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研究生:張育嘉
研究生(外文):Yu-Jia Chang
論文名稱:利用Alanine置換方式研究凝血第九因子的功能
論文名稱(外文):Functional analysis of factor IX by alanine scanning mutagenesis
指導教授:吳華林
指導教授(外文):Hua-Lin Wu
學位類別:博士
校院名稱:國立成功大學
系所名稱:基礎醫學研究所
學門:醫藥衛生學門
學類:醫學學類
論文種類:學術論文
畢業學年度:92
語文別:中文
論文頁數:142
中文關鍵詞:凝血因子第九因子血友病
外文關鍵詞:factor IXalanine scanning
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凝血第九因子(factor IX)在凝血機制上扮演重要的角色,本論文分別針對第九因子的第二個類表皮生長因子功能區(EGF-2功能區,第85至127個胺基酸)及蛋白酶活性功能區的胺基端(胺基酸序列為第181至310個胺基酸)的角色,利用Ala置換的方式作詳細的分析。於人類第九因子的第二個類表皮生長因子功能區中選擇28個胺基酸位置,利用定點突變法將原胺基酸逐一置換成Ala,並以人類胚胎腎臟293(HEK-293)細胞表現突變型第九因子重組蛋白。經過抗生素篩選後,篩選出持續及高量表現重組第九因子蛋白的單一細胞株,利用酵素免疫連結法,測定細胞培養液含第九因子知表現量,發現置換在Gln97、Phe98、Try115、Leu117的突變型第九因子蛋白質無法在細胞培養液中被偵測到,其他的突變型第九因子皆可在培養液中偵測到蛋白質。經過大量表現收集培養液後,經管柱層析法將重組第九因子純化。利用得到高度純化的重組第九因子進行功能性試驗。由凝血酶元時間(APTT)測定發現,置換Asn89、Asn92、Gly93、Val107等位置為Ala時,完全測不到凝血活性。以酵素動力學的方式測定第九因子與輔因子的結合常數,發現V107A與活化態第八因子(factor VⅢa)的結合常數較野生型(wild-type)升高近35倍;於競爭性實驗發現,N89A與N92A完全喪失與野生型第九因子競爭活化態第八因子的能力,而G93A則是競爭能力降低,其抑制常數(Ki)為野生型第九因子的6倍,以上顯示Asn89、Asn92、Gly93、Val107與活化態第八因子的結合很重要。由酵素動力學實驗發現,突變在Thr87至Gly93及Asn101至Val107兩個區域內的突變型第九因子,於輔因子不存在時,與第十因子的結合常數(Km)增加2-10倍。但在活化態第八因子的存在下,所有的突變型第九因子的催化效率皆與其專一活性一致。由以上的結果顯示出,第九因子的第一個loop(Asn89至Arg94)主要是牽涉與活化態第八因子的結合;而第三個loop連接β-sheets的位置(區域Ser102至Val108)主要是牽涉到與第十因子的結合上。
對於蛋白酶活性功能區的胺基端(包含胺基酸序列自第181至310個胺基酸)上則選擇了20個不同的位置作胺基酸的置換,經表現純化後,進行定性實驗時發現,突變在Lys201、Asp203、Lys228、Arg252及Asp276等位置的胺基酸時,單株抗體A5對於這些突變第九因子的辨識程度不同。由於單株抗體A5被廣泛用在第九因子結構與功能間關係的研究上,過去的研究認定單株抗體A5其識別區是第九因子的重鏈第181到310個胺基酸的區域,故本論文第二部分主要針對這些胺基酸與單株抗體A5間的作用做進一步的釐清。當置換Lys228及Arg252時,單株抗體A5完全無法辨識突變的第九因子。利用生物感應器(biosensor)來即時性地偵測第九因子蛋白與單株抗體A5間的作用時發現,當鈣離子存在的情況下,野生型第九因子與單株抗體A5間的作用比沒有鈣離子時的作用增加十倍。而單株抗體A5與第九因子間的結合係數(KD)分別是:野生型第九因子為6.0 x10-9M 、K201A/D203A為1.4 x10-8M、 D276A為2.0 x10-8M。K201A/D203A及D276A的解離係數增加,則是由於解離速率常數(dissociation constant)增加的緣故,意味著這些突變第九因子與單株抗體A5的結合力降低。而K228A及R252A則由於與單株抗體A5結合作用非常小,由生物感應器的轉感圖無法計算其結合常數。由蛋白質立體結構分析發現,Lys201、Asp203、Lys228、Arg252及Asp276在立體結構上是位於同一平面上,而此作用面與第九因子和第八因子作用的區域位於第九因子的對側 。
The role of EGF-2 domain and amino-terminal of protease domain of factor IX is unclear. This study is mainly focused on these two regions and characterized their function by alanine scanning mutagenesis. Twenty-eight positions of the second EGF (EGF-2) domain of factor IX were chosen to be replaced by alanine. Four positions of Q97, F98, Y115 and L117 , those are critical for secretion of factor IX. Of the remaining mutations, four mutants (V86A, E113A, K122A, S123A) are as active as wild-type factor IX (IXwt), sixteen (D85A, K100A, N101A, D104A, N105A, R116A, E119A, T87A, I90A, K91A, R94A, E96A, S102A, K106A, T112A, and N120A) retain reduced but detectable activity, whereas four (N89A, N92A, G93A, and V107A) are nearly inert in the clotting assay. Both factor XIa and the factor VIIa-tissue factor (TF) complex effectively catalyzed the activation of these mutants except N89A. The mutant V107A failed to form the factor tenase complex with factor VIIIa owing to a 35-fold increase in Kd. The mutants N89A and N92A did not compete with factor IXwt for factor VIIIa binding, and G93A exhibited a 6-fold increase Ki in the competitive binding assay. It appears that mutations at these positions have significantly affected the interaction between factor IX and factor VIIIa, while other mutations had little effect on the binding of factor IX to factor VIIIa. Mutations in two regions, T87-G93 and N101-V107, significantly increased the Km of factor IXa (2-10 folds) in cleavage of factor X in the absence of factor VIIIa. In the presence of factor VIIIa, the catalytic efficiency of each mutant toward factor X paralleled its clotting activity. Briefly, we propose two relatively distinctive functions of factor IX for two adjacent regions in the EGF-2 domain: the first loop region (residues 89-94) is involved with the binding of its cofactor, factor VIIIa, and the third loop with connected β-sheets (residues 102-108) is involved in the proper binding to the substrate, factor X.
