the primary active hormone of the renin/angiotensin system (RAS) and has been implicated in various cardiovascular diseases. Numerous structure activity relationship studies have identified Asp(1), Arg(2), and His(6) of Ang II to be critical for its biological activity and receptor binding. From the reactions of Ang II with lipid peroxidation-derived aldehydes, 4-oxo-2(E)-nonenal (ONE) or 4-hydroxy-2(E)-nonenal (HNE), we have identified the major modifications to the N-terminus, Asp(1), Arg(2), and His(6) of Ang II by liquid chromatography/mass spectrometry (LC/MS) and matrix-assisted laser desorption ionization-time-of-flight/MS (MALDI-TOF/MS). The identities of ONE- and HNE-modified Ang II were confirmed by tandem mass spectrometry (MS/MS) and postsource decay (PSD)-TOF/MS before and after the reaction with sodium borohydride. In the reaction with ONE, a pyruvamide-Ang If that formed via oxidative decarboxylation of N-terminal Asp was detected as the most abundant product after 48 h of incubation. It was followed by Arg-modified [Arg(2)(ONE H(2)O)]-Ang II and the N-terminal-modified 4-ketoamide form of [N-ONE]-Ang II. The Michael addition products of [His(6)(HNE)]-Ang II were the most abundant products in the beginning of the reaction with HNE, followed by the dehydrated Michael addition products of [His(6)(HNE H(2)O)]Ang II. [His(6)(HNE)]-Ang II was dehydrated to [His(6)(HNE H(2)O)]-Ang H during the prolonged incubation, and [His(6)(HNE H(2)O)]-Ang II became the major products after 7 days. The model reactions of N(alpha)-tert-butoxycarbonyl (tBoc)-Arg with ONE and tBoc-His with HNE were performed and compared with the Ang II reaction. tBoc-Arg readily reacted with ONE to produce a compound analogous to [Arg(2)(ONE H(2)O)]Ang II, which confirmed Arg as one of the important target nucleophiles of ONE. However, tBoc-His exclusively formed a Michael addition product upon the reaction with HNE. The unexpected formation of [His(6)(HNE H(2)O)]-Ang II can be explained by the proximity of His(6) to C-terminal carboxylate in the specific conformation of Ang II, which facilitates the dehydration of Michael addition products. Therefore, our results suggest a possible discrepancy in the adduction chemistry of ONE and HNE for model amino acids and endogenous bioactive peptides, which is governed by the microenvironment of peptides, such as the specific amino acid sequence and conformation. Such stable ONE- and HNE-derived modifications to Ang H could potentially modulate its functions in vivo by disrupting the interaction with Ang H type 1 (AT(1)) receptor and/or inhibiting the enzyme activity of aminopeptidase A (APA), which cleaves the N-terminal Asp residue of Ang II to generate Ang III.