臺灣博碩士論文加值系統

雞母珠毒蛋白(Abrin)是由A 鏈及B 鏈以一對雙硫鍵連結所組成,B 鏈能與
細胞表面 的受體結合,使Abrin 進入細胞中,A 鏈具有N-glycosidase 的
活性,當進入細胞質後,能水解真核細胞核醣體上60S 次單元中28S rRNA
第4324 位置上腺嘌呤核甘酸的醣甘鍵,而抑制蛋白質生合成.
雞母珠毒蛋白已知具有四種Isoforms,對蛋白質生合成抑制能力不盡相同,
其中 Abrin-a是活性最強且含量最多的一種. 比較Abrin-a A 鏈與其它
RIPs 的胺基酸序列,其中有13個胺基酸完全保留一致,而只有5 個胺基酸
的側鏈是位於Abrin-a A 鏈可能的活性區位中,分別為 Tyr74, Tyr113,
Glu164, Arg167 及Trp198.利用Site-directed mutagenesis 將Abrin-a
A 鏈活性位置之四個胺基酸進行突變 ,並且利用大腸菌進行表現.結果發
現突變種Y74F, Y113F 及W198F 抑制蛋白質生合成的能力只些微降低,而
E164Q 則大幅下降1,600 倍.以酵素動力學進行分析, Y73F, Y113F 及
W198F 主要是造成Km值上升,而E164A造成kcat值降低, 因此Glu164可能作
為催化反應之用 而Tyr74, Tyr113 及Trp198 與受質的結合有關.其中
W198F 無法與Abrin-a B 鏈重新組合分析其胰蛋白脢耐受性及圓二色光譜
發現有明顯的結構變化,此變化可能是由於失去Trp198 與Leu242之間的氫
鍵所引起.將Leu242 突變為Ala 或是加以刪除,同樣也無法與B 鏈重新組
合.因此推測Trp198 是透過Leu242 來維持A 鏈C 端的構形,使A 鏈和B 鏈
能完整地結合.將Abrin-a A 鏈C 端5 個氨基酸刪除,活性並未改變,且其
長度恰與C 端最短的RIPs Mirabilis antiviral protein 一樣.而刪除C
端6 個胺基酸則活性下降149倍,因此C 端末5 個胺基酸並非維持活性所必
需,Val246 則是維持A 鏈完整的重要胺基酸.將異雞母珠毒蛋白A 鏈N 端
差異較大的5 個胺基酸刪除並不影響其活性,而再刪除一個β-sheet (N
端第5-8 個氨基酸) 則活性下降46 倍,且因構形改變而易沉澱出來.可能
在大腸菌pGEX-2T 表現系統中, Glutathione S-transferase 會干擾N 端
刪除突變種的構形. 綜合以上結果,雞母珠毒蛋白A 鏈三度空間結構的
Domain I (1-109 胺基酸位置)功能尚未清楚; Domain II (110-197 氨基
酸位置) 負責其N-glycosidase activity, 而 Domain III (198-251 胺
基酸位置)是用以與B 鏈結合.在其N端及C 端可個別刪除5 個胺 基酸,以
作為製備更有效的免疫毒素之用.

Abrin is a toxic protein consisting of two subunits, an
enzymatic A chain with N-glycosidase activity and a B chain with
lectin activity, linked by a disulfide bond. Abrin possesses
four isoforms, abrin-a, -b, -c and -d, whichhave have different
degrees of inhibitory effect on protein biosynthesis. Abrin-a is
the most abundant and toxic one. Alignment of the amino acid
sequencesof eleven RIPs shows that only thirteen residues are
identical and five of them, Tyr74, Tyr113, Glu164, Arg167 and
Trp198, are located in the active siteof abrin-a A chain. Site-
directed mutagenesis was performed by PCR to study how the
identity amino acid residues around the active site of abrin-a
chain are involved in enzyme catalysis, enzyme-substrate
recognition and reassoci-ation of Aand B chains. The protein
biosynthesis inhibitory activity of Y74F, Y113F and W198F was
decreased moderately to that of wild type abrin-a A chain, while
that of E164Q, dramatically. Kinetic analysis revealed that the
kcat value ofY74F, Y113F and W198F was similar to that of wild
type, while Km value of these mutants was increased
significantly. These observations suggest that Glu164is
directly involved in catalytic function, and Tyr74, Tyr113 and
Trp198, insubstrate binding.W198F did not have the ability to
reassociate with abrin-aB chain to form heterodimer, while Y74F,
Y113F and E164Q did. Trypsin digestion and CD spectra analyses
revealed that W198F sh-owed a significant conformational change,
suggesting that a hydrogen bi-nding between Trp198 and Leu242
was disrupted when Trp198 was converted to Phe, and could lead
to the loss of reassociation between A and B cha-ins due to the
conformational change of W198F. Two mutants, L242A andL242
deleted, were unable to reassociate with abrin-a B chain.
Therefore, the two conserved residues, Trp198 and Leu242,are
crucial for the proper fold-ing of the C-terminal hydrophobic
region that facilitates the formation of heterodimer, abrin.
When five residues were deleted from the C-terminus of abrin-a A
chain (△C247-251), the inh-ibitory activity of protein
bioynthesis was simolar to that of wild type. In △C247-251,
the length of C-terminus is the same as that of Mirabilis
antiviral protein (MAP), which has the shortest length of the C-
terminus in RIPs. However, when six residues were dleted from
the C-terminus from theC-terminus of abrin-a A chain, the
inhibitory activity of protein biosynthesis was reduced 149-fold
lower than that of wild type. It suggests that these five
residues at C-terminus of abrin-a A chain are not essential
forN-glycosidase activity, and Val246 may play aimportant role
in maintaining the proper conformation of abrin-a A chain.Five
residues located at the N-terminus of abrin-a A chain are not
essential for N-glycosidase activity of abrin-a A chain and
those of four isoabrins have quite differentsequences. When the
first β-sheet of abrin-a A chain was further deleted, the
activity of GST-△N1-9 was 46-fold lower than that of wild type.
It is interesting to note that the fusion proteins, GST-△N1-5
and GST-△N1-9, were not dgested by thrombin. It suggests that
GST may affect the folding of the mutatd fusion proteins. In
conclusion, domain II of abrin-a A chain contains the active
site and domain III is responsible for the reaLociation of A and
B chains. The function of domain I is stillnot clear. Five
residues located at the N-erminus and at C-terminus are
dispensable and the polypeptides with these deltions may be
useful for preparing valuable immunotoxins.

封面
目錄
頁次
一、中文摘要
二、英文摘要
三、縮寫
四、緒論
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
參考文獻
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