臺灣博碩士論文加值系統

核醣體抑制蛋白(ribosome-inactivating proteins; RIPs)是一群可以專一性抑制真核細胞核醣體作用的蛋白質，它具有N-glycosidase活性，可專一性地將28S rRNA序列中第4324腺嘌呤之glycosidic bond水解，進而抑制蛋白生合成之延長(elongation)步驟。到目前為止核醣體抑制蛋白可分為兩類: (1)第一型RIPs(type I RIPs)─由單一多胜月太 鏈組成，分子量介於25-30 kDa之間且具有鹼性等電點; (2)第二型RIPs (type II RIPs)─為兩分子多胜月太 鏈構成，由一分子與第一型RIPs相似具有N-glycosidase活性之多胜月太 鏈(A 鏈)以單一雙硫鍵連接另一分子可結合至細胞膜表面半乳糖的多胜月太 鏈(B 鏈)所組成。在本實驗中，我們由準人瓜(Sechium edule Swartz)種子純化出一種可抑制蛋白生合成的蛋白質，我們將之命名為sechiumin。經由SDS-PAGE分析發現sechiumin由單一多胜月太 鏈所構成，分子量約為27 kDa，其抑制非細胞系統(cell free system)蛋白質生合成之IC50為0.8 nM，遠低於抑制完整細胞蛋白生合成之IC50(5000-6000 nM)。將sechiumin與老鼠肝臟抽出之核醣體一起作用，再以aniline處理後由含有7M尿素之膠電泳分析可發現有一特殊的RNA片段(約420個nucleotides)被釋放出來。由以上這些結果可推測sechiumin是一種第一型RIP。
運用RACE(rapid amplification of cDNA ends)方法，我們已選殖出sechiumin cDNA。sechiumin cDNA全長951 bp， 其中open reading frame有855 bp，推測可以合成具有285個胺基酸之蛋白質，在N端序列之前有一段含有21個胺基酸之signal peptide，而C端則有19個胺基酸推測經由修飾作用(modification)之後是不存在於成熟sechiumin蛋白中，因此成熟的sechiumin蛋白由245個胺基酸組成。與其它RIPs胺基酸序列比對，有12個保留性(conserved)胺基酸(其中包含推測的活化位置)是完全一致的。Sechiumin cDNA經由pET載體表現系統以His-tag融合蛋白型式在大腸桿菌中大量表現,而這些重組蛋白仍具有高度生物活性，其抑制非細胞系統蛋白生合成之IC50為0.9 nM。
利用site-directed mutagenesis將sechiumin可能的活性位置胺基酸Glu160突變成Gln，Arg163突變成Leu或兩者皆突變，結果對無細胞蛋白質生合成抑制活性而言，E160Q與R163L活性分別降低了150倍和457倍，而E160Q.R163L活性更降低了1222倍。我們推測sechiumin的Glu160與Arg163如同其它RIPs一樣與催化作用活化位置有關。
Sechiumin N端與C端胺基酸對其活性之影響以一系列刪除實驗加以探討。將C端2個胺基酸刪除後活性並沒有改變，而刪除C端4個胺基酸活性下降88倍，刪除C端7個胺基酸則重組蛋白幾乎完全失去活性。因此我們推測C端末2個胺基酸並非維持活性所必需，而C端Tyr242至Leu239則是維持sechiumin生物活性的重要胺基酸區域。將sechiumin N端2個胺基酸刪除後並不影響其活性，刪除N端4個胺基酸活性下降22倍，而刪除N端6個胺基酸完全破壞第一個 -sheet (N端第3-5個胺基酸)則活性下降約50倍。因此由sechiumin N端與C端胺基酸刪除實驗得知，將sechiumin N端或C端刪除2個胺基酸並不會影響其活性，將來可作為製備免疫毒素之用。

RIPs are a group of plant enzymes that can inhibit polypeptide-chain elongation by inactivating the ribosomes. The molecular mechanism of the inhibition is identified as a specific N-glycosidase activity that hydrolytically cleaves the N-glycosidic bond of adenine at position 4324 (A4324) of 28S rRNA. RIPs are divided into two groups: (1) type I RIPs, single-chain polypeptide with molecular mass ranging from 25 to 30 kDa; (2) type II RIPs, heterodimer proteins consisting of an A-chain with enzymatic activity equivalent to the type I RIPs and a lectin-like B-chain. By binding to the D-galactose of the cell-membrane receptors, the B-chain of type II RIPs facilitates the A-chain to enter the cytoplasm and irreversibly inactivate ribosomes.
A ribosome-inactivating protein (RIP), sechiumin, was purified from the seeds of edible gourd, Sechium edule Swartz, with an apparent relative molecular mass of 27 kDa. It inhibits the protein synthesis of rabbit reticulocyte lysate strongly with an IC50 of 0.8 nM, but has a much lower inhibitory effect on that of intact HeLa cells, with an IC50 of 5500 nM. Sechiumin has a highly specific RNA N-glycosidase activity towards 28S rRNA, as the A-chain of abrin does. It suggests that sechiumin is one of the type I RIPs
The cDNA of sechiumin has been cloned and expressed by pET expression system in Escherichia coli. The sechiumin cDNA has 951 nucleotides, encoding a protein with 285 amino acids. The amino acid sequence deduced from the cDNA reveals that the first 21 N-terminal amino acid residues constitute a signal peptide and the 19 C-terminal amino acid residues were proposed to be removed by the post-translational modification as found in other type I RIPs, trichosanthin. Sechiumin has nearly 60 % homology to several type I RIPs purified from Cucurbitaceae family. Two amino acid residues, Glu160 and Arg163, found at the putative active site of sechiumin are known to be catalytically active in ricin and abrin. The recombinant sechiumin was obtained as an insoluble protein, and the preparation of active soluble form was achieved by renaturing the denatured protein.
Site-directed mutagenesis was utilized to identify the potential active site of sechiumin with the mutants E160Q, R163L and E160Q.R163L, respectively. The IC50 of E160Q and R163L was found to decrease 150-fold and 457-fold to that of wild type re-sechiumin, respectively, by using rabbit reticulocyte lysate protein translation inhibition assay, while E160Q.R163L decreased 1222-fold. It suggested that Glu160 and R163 might play an important role in active site.
Deletion of four residues from the C-terminus of sechiumin, decreased its protein biosynthesis inhibitory activity moderately. Deletion of seven residues from the C-terminus of sechiumin caused a great loss in its activity due to the removal of a highly conserved hydrophobic residue, Leu239. N-terminal deletion analysis revealed that the mutations of sechiumin with the deletion of the first 4 or 6 amino acid residues from N-terminus including the firstβ-sheet which contains a highly conserved hydrophobic residue, Phe4, led to the inactivation of the RIP. The results indicate that the two residues located either from N- or C-terminus of sechiumin are dispensable for its inhibitory activity of protein biosynthesis. The polypeptides with these deletions generated from this study may be useful for the preparation of valuable immunotoxins.

封面
目錄
一、中文摘要
二、英文摘要
三、縮寫
四、緒言
五、實驗材料
六、實驗方法
七、實驗結果
八、討論
九、圖表及圖表說明
十、參考文獻
十一、附錄
其他

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