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http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/59881完整後設資料紀錄
| DC 欄位 | 值 | 語言 |
|---|---|---|
| dc.contributor.advisor | 翁啟惠(Chi-Huey Wong) | |
| dc.contributor.author | Yen-Wen Huang | en |
| dc.contributor.author | 黃彥文 | zh_TW |
| dc.date.accessioned | 2021-06-16T09:42:56Z | - |
| dc.date.available | 2019-02-16 | |
| dc.date.copyright | 2017-02-16 | |
| dc.date.issued | 2017 | |
| dc.date.submitted | 2017-02-04 | |
| dc.identifier.citation | (1) Fuster, M. M.; Esko, J. D. (2005) The sweet and sour of cancer: glycans as novel therapeutic targets. Nature reviews. Cancer 5(7):526-542.
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Proceedings of the National Academy of Sciences of the United States of America 92(1):97-101. | |
| dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/59881 | - |
| dc.description.abstract | N-醣基化是一種很重要的轉譯後修飾，這種修飾可以調控醣蛋白多種功能並且影響醣蛋白藥物的發展。N-醣基化的發生是當胺基酸在內質網合成時，此時寡醣轉接酵素OST會辨認共識蛋白質序列NXS/T，並且接上寡醣前驅物。但是並不是所有的共識蛋白質序列會被接上寡醣，大約只有65%會被接上，其中真正的機制還尚未清楚。過去研究指出，三個胺基酸片段為OST最簡單的受質，並且形成β-turn 和Asx-turn這兩種可區分的形狀，此外，之前研究發展出一個預測的方式，包括Phe-X-Asn-X-Thr和Phe-X-X-Asn-X-Thr的序列，讓我們有更進一步的了解。為了要深入的了解在醣基化的Asn附近其他胺基酸，是否會影響醣基化的效率，我們使用人類T細胞上抗原蛋白CD2的黏著片段當作本實驗的模型，我們使用階梯式的掃描方式，來檢視醣基化Asn(0)附近其他胺基酸(-2,-1,+1,+2) 是否會影響其效率。我們發現帶有芳香環官能基特別是Trp和硫官能基的氨基酸在醣化Asn上游前兩個位置(-2)可以促進醣化的效率，然而帶有正電的氨基酸具有相反的效果，官能基具有硫,氫氧,脂肪族官能基的氨基酸在-1的位置有高的醣化效率，尤其Cys可以恢復醣化效率即使在Arg在的-2位置，小分子量的氨基酸和Ser在+1的位置可以有效的促進醣化的效率。根據這些不同胺基酸片段組合所產生的效率比例，我們能利用SAS軟體建立一種運算方式，是根據胺基酸組合的片段來預測醣基化的效率。為了證明優化片段能夠促進醣化的效率，我們在其他具有N-醣化的蛋白中將優化片段置換原本的片段，結果促進醣化效率和人類CD2模型的結果一致。最後使用電腦模擬的方式可以發現優化片段和人類OST亞基具有高度的親和力和相互作用。我們的發現提供一個預測N-醣基化效率的指引，可以針對我們有興趣的醣化蛋白上的醣化位置去做基因工程，改變成促進或是抑制醣化的效率，此實驗讓我們對於OST所催化的醣化有更進一步的了解。 | zh_TW |
| dc.description.abstract | N-glycosylation is an important co-translational modification that regulates diverse glycoprotein functions and influences the development of glycoprotein pharmaceuticals. N-glycosylation happens in endoplasmic reticulum (ER) when nascent peptide synthesis. The oligosaccharyltransferase (OST) recognizes the consensus sequon Asn-X-Ser/Thr (NXS/T) to link dolichol-linked precursor oligosaccharide. However, only 65% of the consensus sequences are be glycosylated and this mechanisms are still unclear. It is known that tripeptides, NXS/T, are the substrate of OST and form two distinct structures as β-turn and Asx-turn. Furthermore, previous work on the development of a predictive rule to identify a sequon for N-glycosylation, including the aromatic sequons Phe-X-Asn-X-Thr and Phe-X-X-Asn-X-Thr has advanced our understanding of N-glycosylation. To further investigate the influence of sequence variation on N-glycosylation efficiency in the context of a pentapeptide enhanced aromatic sequon and to use human CD2 adhesion domain (hCD2ad) to screen the -2, -1, +1 and +2 residues flanking Asn at position 0 in a stepwise manner to identify the optimal sequon for N-glycosylation. It was found that aromatic, especially the Trp residue, and sulfur-containing residues at the -2 position upstream of the sequon Asn residue improved N-glycosylation efficiency, while positive-charge residues such as Arg had a negative effect. The thiol, hydroxyl, and aliphatic residues at the -1 position had higher N-glycosylation efficiency, and Cys, in particular, restored the negative effect of Arg at the -2 position. Small residues and Ser at the +1 position downstream of Asn increased the likelihood of N-glycosylation. Based on the degree of glycosylation of various sequences, we devised an algorithm for prediction of N-glycosylation efficiency using the SAS software. As a proof-of-concept, we introduced the optimized sequons to other glycoproteins and found enhancement in glycosylation, with a modeling support to show the high-affinity interactions between the optimized sequence on hCD2ad and an OST subunit. Our findings in this study provide a better understanding of the OST-catalyzed N-glycosylation and a predictive guide for glycoprotein design to introduce or suppress N-glycosylation at a site of interest. | en |
