探討糖奈米粒子於抑制β-乳球蛋白類澱粉纖維形成之影響

Examining the Effects of Sugar-Terminated Nanoparticles on Amyloid Fibril Formation of β-Lactoglobulin

林千鈺(Chien-Yu Lin)

指導教授 ： 王勝仕

國立臺灣大學/工學院/化學工程學研究所/碩士(2019年)

https://doi.org/10.6342/NTU201901481

摘要

目前有許多疾病與蛋白質/肽錯誤折疊所形成之類澱粉纖維聚集有關，例如：阿茲海默症、帕金森氏症及亨廷頓氏症等。然而，許多文獻指出於蛋白質溶液中添加小分子或是奈米粒子將有助於修復蛋白質之錯誤折疊，甚至抑制纖維狀聚集成核以及成長。本研究使用β-乳球蛋白作為研究目標並於高溫且酸性條件之下培養成類澱粉纖維，雖然此類蛋白質本身並非為致病蛋白質，但透過實驗了解其所形成類澱粉纖維之行為以及特性，仍可對於抑制劑如何影響蛋白質纖維化之機制有更深入的了解，也可以應用於設計其他分子抑制劑。然而，抑制劑之選擇分別為蔗糖及果糖所形成之糖奈米粒子(sugar terminated nanoparticle)。
本研究結果顯示，無論是蔗糖或是果糖奈米粒子皆能明顯地降低及延緩β-乳球蛋白形成類澱粉纖維之程度。另外，亦發現於此滲透物奈米粒子之存在下，能有效地穩定β-乳球蛋白之二級結構；且蔗糖分子與果糖分子之對照組別卻不具此性質，甚至造成β-乳球蛋白結構損失更為嚴重。而欲了解所使用之糖奈米粒子影響此蛋白質三級結構之變化，則以ANS螢光分析方法檢測，結果發現此兩種糖類抑制劑可有效地降低β-乳球蛋白疏水區域裸露開展之程度。亦以散射實驗和電泳檢測所形成之聚集物之大小，發現添加高濃度糖奈米粒子能有效地降低蛋白質聚集程度。
除此，於本研究中試圖以螢光淬滅法分析其之間所形成之交互作用力，並試圖了解於不同溫度之下抑制劑與蛋白質之間的結合常數等參數。於實驗結果發現糖類抑制劑與蛋白質混合後將導致整個系統處於放熱狀態，進而推測氫鍵於蛋白質和奈米粒子之間的交互作用中具有具有相當重要之影響，且果糖奈米粒子組別於三種溫度下之結合常數皆比另一系統來得高，推測果糖奈米粒子與β-乳球蛋白之間的相互作用是較有利的，或β-乳球蛋白對果糖奈米粒子具有較高的親和力。
本實驗所合成之蔗糖及果糖奈米粒子其作為抑制劑之效果皆遠大於其為分子狀態時來得有效，顯示此糖奈米粒子之結構能提供多價鍵結，進而減少β-乳球蛋白類澱粉纖維之形成。我們期望此研究結果能有助於開發一具潛力之抑制劑以針對類澱粉纖維相關疾病。

關鍵字

類澱粉纖維症 ； 滲透物 ； 奈米粒子 ； 抑制劑 ； 糖類 ； β-乳球蛋白 ； 聚集

並列摘要

Currently, accumulation of proteins/peptides has been found to be associated with many diseases, such as Alzheimer's disease, Parkinson's disease, and Huntington's disease. It has been shown that the addition of small molecules or nanoparticles can interfere with amyloid fibril formation. In this study, β-lactoglobulin (β-LG) was used to form amyloid fibrils under high temperature and acidic conditions. Although the protein itself is not pathogenic, understanding the behavior and characteristics of its amyloid aggregation form could provide a better understanding of how inhibitors affect protein fibrillogenesis. Moreover, the knowledge gained here could also aid in designing new inhibitory molecules. Herein, the sugar (sucrose or fructose)-terminated nanoparticles were used as the protein aggregation inhibitors. Our results showed that both sucrose and fructose nanoparticles could significantly retard and/or decrease β-LG fibrillation. In addition, results showed that the secondary structure of β-LG was stabilized in the presence of the sugar-based nanoparticles under 80˚C and pH 2.0 conditions. However, this secondary structural stabilization behavior was not observed when the two sugars in their molecular form were added. Using ANS binding assay, we found that the sugar-terminated nanoparticles effectively reduced the degree of exposure of the β-LG hydrophobic area. Furthermore, as revealed by light scattering experiment and electrophoresis, the size of aggregates was observed to significantly decrease as the sugar nanoparticles at higher concentrations were added.
We also went ahead to analyze the interaction/binding between sugar nanoparticles and β-LG by fluorescence quenching experiments at different temperatures. Several points could be made from the fluorescence quenching results: (1) The reaction between proteins and sugar terminated nanoparticles was determined to be exothermic. (2) Hydrogen bonding may play an important role in the interaction between protein and nanoparticles. (3) The binding constants of the fructose nanoparticle group at three temperatures were all considerably higher than those of the sucrose nanoparticle system, implying that the interaction between fructose nanoparticles and β-LG was favorable or β-LG has a high affinity for fructose nanoparticles.
As revealed by several spectroscopic tools and biophysical methods, the sucrose and fructose-terminated nanoparticles synthesized in this study were found to be more effective in inhibiting β-LG fibril formation, as compared to their molecular form., This result suggested that the sugar nanoparticle structure could provide multivalent bonds, thereby reducing the formation of β-LG amyloid fibrils. While more research is warranted to decipher the underlying mechanism of action of sugar-based nanoparticles against protein fibril formation, we believe the outcome from this work may aid in the development of potential inhibitory agents against the diseases associated with amyloid fibrillogenesis.

並列關鍵字

amyloidosis ； osmolyte ； nanoparticle ； inhibitor ； sugar ； β-lactoglobulin ； aggregation

參考文獻

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