Monoclonal antibody A-5 (Mab A-5) has been widely used in structure-function relationship studies in relation to factor IX. It recognizes the catalytic domain of human factor IX. Regions important for binding Mab A-5 have previously been localized to the amino terminus of factor IX, encompassing amino acid residues 181-310. We have selected 19 positions in this region for alanine-scanning mutagenesis and used these proteins we found that Mab A-5 failed to react with the recombinant factor IX mutants with substitutions at positions 228, and 252. Mab A-5 also reacted to a lesser extent to FIXD276A (factor IX with alanine substitution for aspartic acid at residue 276) and to factor IX with double alanine substitutions at residues 201 and 203 (FIXK201A/D203A). The lost or reduction of binding of these factor IX mutants to Mab A-5 was further confirmed by competition experiments and by direct binding assays. We found that the binding kinetics were in favor of the presence of calcium, and the apparent KD values were 6.0 x10-9 M, 1.4 x10-8 M, and 2.0 x10-8 M, for wild-type FIX, FIXK201A/D203A, and FIXD276A, respectively. The increased KD values of FIXK201A/D203A and FIXD276A are mainly owing to the increased dissociation constants. FIXK228A and FIXR252A failed to elicit any binding ability to Mab A-5. Computer modeling showed that these residues constitute a surface opposite to the factor VIIIa binding surface and this side of factor IX. We conclude that the epitope for Mab A-5 is on a surface composed of residues 228, 252, 276, and 201 or 203. This surface, although may not be important for binding cofactor, factor VIII, is a strong antigenic determinant.
中文摘要 v
英文摘要 vii
誌謝 x
圖目錄 xi
表目錄 xii
第一章 緒 論 1
1.1 止血作用(haemostasis) 2
1.2 凝血因子(coagulation factors) 4
1.3 血友病(hemophilia) 4
1.4 凝血第九因子(coagulation factor IX) 5
1.4.1 凝血第九因子基因與蛋白質結構 5
1.4.2 第九因子的Gla功能區 6
1.4.3 類表皮生長因子功能區(epidermal growth factor-like domain) 8
1.4.4 活化�A�B鏈(activation peptide) 9
1.4.5 活化態第九因子的重鏈(heavy chain of factor IXa) 10
1.4.6 第九因子的活化 (Activation of factor IX) 11
1.4.7 第九因子的功能特性 (Function of factor IX) 13
1.4.8 活化態第九因子與活化態第八因子的作用區(Interaction of factor IXa and factor VIIIa) 14
第二章 研究動機 15
第三章 實驗方法 20
3.1 限制�@切割(restriction enzyme digestion) 21
3.2 勝任細胞的製備(preparation of competent cells) 21
3.3 質體DNA的形質轉移(plasmid DNA transformation) 22
3.4 小量質體DNA萃取法(mini-preparation) 22
3.5 大量質體DNA萃取法(CsCl2 method) 23
3.6 瓊脂膠電泳分析(agarose gel electrophoresis) 24
3.7 核酸定序分析(DNA sequencing) 24
3.8 菌種的保存 25
3.9 定點突變法 25
3.10 哺乳動物細胞的轉感作用(transfection) 26
3.11 酵素免疫連結法(ELISA) 27
3.12 細胞株的儲存 28
3.13 重組第九因子的大量表現及純化 28
3.14 重組第九因子的定量 29
3.15 蛋白質電泳分析(SDS-PAGE) 30
3.16 重組第九因子的專一活性測定(凝血時間測定, clotting time) 31
3.17 重組第九因子的活化 32
3.18 活化態重組第九因子酵素動力學分析 35
3.19 測定重組第九因子與輔因子的結合常數 37
3.20 競爭性分析(competition assay) 39
3.21 單株抗體與第九因子的抗原性分析 41
3.22 利用表面薄膜共振法(surface plasmon resonance assay)測定兩蛋白質間的結合作用 42
第四章 結果 44
4.1 突變型與野生型第九因子的表現及純化 45
4.2 第九因子的活性測定 46
4.3 第九因子的活化 47
4.4 酵素動力學方式測定第九因子的活性 48
4-5 單株抗體A5在第九因子重鏈上的抗原辨識區的分析
52
4-6 第九因子與單株抗體A5間的交互作用 52
4-7 第九因子蛋白質分子模擬 53
第五章 討論及未來發展 54
第六章 圖與表 65
第七章 參考文獻 95
附錄 117
已發表的相關論文 128
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