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Title: | 微脂體平台於流感病毒標靶及細菌 Lipid II 翻轉酶之研究 Investigation of Liposomal Platform for Targeting Influenza Virus and Bacterial Lipid II Flippase Evaluation |
Authors: | 王筱雯 Hsiao-Wen Wang |
Advisor: | 梁碧惠 Pi-Hui Liang |
Keyword: | 血球凝集素抑制劑,蛋白質微脂體,MurJ,Lipid-II,螢光共振能量轉移分析, Hemagglutinin inhibitor,proteoliposome,MurJ,Lipid-II,FRET analysis, |
Publication Year : | 2018 |
Degree: | 博士 |
Abstract: | 微脂體能形成人造膜的系統,用於研究蛋白質功能和藥物輸送,其組成取決於於膜的物化性質、組成成分的特性、尺寸、以及表面電荷。因此,我們利用微脂體平台來研究生物學行為和藥物輸送。
病毒與宿主細胞之間的特異性辨識是取決於兩種膜結合的糖蛋白,血凝素 (HA) 和神經氨酸酶 (NA)。 Zanamivir 和 Oseltamivir 為流感病毒的神經氨酸酶抑制劑,已被廣泛使用,目前少有關於 HA 抑制劑的研究。本論文,我們合成了含唾液酸之參醣及伍醣,也將其共價鍵結於DLPE (1,2-dilauroyl-sn-glycero-3-phosphoethanol-amine) 脂質上。這些醣脂與 DPPC (1,2-dihexadecanoyl-sn-glycero-3-phosphocholine) 和膽固醇在適當的比例混合後形成含有唾液醣之微脂體。高密度之唾液參醣在抑制 A / WSN / 33 / H1N1 流感病毒進入細胞之活性為 EC50 70-180 μM 。低密度之含唾液伍醣之微脂體則具有弱的抑制流感病毒進入人類細胞的能力,但其抑制紅血球凝集則更為增強。 由於多重抗藥性細菌的問題日益嚴重,所以發展新型抗生素是至關重要的。肽聚醣的合成過程皆為具有潛力的抗生素發展標的。在肽聚醣生合成中, MurJEC 在革蘭氏陰性菌中是保守存在的翻轉酶,其造成 Lipid II 穿過細胞質膜後提供 disaccharide-pentapeptide 單體並且產生 polymerization 和 cross-linking 而形成肽聚醣。至今僅有體內實驗證實 MurJ 能翻轉 E.coli 之 Lipid II,尚未有 MurJEC 體外重組系統的相關證據。為了模擬 Lipid II 和 MurJEC 之間的生物學相關的相互作用,我們發展一種 ”外到內” 之蛋白微脂體模型將 MurJEC 重組於單層巨型微脂體 (GUV) 中,並且證明 MurJEC 可辨識並翻轉 NBD-Lipid II(7) 。從我們的人造膜系統中,證實 GlcNAc-MurNAc 雙醣和 pyrophosphoryl-undecaprenol 會影響 MurJ 翻轉的辨識能力。 MurJ 疏水區的突變會影響運輸活性。在本研究中,我們利用 FRET-based liposome 作為能即時監測蛋白微脂體的螢光淬熄的工具。 Ag@CeO2 作為淬熄劑包覆在微脂體內部,此方式能簡化並且改進的蛋白微脂體的測定,獲得 MurJEC 翻轉 NBD-Lipid II(7) 的翻轉平衡常數 Km 為 6.7 nM,平均每個 MurJEC 蛋白每分鐘翻轉 0.1 個 NBD-Lipid II(7)。 Liposomes provide an artificial membrane system to study protein function and drug-delivery. Development of liposomal platform needs to understand and investigate the physicochemical properties of membrane and the nature of liposomal component, as well as to fine-tune the particle size and surface change. In this thesis, two applications were conducted, 1) targeting influenza virus by sialic acid decoyed liposome, 2) development of proteoliposome to study bacterial flippase activity of Lipid II. The specific recognition between influenza virus particles and the host cells depended on two membrane-bound glycoproteins, named hemagglutinin (HA) and neuraminidase (NA). Zanamivir and oseltamivir have been demonstrated to be potent neuraminidase inhibitors against influenza virus. Until now, very few studies have gained achievemet on HA inhibiton. Herein, both S-sialyl bearing trisaccharides and pentasaccharides were synthesised and conjugated with phospholipid-DLPE (1,2-dilauroyl-sn-glycero-3-phosphorylethanolamine). These DLPE containg oligosaccharides combined with DPPC (1,2-dihexadecanoyl-sn-glycero-3-phosphocholine) and cholesterol with appropriate molar ratio, providing sialyl-contaning liposome to target HA of influenza virus. High density of S-Neu5Ac-α2-6-LacNAc containing liposome was shown to have an EC50 of around 70-180 μM in the A / WSN / 33 / H1N1 virus entry inhibitory assay. Low density of S-Neu5Ac-α2-6-di-LacNAc containing liposome displayed weak inhibiton in virus entry inhibition, but had stronger inhibition in the hemagglutination inhibition assay. As the problem of multi-drug resistant bacteria deteriorated, the development of novel classes of antibiotics is important. Peptidoglycan synthesis pathway is an potential target for antibiotics. In peptidoglycan biogenesis, the flippase translocates Lipid II across the cytoplasmic membrane to deliver the disaccharide-pentapeptide monomer as the building block for polymerization and cross-linking. In vivo systems have demostrated MurJ as a flippase of Lipid II in the Gram(-) E.coli. None in vitro reconstitution system of MurJ has been reported. In order to investigate the interaction of Lipid II and MurJ, we developed an “out to in” proteoliposomal model in giant unilamellar vesicles (GUVs) and demonstrated that MurJ was able to recognize and transport NBD-Lipid II(7) efficiently. The substrate specificity study of NBD-Lipid II(7) towared MurJ identified that the structure of the GlcNAc–MurNAc and pyrophosphoryl-undecaprenol were required for the transport activity of the MurJ. Mutation of the hydrophobic groove of MurJ reduced the transporting activity of MurJ. Furthermore, we also developed a FRET-based liposome to monitore the transport kinetic of NBD-Lipid II(7) and MurJ. Encapsulated Ag@CeO2 as a quencher in the liposome, it allowed us to real-time monitor the translocation of NBD-Lipid II(7) by MurJ. The Km of NBD-Lipid II(7) to MurJ was 6.7 nM and maximal translocation rate was 0.1 Lipid II/min/MurJ. |
URI: | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/69976 |
DOI: | 10.6342/NTU201800029 |
Fulltext Rights: | 未授權 |
Appears in Collections: | 藥學系 |
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