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標題: | 鄰近胺基酸對蛋白質N-醣基化效率的影響 Effect of Neighboring Residues on Protein N-glycosylation Efficiency |
作者: | Yen-Wen Huang 黃彥文 |
指導教授: | 翁啟惠(Chi-Huey Wong) |
關鍵字: | 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, N-glycosylation,NXS/T sequon,Site-direct mutagenesis,Saturation mutagenesis,SAS Prediction model,computer simulation,glycoprotein-engineer, |
出版年 : | 2017 |
學位: | 博士 |
摘要: | 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。 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. |
URI: | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/59881 |
DOI: | 10.6342/NTU201700310 |
全文授權: | 有償授權 |
顯示於系所單位: | 生化科學研究所 |
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