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| DC 欄位 | 值 | 語言 |
|---|---|---|
| dc.contributor.advisor | 徐尚德(Shang-Te Hsu) | |
| dc.contributor.author | Ning-En Chang | en |
| dc.contributor.author | 張寧恩 | zh_TW |
| dc.date.accessioned | 2023-03-19T23:50:00Z | - |
| dc.date.copyright | 2022-08-31 | |
| dc.date.issued | 2022 | |
| dc.date.submitted | 2022-08-25 | |
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Glycan clustering stabilizes the mannose patch of HIV-1 and preserves vulnerability to broadly neutralizing antibodies. Nat Commun 6, 7479, doi:10.1038/ncomms8479 (2015). Watanabe, Y., Allen, J. D., Wrapp, D., McLellan, J. S. & Crispin, M. Site-specific glycan analysis of the SARS-CoV-2 spike. Science 369, 330-333, doi:doi:10.1126/science.abb9983 (2020). | |
| dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/86339 | - |
| dc.description.abstract | 人類冠狀病毒屬於網巢病毒目，其可藉由動物傳播給人類，為一種人畜共通的病原體。冠狀病毒主要通過病毒細胞膜上高度醣基化的棘蛋白與宿主細胞上的受體結合，促進病毒與宿主細胞膜的融合，達到病毒入侵的目的。醣基化修飾是一種蛋白質的轉譯後修飾，在棘蛋白的建構中扮演非常重要的角色。因此，了解醣基化棘蛋白的結構與分子動態對受體免疫辨識的影響將有助於後續的預防以及抗病毒治療的發展。在本研究中，我們主要討論屬於乙型冠狀病毒屬的三種人類冠狀病毒，包括嚴重急性呼吸道症候群冠狀病毒(SARS-CoV)、中東呼吸症候群冠狀病毒(MERS-CoV)以及人類冠狀病毒HKU1(hCoV-HKU1)。我們通過哺乳動物細胞蛋白質表達系統製備重組棘蛋白，並藉由差示掃描量熱法(DSC)以及差示掃描螢光法(DSF)分析在不同物化條件下的蛋白質穩定性，接著利用冷凍電子顯微鏡(cryo-EM)解析出受體結合區域在中東呼吸症候群冠狀病毒棘蛋白中的不同構型。為了更進一步瞭解蛋白質表面N-醣基化修飾的化學組成和結構，我們使用質譜儀鑑定在特定醣化位點上的不同醣型，並在GlycoSHIELD的幫助下建立棘蛋白的醣化模型，定量描述N-醣基化修飾對於棘蛋白表面所形成的遮蔽效應，結果表明棘蛋白表面的局部抗原性與醣基化修飾所形成的遮蔽效應間呈現負相關。綜上所述，我們的研究建立一個完整的流程探討醣基化修飾在棘蛋白表面的影響，並且具有預測潛在受體與輔助受體結合區以及抗原決定位的能力。 | zh_TW |
| dc.description.abstract | Human coronaviruses (hCoVs) belong to the order Nidovirales, which are zoonotic pathogens that evolve to infect humans. CoVs use highly glycosylated spike proteins to bind to host receptor(s), the first step towards host membrane fusion and viral entries. As the post-translationally modified glycans constitute a significant part of the overall structures of the spike proteins, understanding their structures and dynamics in the context of receptor and immunity recognition is essential for developing preventions and antivral treatments. Here, we focused on three hCoV spike proteins in the genus Betacoronavirus, including SARS-CoV, MERS-CoV and hCoV-HKU1. We produced the recombinant spike proteins by a mammalian protein expression system. The folding stabilities over a range of experimental conditions were evaluated by differential scanning calorimeter (DSC) and differential scanning fluorimeter (DSF). We also employed single particle analysis by cryo-electron microscopy (cryo-EM) to determine four distinct structures of the MESR-CoV spike protein that differed in the different conformations of the receptor-binding domain (RBD). To characterize the chemical compositions and structures of the surface glycans, we used mass spectrometry to determine site-specifically the glycoforms of individual N-linked glycans. We further implemented a modelling procedure by GlycoSHIELD to quantitatively describe the shielding effect of the N-glycans on the spike proteins. Our results revealed that the local antigenicity of the spike proteins negatively correlated with the extent of protein surface shielding by the N-glycans. Collectively, our study provided a robust workflow to determine the molecular structure of CoV spike proteins in the context of the shielding effect of glycosylation with the ability to predict potential (co)receptor binding sites and epitopes for antibody binding. | en |
| dc.description.provenance | Made available in DSpace on 2023-03-19T23:50:00Z (GMT). No. of bitstreams: 1 U0001-2308202218454300.pdf: 31669761 bytes, checksum: a636401c573eb4ceac7eec32e8974dcc (MD5) Previous issue date: 2022 | en |
