肝素寡糖与β-淀粉样蛋白相互作用的核磁共振研究

发布时间：2018-04-15 19:12

本文选题：肝素寡糖 + β-淀粉样蛋白　；参考：《山东大学》2017年硕士论文

【摘要】：阿尔兹海默症(Alzheimer's disease,AD)是一种进行性发展的神经功能障碍性疾病,老年人是其主要的发病人群。随着人口老龄化情况加剧,阿尔兹海默症已经逐渐成为严重危害老年人健康的常见病。β-淀粉样蛋白(β-amyloid peptide,Aβ)是AD病理性标志淀粉样斑块的重要组成部分,tau蛋白是神经元纤维缠结(neurofibrillarytangles,NFTs)的主要组成部分,而淀粉样斑块和NFTs是神经病理学诊断AD的标准。但是,NFTs在很多神经退行性疾病中观察到,因此,β-淀粉样蛋白成为研究AD的主要物质。人类终身产生Aβ,它是一种正常的生理代谢产物,其不正常的病理变化是引发AD的重要原因。研究发现,糖胺聚糖(glycosaminoglycan,GAG)介导了Aβ的聚集并导致神经毒性的发生,硫酸化的GAG能够有效促进Aβ的纤维化聚集,并且稳定已形成的老年斑,但是未硫酸化的透明质酸(hyaluronic acid,HA)不能够促进Aβ聚集。而肝素寡糖与Aβ结合可以有效的抑制Aβ的聚集并减少其神经毒性。肝素寡糖不具有毒性且能够通过血脑屏障(blood brain barrier,BBB)直接作用于Aβ。肝素寡糖混合物C3是一种低分子肝素(low molecular weight heparin,LMWH)衍生物,它是一种Aβ聚集抑制剂,已在临床治疗中应用。然而该药物是一种混合物,由于肝素可以与近百种蛋白质相互作用并发挥不同的生理活性,混合物可能存在潜在的副作用,因此,需进一步明确研究肝素寡糖与Aβ之间相互作用的构效关系。本课题旨在研究不同硫酸化程度及硫酸化位点的肝素寡糖与Aβ之间的相互作用的差异性,从分子水平上研究明确结构的寡糖与Aβ40结合的构效关系。运用质谱(mass spectrum,MS)和核磁共振波谱(nuclear magnetic resonance,NMR)分析方法分别对制备的三种肝素四糖dp4-1、dp4-3、dp4-4和市售的磺达肝癸钠(肝素五糖片段)进行了结构表征,确定了三种肝素四糖结构分别为:ΔUA2S(1 →4)GlcNS6S(1 →4)IdoA2S(1 →4)GlcNS,ΔUA2S(1 →4)GlcNS6S(1 →4)GlcA(1→4)GlcNS6S,ΔUA2S(1→4)GlcNS6S(1→4)IdoA2S(1→4)GlcNS6S.五糖的结构为GlcNS6S(1 →4)GlcA(1→4)GlcNS3S6S(1→4)IdoA2S(1→4)GlcNS6SOMe。利用凝胶迁移速率分析(gel mobility shift assay,GMSA)优化寡糖与Aβ相互作用的最适条件,根据实验结果分析肝素四糖与Aβ相互作用强弱顺序为:dp4-4dp4-1dp4-3。利用NMR分析方法研究寡糖与Aβ结合的明确位点,结果表明肝素寡糖结构中IdoA2S为必要的结合位点,而GlcNS6S中的6S对两者的相互作用起到增强的作用,而磺达肝癸钠中具有特异性抗凝活性的3S结构并未参与两者相互作用。
[Abstract]:Alzheimer's disease (AD) is a progressive neurologic disorder in which the elderly are the main ones.As the population ages,Alzheimer's disease has gradually become a common disease that seriously endangers the health of the elderly. 尾 -amyloid peptide A 尾 is an important component of amyloid plaques in AD. Tau protein is the main component of neurofibrillary tanglesn (NFTs).Amyloid plaques and NFTs are the criteria for neurologic diagnosis of AD.However, NFTs have been observed in many neurodegenerative diseases. Therefore, 尾-amyloid protein has become the main substance in the study of AD.Human life produces A 尾, which is a normal physiological metabolite, and its abnormal pathological changes are the important causes of AD.It has been found that glycosaminoglycan (Gage) mediates the aggregation of A 尾 and leads to neurotoxicity. Sulfated GAG can effectively promote the accumulation of A 尾 fibrosis and stabilize the formation of senile plaques.But hyaluronic acid (HA), which was not sulfated, did not promote A 尾 aggregation.The binding of heparin oligosaccharide with A 尾 can effectively inhibit the aggregation of A 尾 and reduce its neurotoxicity.Heparin oligosaccharide is not toxic and can directly act on A 尾 through blood-brain barrier blood brain barrier.Heparin oligosaccharide mixture C3 is a low-molecular-weight heparin molecular weight weight LMWH.It is an A 尾 aggregation inhibitor and has been used in clinical treatment.However, the drug is a mixture, and because heparin can interact with nearly 100 proteins and perform different physiological activities, the mixture may have potential side effects, so,The structure-activity relationship between heparin oligosaccharide and A 尾 should be further studied.The purpose of this study was to study the difference of the interaction between heparin oligosaccharides and A 尾 at different sulfation levels and sites, and to study the structure-activity relationship between A 尾 40 and heparin oligosaccharides at molecular level.The structures of three kinds of heparin tetrasugar, dp4-1, dp4-3, dp4-4 and sulfodecyl sodium (heparin pentaccharide fragments), were characterized by mass mass spectrometry and nuclear magnetic resonance NMRs, respectively.Three types of heparin tetracosaccharide structures were identified as 螖 UA2S(1 4)GlcNS6S(1 4)IdoA2S(1 4GlcNS4GNC, 螖 UA2S(1 4)GlcNS6S(1 4)GlcNS6S(1 4)GlcA(1 4GlcNS6S, 螖 UA2S(1 4)GlcNS6S(1 4)IdoA2S(1 4GlcNS6S.The structure of pentaccharide is GlcNS6S(1 4)GlcA(1 4)GlcNS3S6S(1 4)IdoA2S(1 4 GlcNS6SOMe.The optimum conditions for the interaction of oligosaccharides with A 尾 were optimized by gel migration rate analysis. The order of the interaction between heparin tetrasugar and A 尾 was:: dp4-4dp4-1dp4-3.NMR analysis was used to study the specific site of oligosaccharide binding to A 尾. The results showed that IdoA2S was the necessary binding site in the structure of heparin oligosaccharide, while 6s in GlcNS6S enhanced the interaction between the two.However, the 3s structure with specific anticoagulant activity was not involved in the interaction.
【学位授予单位】：山东大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R749.16;O657.2

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