绵蜇源ACE C-结构域选择性抑制肽的筛选及抑制机理研究

The high moisture of jellyfish brings certain difficulties to its large-scale processing and storage. Processing modes at present cause the loss of a large number of active components. Our recent research indicated that angiotensin converting enzyme (ACE) inhibitory peptides could be produced during the hydrolysis of jellyfish. ACE contains two active areas, the N- and C-domain, which are both catalytically active. For human ACE, the two domains are about 60% homologous in amino acid sequences. Even so, there are differences in performance and thermostability of the C- and N-domain. The C-domain, which is the predominant site of angiotensin I cleavage to angiotensin II in vivo, is supposed to play a major role in blood pressure regulation. It has been postulated that application of the C-domain selective inhibitor could reduce blood pressure and lower the side effects, which may be attributable to the uneffected activity of the N-domain. Selective inhibitors of ACE C-domain should be valuable in regulation of blood pressure. In the present study, we aim at screening of the selective inhibitory peptides from jellyfish (Rhopilema esculentum Kishinouye) hydrolysates and determine their amino acid sequence. Using different methods and techniques such as the isothermal titration calorimetry, fluorescence spectra, circular dichroism spectra, atomic force microscope etc., the binding constant, the interaction force type of ACE C-domain and inhibitor can be calculated and the conformation the C-domain can be observed. The activity of N-domain after treatment of C-domain selective inhibitory peptides can be investigated. The performance of blood pressure regulation in vivo, and the expression of eNOS, PPAR-γ,ET-1 and p-eNOS would be investigated and from which, we can speculate the blood pressure regulation mechanism combined with previous conformation and interaction information.

绵蜇极高的含水量给大规模加工、贮藏带来一定的困难，目前的加工方式易造成活性成分流失。本课题组前期研究发现，绵蜇蛋白酶解可产生血管紧张素转化酶(ACE)抑制肽。文献报道ACE由两个功能不同的结构域组成，其中C-结构域在血压调控方面起着重要的作用，如何探明绵蜇源抑制肽与ACE之间的作用机理并有针对性地抑制C-结构域活性是目前亟待解决的问题。本项目将综合运用等温滴定量热法、圆二色谱法、原子力显微镜技术和分子动力学模拟等手段，研究绵蜇源抑制肽对ACE C-结构域构象变化的影响，明确其结合位点及结构域之间的相互作用模式。通过动物实验验证抑制肽的降血压活性并测定大鼠肺中内皮型一氧化氮合酶(eNOS)、过氧化物酶体增殖物激活受体-γ(PPAR-γ)、内皮素-1(ET-1)和磷酸化eNOS表达量变化，推测其降压作用机制，最终阐明绵蜇ACE C-结构域选择性抑制肽的作用机理，为高效利用绵蜇蛋白提供科学依据。

本项目筛选出具有血管紧张素转化酶(ACE) C结构域选择性抑制作用的肽段，并进行了相关构效关系和抗氧化活性研究，通过光谱学和分子模拟技术初步探索了ACE与其抑制肽之间的相互作用机制。主要进行了以下工作：在最优的酶解条件下，从绵蜇和皮氏蛾螺蛋白酶解产物中提取分离出三个活性肽段，通过HPLC-Q Tof解析其氨基酸序列分别为Ser-Tyr (SY)、Ile-Val-Thr-Asn-Trp-Asp-Asp-Met-Glu-Lys (IVTNWDDMEK)和Val-Gly-Pro-Ala-Gly-Arg-Pro-Gly (VGPAGPRG)。通过特异性荧光底物法测定所分离肽段对ACE两个活性结构域的选择性抑制率；以SY为基础，选择C端氨基酸为酪氨酸，N端分别为脂肪族(丙氨酸Ala 、亮氨酸Leu 、缬氨酸Val 、异亮氨酸Ile )、酸性(谷氨酸Glu)、碱性(精氨酸Arg)、芳香族氨基酸(苯丙氨酸Phe)的二肽进行构效关系研究，考察了氨基酸残基种类对抑制肽选择性抑制作用的影响，发现N端为脂肪族类的氨基酸则大多数表现出对ACE的C-结构域的抑制显著高于对N-结构域的抑制活性。通过红外光谱、紫外光谱和圆二色谱分析ACE在几个肽段作用后的构象变化，发现所研究肽段对ACE的构象均有影响。利用分子模拟软件进行分子对接模拟，推测ACE的活性结构域与选择性抑制肽的结合模式，发现所研究肽段中，IY、LY对ACE C-结构域有更高的亲和力，理论研究结果与体外活性实验结果相一致。另外，本项目还对ACE结构域选择性抑制肽IVTNWDDMEK和VGPAGPRG的抗氧化性及对人脐静脉血管内皮细胞保护作用进行了研究，发现两种肽均可保护人脐静脉内皮细胞免受H2O2 诱导的细胞损伤，其中高剂量(800ug/mL)的IVTNWDDMEK的保护作用最强。