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標題: | 登革病毒外套膜蛋白醣基修飾及其與凝集素結合之探討 Identification of Glycan Structures on Dengue Virus Envelope Protein and Its Interaction with Lectin Receptors |
作者: | Jia-Huei Lyu 呂家慧 |
指導教授: | 翁啟惠(Chi-Huey Wong) |
關鍵字: | 登革病毒,外套膜蛋白,凝集素,醣基化,醣蛋白, Dengue Virus,Dengue E protein,Glycoforms,Lectin,glycan, |
出版年 : | 2011 |
學位: | 碩士 |
摘要: | 登革熱是一種以蚊子〈埃及斑蚊、白線斑蚊〉為傳播媒介的傳染性疾病,在台灣等熱帶及亞熱帶地區相當盛行,其中少數患者會引發致命的登革出血熱和登革休克症候群。登革病毒的表面是由兩種醣蛋白所組成,分別是外套膜蛋白(E)及膜蛋白(M),在其他黃病毒的外套膜蛋白中,只有在胺基酸153或154的位置上被醣基化,但在登革病毒中,除了在153號的位置外,在67位置也被修飾上醣基,由於登革病毒上的外套膜蛋白與其進行宿主細胞的感染很有關係,因此,外套膜蛋白上的醣基也是在感染過程中,被視為一個讓宿主細胞辨識的重要因子。
先前研究發現登革病毒會感染樹突細胞,而此細胞上的凝集素DC-SIGN可以直接跟病毒上的醣作結合,因此我們認為,探討登革病毒上醣的結構是很重要的。我們表現了兩種醣基化程度不同的登革病毒外套膜蛋白,分別是表現在細胞內的外套膜蛋白及被分泌出細胞外的外套膜蛋白,除此之外,我們利用蚊子細胞〈C6/36〉及人類白血病細胞〈K562〉來得到登革病毒,然後使用質譜儀分析病毒外套膜蛋白上醣的結構。結果顯示,表現在細胞內的病毒外套膜蛋白上的醣基為高甘露醣態,而分泌型的病毒外套膜蛋白上的醣基則為複合型。在病毒粒子方面,蚊子細胞所表現的病毒外套膜蛋白上的醣基為寡甘露醣態,而在人類白血病細胞所表現的病毒外套膜蛋白上的醣基為複合型,而在被登革病毒感染的細胞中所得到的外套膜蛋白,其上所修飾的醣基為高甘露醣態。我們發現外套膜蛋白在不同細胞所得到醣基化程度是不同的,而這些醣化差異可能影響其與宿主細胞受體結合的能力。為了更了解其受體與登革熱病毒上外套膜蛋白的結合,我們使用四十三種凝集素來篩選,發現其中有一些凝集素能與外套膜蛋白專一性地結合。這些新發現可能對於治療登革熱的藥物及疫苗的開發有很大的幫助。 Dengue has been regarded as one of the most important mosquito-borne emerging infectious diseases especially in tropical and subtropical regions. Dengue infection may cause dengue fever (DF) or develop a life-threatening illness, dengue hemorrhagic fever (DHF)/dengue shock syndrome (DSS). Dengue virus (DENV) particle contains two surface proteins, the envelope (E) and membrane (M) proteins. The E protein of most flaviviruses is modified by N-linked glycosylation at residue 153/154. In the case of DENV, comparing to other flaviviruses there is an additional N-linked glycosylation site at residue 67. Because DENV E protein is responsible for viral entry and attachment, the glycans on viral particle can be important for infection and recognition. It is reported that DENV infection of dendritic cells (DCs) is mediated by a glycan-dependent binding of DENV to DC-SIGN (DC-specific ICAM3-grabbing non-intergrin). Therefore, it is interesting to identify the glycan structures on DENV. To do this, we constructed plasmids to overexpress the full-length DENV2 (serotype 2) E protein in mammalian cells. Our results showed that there were two different glycosylated E proteins produced: full-length and secretory E proteins. We also isolated the viral particles and E proteins from both mosquito intestinal (C6/36) and human leukemia cells (K562), and used LC-MS/MS to determine the glycan structures on E protein. In protein overexpression system, we found that the N-glycans on the full-length E protein were high-mannose type, while secretory E protein bore complex type N-glycans. The N-glycans of E protein on C6/36 cell-derived DENV2 particles and in DENV2-infected C6/36 cell were mainly paucimannose and high mannose type, respectively. DENV2-infected K562 cell were mainly complex type glycan. Detailed studies with E protein and viral particles show that the glycans are distinct in different cell types. These differences may account for the binding specificity to the receptors. To explore the immune receptors that can recognize DENV, we used 43 immune receptors to screen potential receptors. The lectin binding assay showed that BIMLC, DC-SIGN, DC-SIGNR, Dectin-2, Langerin, L-Ficollin, mKCR, and NKP44 could specifically bind to DENV E protein. These findings may have implications on drug design and vaccine development to fight DENV infection. |
URI: | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/24642 |
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