南瓜酸性多糖的结构解析及其与功能蛋白的相互作用
发布时间：2018-01-05 05:37
本文关键词：南瓜酸性多糖的结构解析及其与功能蛋白的相互作用 出处：《中国农业大学》2017年博士论文 论文类型：学位论文
【摘要】：本文以南瓜为原料,分离纯化出两种具有不同结构特征的酸性多糖,分别为水提南瓜酸性多糖WAP和碱提南瓜渣酸性多糖AAP,并对WAP和AAP的结构进行了解析。对WAP进行了硫酸化修饰,以肝素为参照物研究了硫酸化南瓜多糖SWAP与阿尔兹海默症关键蛋白tau的相互作用方式和作用位点。研究了 SWAP与抗凝血相关蛋白抗凝血酶III和肝素辅助因子II的相互作用,探索其抗凝血活性和作用途径。研究了碱提酸性多糖AAP与半乳凝集素-3的相互作用,并在细胞水平对其半乳凝集素-3抑制活性进行了验证。(1)南瓜冻干粉经热水浸提,80%乙醇沉淀,得到水提粗多糖WP,粗多糖WP经离子交换层析和乙醇二次沉淀得到主要酸性组分WAP,分子量为8.32×104Da。单糖组成分析得出WAP以半乳糖醛酸为主,酯化度高,为67.91%。通过核磁共振对WAP的结构进行了鉴定,确定WAP为α-(1-4)-糖苷键连接的聚半乳糖醛酸,原子力显微镜测得WAP在水溶液中呈链状分布,少分支,平均链长为160.8 nm,链高为0.28～1.4nm。(2)不溶性南瓜残渣冻干粉经碱液浸提,80%乙醇沉淀,得到碱提粗多糖AP,粗多糖AP经离子交换层析和乙醇二次沉淀得到主要酸性组分AAP,分子量为2.26×104 Da。单糖组成分析得出AAP为杂多糖,单糖组成为鼠李糖、半乳糖醛酸、半乳糖和阿拉伯糖(7.4:25:28:2.6),酯化度低,仅为5.4%。通过核磁共振对AAP的结构进行鉴定,确定AAP主链为以α-(1-4)-糖苷键连接的半乳糖醛酸和以[→4)-α-D-半乳糖醛酸-(1→2)-α-L-鼠李糖-(1→]方式交替连接的半乳糖醛酸和鼠李糖构成;侧链为β-(1-4)-糖苷键连接的聚半乳糖和少量的阿拉伯糖。(3)对南瓜多糖WAP进行硫酸化修饰,得到硫酸化南瓜多糖SWAP,硫酸根含量为16.7%,取代度1.81,分子量为2.47×104Da。经核磁共振鉴定,推测WAP的硫酸化修饰主要发生在半乳糖醛酸的C-2和C-3位。利用表面等离子共振测得硫酸化南瓜多糖SWAP与tau K18的结合强度Ki=1.15×10-8M,高于肝素(KD=2.03×10-7M)。核磁共振实验发现SWAP与tauK18主要的结合位点与肝素基本吻合,为tau K18的R2结构域,表明SWAP能够很好地仿效肝素与tau K18蛋白结合,体现了 SWAP潜在的抑制tau蛋白转移的活性。(4)利用表面等离子共振技术研究了硫酸化南瓜多糖SWAP与抗凝血酶III和肝素辅助因子II的相互作用,SWAP与抗凝血酶III结合强度Ki=1.71×10-4M,低于肝素,为弱相互作用;与肝素辅助因子II的结合强度Ki=1.39×10-7M,与肝素接近,为强相互作用;表明SWAP主要通过肝素辅助因子II介导发挥对凝血酶的抑制作用。SWAP能够明显地延长活化部分凝血酶时间和凝血酶原时间,对凝血酶时间没有显著的影响,表明SWAP主要通过内源性途径发挥抗凝血作用。碱提酸性多糖AAP能够与半乳凝集素-3结合,结合强度KD为1.26×10-6 M,AAP能在细胞水平作为Gal-3抑制剂阻抑红细胞凝集。
[Abstract]:In this paper, two kinds of acidic polysaccharides with different structural characteristics were isolated and purified from pumpkin. They were water extracted pumpkin acid polysaccharide WAP and alkaline pumpkin residue acid polysaccharide AAP. The structures of WAP and AAP were analyzed and WAP was modified by sulfation. The interaction between sulfated pumpkin polysaccharide (SWAP) and Alzheimer's disease (tau) was studied using heparin as a reference material. Interaction of SWAP with antithrombin III and heparin cofactor II. The anticoagulant activity and pathway were explored. The interaction between alkaline extraction of acidic polysaccharide AAP and galactoagglutinin-3 was studied. The inhibitory activity of galactoagglutinin-3 was verified at the cell level. 1) Pumpkin freeze-dried powder was precipitated by 80% ethanol in hot water to obtain the crude polysaccharide WP. The main acidic component WAP was obtained by ion exchange chromatography and ethanol secondary precipitation. The molecular weight of WP was 8.32 脳 104 Da.The single sugar composition analysis showed that galacturonic acid was the main component of WAP. The esterification degree was 67.91. The structure of WAP was identified by nuclear magnetic resonance (NMR). It was confirmed that WAP was the polygalacturonic acid connected by 伪 -nc-1-4C-glucoside bond. Atomic force microscopy (AFM) showed that WAP distributed in aqueous solution as a chain, with less branching and an average chain length of 160.8 nm. The chain height was 0.28 ~ 1.4nm.m-2) the lyophilized powder of insoluble pumpkin residue was precipitated by 80% ethanol extracted from alkali solution, and the crude polysaccharide AP was obtained by alkali extraction. The main acidic component of AP was obtained by ion exchange chromatography and ethanol secondary precipitation. The molecular weight of AP was 2.26 脳 10 ~ 4 Da.Monosaccharide composition analysis showed that AAP was heteropolysaccharide. The monosaccharide consists of rhamnose, galacturonic acid, galactose and arabinose (7.4: 25: 28: 2.6), and the esterification degree is low, only 5.4%. The structure of AAP was identified by NMR. It is determined that the main chain of AAP is galacturonic acid connected with 伪 -tau1-4-glucoside bond and that the main chain is galacturonic acid with. [鈫，
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