| dc.description.provenance | Made available in DSpace on 2021-06-16T09:42:56Z (GMT). No. of bitstreams: 1 ntu-106-D01B46013-1.pdf: 3715201 bytes, checksum: b28267ac3dce61ef6cba689f26d007a3 (MD5) Previous issue date: 2017 | en |
| dc.description.tableofcontents | CHAPTER 1: Introduction.................................1
1.1 What is N-glycosylation?............................2 1.2 N-glycosylation Sequons.............................3 1.3 N-glycosylation and Protein Stability...............4 1.4 Rationale and Significance..........................5 CHAPTER 2: Materials and Methods........................7 2.1 Establishment of Saturation Mutagenesis Library of hCD2ad..............................................8 2.2 Binding Interaction between OST and Acceptors.......9 2.3 Cell Culture.......................................10 2.4 Protein Expression and Western Blotting............10 2.5 Statistically analysis for mutagenesis library.....11 CHAPTER 3: Results.....................................14 3.1 Establishment of Pentapeptide Sequon Library.......15 3.2 Establishment of Screening Methods: High Throughput and Stepwise Screening.............................16 3.3 Aromatic Residues and Sulfur-containing Residues At the -2 Position Enhance the N-Glycosylation Efficiency, While Positively Charged Residues Show Inhibition.........................................18 3.4 Cys at the -1 Position Increased the N-Glycosylation Efficiency.........................................19 3.5 Hydoxyl and Small Amino Acids at +1 Position Enhanced the Glycan Occupancy...............................20 3.6 The Correlation Between the Characteristics of Pentapeptides Library and N-glycosylation Efficiency.........................................21 3.7 Determinants and the Algorithm for Prediction of the N-glycosylation Efficiency.........................22 3.8 Expansion of the Predictive Sequons to Other Glycoproteins......................................27 3.9 Computer Simulation of Docking Between Optimized Sequon and Human OST subunit.......................29 CHAPTER 4: Discussion..................................31 CHAPTER 5: Figures.....................................38 CHAPTER 6: Supplementary figures.......................63 CHAPTER 7: Tables......................................66 CHAPTER 8: Refences....................................73 CHAPTER 9: Apendix.....................................79 | |
| dc.language.iso | en | |
| dc.subject | 醣蛋白基因工程 | zh_TW |
| dc.subject | N-醣基化 | zh_TW |
| dc.subject | NXS/T片段 | zh_TW |
| dc.subject | 飽和點突變 | zh_TW |
| dc.subject | SAS統計軟體 | zh_TW |
| dc.subject | 預測醣化效率模型 | zh_TW |
| dc.subject | 電腦模擬 | zh_TW |
| dc.subject | SAS Prediction model | en |
| dc.subject | glycoprotein-engineer | en |
| dc.subject | NXS/T sequon | en |
| dc.subject | computer simulation | en |
| dc.subject | Site-direct mutagenesis | en |
| dc.subject | N-glycosylation | en |
| dc.subject | Saturation mutagenesis | en |
| dc.title | 鄰近胺基酸對蛋白質N-醣基化效率的影響 | zh_TW |
| dc.title | Effect of Neighboring Residues on Protein N-glycosylation Efficiency | en |
| dc.type | Thesis | |
| dc.date.schoolyear | 105-1 | |
| dc.description.degree | 博士 | |
| dc.contributor.oralexamcommittee | 楊懷壹(Hwai-I Yang),吳盈達(Ying-Ta Wu),吳宗益(Chung-Yi Wu),王惠鈞(Andrew Hui-Jun Wang) | |
| dc.subject.keyword | N-醣基化,NXS/T片段,飽和點突變,SAS統計軟體,預測醣化效率模型,電腦模擬,醣蛋白基因工程, | zh_TW |
| dc.subject.keyword | N-glycosylation,NXS/T sequon,Site-direct mutagenesis,Saturation mutagenesis,SAS Prediction model,computer simulation,glycoprotein-engineer, | en |
| dc.relation.page | 83 | |
| dc.identifier.doi | 10.6342/NTU201700310 | |
| dc.rights.note | 有償授權 | |
| dc.date.accepted | 2017-02-05 | |
| dc.contributor.author-college | 生命科學院 | zh_TW |
| dc.contributor.author-dept | 生化科學研究所 | zh_TW |
| 顯示於系所單位： | 生化科學研究所 | |
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