| dc.description.tableofcontents | 中文摘要 i ABSTRACT ii CONTENTS iv LIST OF FIGURES vi LIST OF TABLES viii Abbreviations ix Chapter 1 INTRODUCTION 1 1.1 Human coronaviruses 1 1.2 Structural features of spike (S) protein 2 1.3 Protein N-glycosylation 5 1.4 Cryo-EM, mass spectrometry and MD simulation in glycosylated S proteins 6 1.5 Specific aims 8 Chapter 2 MATERIAL AND METHODS 9 2.1 Construct design 9 2.2 Protein expression and purification 9 2.3 Differential scanning fluorimeter (DSF) 10 2.4 Differential scanning calorimetry (DSC) 10 2.5 Negative staining electron microscopy (NSEM) analysis 11 2.6 Cryo-EM grid preparation and Data collection 11 2.7 Image processing and 3D reconstruction 12 2.8 Model building and refinement 13 2.9 In-gel proteolytic digestion of the S proteins (Digestion protocol A) 14 2.10 In-solution proteolytic digestion of the S proteins (Digestion protocol B) 15 2.11 In-solution proteolytic digestion of the S proteins (Digestion protocol C) 15 2.12 Glycopeptide analysis by liquid chromatography-mass spectrometry 16 2.13 Glycopeptide identification and quantification 16 2.14 Representative Glycoforms selection of the S proteins 17 2.15 Atomic model building of fully glycosylated S proteins by CHARMM-GUI 19 2.16 Glycan shield modeling with GlycoSHIELD 20 Chapter 3 RESULTS 22 3.1 Expression and validation of S proteins 22 3.2 Structural heterogeneity of the MRES-CoV S protein 27 3.3 The workflow for N-glycopeptide analysis 32 3.4 Site-specific N- glycosylation analysis of the SARS-CoV S protein 35 3.5 Site-specific N glycosylation analysis of the MERS-CoV S protein 46 3.6 Site-specific N glycosylation analysis of the hCoV-HKU1 S protein 54 3.7 Quantifying the N-glycan shielding effects on S proteins by GlycoSHIELD 64 Chapter 4 DISCUSSION 73 4.1 The limitation of structure resolution in our cryo-EM single particle analysis 73 4.2 The inherent variation in N-glycopeptide sample preparation 75 4.3 The interplay between N-glycan maturation and the local environment 76 4.4 The limitations of GlycoSHIELD analysis pipeline 77 4.5 The roles of glycans in modulating S protein conformations 79 REFERENCES 83 APPENDIX 88 | |
| dc.language.iso | en | |
| dc.subject | 醣蛋白 | zh_TW |
| dc.subject | 棘蛋白 | zh_TW |
| dc.subject | 分子模擬 | zh_TW |
| dc.subject | 質譜儀 | zh_TW |
| dc.subject | 醣基化修飾之遮蔽效應 | zh_TW |
| dc.subject | 冷凍電子顯微鏡 | zh_TW |
| dc.subject | cryo-EM | en |
| dc.subject | spike proteins | en |
| dc.subject | molecular modeling | en |
| dc.subject | mass spectrometry | en |
| dc.subject | glycan shielding | en |
| dc.subject | glycoproteins | en |
| dc.title | 透過冷凍電子顯微鏡、質譜儀以及分子模擬探討人類冠狀病毒棘蛋白其結構與功能之關係 | zh_TW |
| dc.title | Structure-function relationships of human coronavirus spike proteins by cryo-EM, mass spectrometry and molecular modeling | en |
| dc.type | Thesis | |
| dc.date.schoolyear | 110-2 | |
| dc.description.degree | 碩士 | |
| dc.contributor.author-orcid | 0000-0001-7841-3751 | |
| dc.contributor.oralexamcommittee | 邱繼輝(Kay-Hooi Khoo),吳昆峯(Kuen-Phon Wu),嚴欣勇(Hsin-Yung Yen) | |
| dc.subject.keyword | 棘蛋白,醣蛋白,醣基化修飾之遮蔽效應,冷凍電子顯微鏡,質譜儀,分子模擬, | zh_TW |
| dc.subject.keyword | spike proteins,glycoproteins,glycan shielding,cryo-EM,mass spectrometry,molecular modeling, | en |
| dc.relation.page | 113 | |
| dc.identifier.doi | 10.6342/NTU202202723 | |
| dc.rights.note | 同意授權(全球公開) | |
| dc.date.accepted | 2022-08-25 | |
| dc.contributor.author-college | 生命科學院 | zh_TW |
| dc.contributor.author-dept | 生化科學研究所 | zh_TW |
| dc.date.embargo-lift | 2024-09-01 | - |
| 顯示於系所單位： | 生化科學研究所 | |
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