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

黃彥文(Yen-Wen Huang)

指導教授 ： 翁啟惠

國立臺灣大學/生命科學院/生化科學研究所/博士(2017年)

https://doi.org/10.6342/NTU201700310

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。

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

並列摘要

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.

並列關鍵字

N-glycosylation ； NXS/T sequon ； Site-direct mutagenesis ； Saturation mutagenesis ； SAS Prediction model ； computer simulation ； glycoprotein-engineer

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