蛋白質的醣化在真核生物體中是一種常見的修飾，同時對蛋白質結構與功能也很重要。
因此科學界企圖找出蛋白質醣化與疾病之間的關聯性，以期望在人類的疾病治療上開啟一個
新方向。
然而就結晶學解析蛋白質結構的角度而言，附著於蛋白質的醣類導致了醣蛋白的結晶不
易。因此本篇論文的目標在於快速大量的純化不同的醣苷內切酶，在受質醣蛋白摺疊完好的
狀態下應用於移除蛋白質上的醣類，並發展理想的醣苷內切酶固定化方式，以利於分離酵素
與醣蛋白受質。
在實驗中，我們利用大腸桿菌表現醣苷內切酶F1、F2、F3、H 及N-醣酰胺酶F 用於進
行去醣化反應。受測受質則採用兩種不同品系A 型流行性感冒病毒的凝血素蛋
(hemagglutinin)，並分別以兩種不同的細胞株表現，使凝血素蛋白帶有兩種不同類型的多醣。
大致的反應效率以鈉十二烷基硫酸鹽聚丙烯酰胺凝膠電泳法觀察，接著受質的整體分子量變
化則透過基質輔助雷射脫附游離飛行時間質譜儀偵測，最後針對個別醣化位的去醣反應效率
則以液相層析串聯式質譜儀決定。
帶有high mannose type glycan 之凝血素蛋白去醣化反應在可溶性及固定化醣苷內切酶H
的作用下，呈現完全的反應性；相較之下帶有complex type glycan 凝血素蛋白去醣化反應即
使在可溶性醣苷內切酶F1、F2 及F3 的作用下，仍呈現反應不完全的現象；N-醣酰胺酶F 則
對兩種不同的醣皆呈現類似程度的反應性。比對不同反應受質之間的差異，我們得知complex
type glycan 凝血素蛋白去醣化反應不完全可能源自於complex type glycan 或是蛋白質構型對
醣苷內切酶所造成的立體障礙。
受質蛋白由於在原始摺疊的狀況下進行去醣化反應，所以其構型不會發生改變，因此可
利用此種反應所產生的蛋白進行結晶學、免疫學、細胞生理學等進一步的研究。

Glycosylation is one of the common modifications in eukaryotic cell and is important for protein
structure and function. Therefore, scientists have been tring to figure out the connection between
protein glycosylation and human diseases.
However, for protein crystallography, the glycans on the protein make it more difficult to get
glycoprotein crystals. Our goal is to utilize endoglycosidases to quickly and massively produce
deglycosylated glycoprotein under native condition. Furthermore, a suitable method was applied
to immobilize these endoglycosidases for rapid isolation of the deglycosylated glycoproteins.
In our experiments, we cloned the endoglycosidase F1, F2, F3 and peptide-N-glycosidase F from
Elizabethkingia meningosepticum and endoglycosidase H from Streptomyces plicatus. All the
endoglycosidase were expressed in E. coli expression system. We adopted two strains of Influenza
A hemagglutinin (HA) as the glycoprotein substrates. Each of HA proteins was expressed by two
different cell type individually in order to generate identical protein with different glycan form.
Three methods were used to exam the deglycosylation reaction level. First, the deglycosylation of
glycoprotein makes protein band down shifted in SDS-PAGE analysis. Second, the exactly
molecular weight decreasing due to deglycosylation is measured by MALDI-TOF Mass analysis.
Finally, the deglycosylation efficiency on each glycosylation site is detected by glycopeptide
analysis method with LC MS/MS.
The deglycosylation result of high mannose type glycan HA is satisfactory, even when the reaction
is performed with immobilized endoglycosidase H. However, the deglycosylation level of the
complex type glycan HA is not enough by using endoglycosidases individually or several
endoglycosidases at the same time. The results of two different type glycan substrates digested by
peptide-N-glycosidase F were similar. We suggest that the uncompleted deglycosylation of
different substrate of HA might be result from the steric barrier caused by bulky complex glycan or
the conformation of substrate.
Our goal is to find a general procedure to generate deglycosylated glycoproteins in a native
condition that is suitable for protein crystallography and other biological research.

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