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系統識別號 U0026-2208201410550000
論文名稱(中文) 臺灣人新奇第五因子基因突變之分子機轉探討
論文名稱(英文) Molecular characterization of novel Factor V mutations in Taiwanese
校院名稱 成功大學
系所名稱(中) 基礎醫學研究所
系所名稱(英) Institute of Basic Medical Sciences
學年度 102
學期 2
出版年 103
研究生(中文) 劉相君
研究生(英文) Hsiang-Chun Liu
學號 S58961573
學位類別 博士
語文別 英文
論文頁數 72頁
口試委員 指導教授-林尊湄
口試委員-沈銘鏡
召集委員-吳華林
口試委員-林淑華
口試委員-蔡少正
口試委員-傅子芳
中文關鍵字 第五因子  體外表現研究  分子模擬  細胞內蛋白質降解  分泌缺陷  解離 
英文關鍵字 Factor V  in vitro expression study  Molecular modeling  Intracellular protein degradation  Impaired secretion  Dissociation 
學科別分類
中文摘要 先天性第五因子缺乏是罕見的遺傳性疾病,在兩個第五因子缺乏的家族中,我們發現了兩個新奇突變位點(Asp68His和His147Arg), Asp68His和His147Arg突變位點均座落在第五因子的A1功能區中,且在不同物種和血漿銅藍蛋白(ceruloplasmin)的序列比對中具有高度的保留性。研究結果發現,Asp68His突變導致第五因子蛋白在上清液的抗原表現量顯著下降,但不影響第五因子的活性比,因此定義為第一型的第五因子突變;由於野生型和Asp68His突變型第五因子的mRAN降解速率相似,因此證實Asp68His突變並不影響第五因子蛋白的轉譯過程;利用分泌途徑抑制劑(brefeldin A)發現Asp68His突變的第五因子蛋白質從細胞內運輸到細胞外的速度較野生型慢;此外我們也發現Asp68His突變會造成第五因子細胞內的降解作用增加,此降解作用會受到抑制劑MG132的調控;分析轉染後的細胞發現Asp68His尚未完成和野株型相同的醣化修飾;更進一步觀察Asp68His突變第五因子蛋白質會局限於細胞的內質網中,無法順利地運輸到高基氏體,因此無法運送到細胞外。相對地, His147Arg突變對於第五因子抗原的表現影響不顯著,但會使得第五因子活性下降為野生型第五因子的33.9±2.2%,因此定義為第二型得第五因子缺乏突變;在凝血酶(thrombin)活化第五因子的實驗中,我們觀察到His147Arg突變並不影響第五因子的活化,但His147Arg突變會降低活化態第五因子重鏈和輕鏈連結的穩定性,進而降低第五因子活性。總而言之,我們的研究主要利用體外功能分析與資訊結構的探討釐清基因序列變異引起蛋白質結構功能改變的相關機制。
英文摘要 Congenital factor V (FV) deficiency is a rare inherited disorder. Two missense mutations, Asp68His and His147Arg, were found in two families of FV deficiency. In Taiwan, both Asp68 and His147 are located in A1 domain of FV, which are highly conserved in homologous sequence positions from other species and in ceruloplasmin. Expression of FV-Asp68His in cell culture system resulted in severe decreased levels of FV protein in the conditioned media. FV-Asp68His contained normal specific FV activity and belonged to type I FV deficiency. Similar mRNA degradation rates were found in FV-wild-type (wt) and FV-Asp68His mRNA showed, indicating the Asp68His mutation does not affect FV expression at the transcriptional level. The lower transport efficiency was observed to the outside of the cell for FV-Asp68His mutant protein compared with that of the FV-wt protein. Furthermore, we showed that the Asp68His mutation resulted in increased intracellular degradation through a MG132-mediated proteasomal degradation pathway. In the transfected cell lysates, FV-wt protein had multiple post-translational modified forms, but the FV-Asp68His protein was less glycosylated. We further observed that the FV-Asp68His protein was retrieved in the ER only and did not undergo transport to the Golgi apparatus, leading to impaired secretion. In contrast, His147Arg mutation was showed only a little reduction antigen level both in cell lysates and conditioned media, but the FV activities of His147Arg mutant significantly decreased to 33.9±2.2% of FV-wt protein and was identified as type II FV deficiency mutation. Form the thrombin activation experiments, we observed that His147Arg mutation didn't affect FV protein activation. His147Arg was decrease in the association of heavy and light chain of FVa protein to reduce the stability. Collectively, our studies emphasize the benefits of using in vitro functional analysis in combination with computer-based structural investigations to understand sequence-structure-function relationships.
論文目次 I. Literature review 1
(1)、 Hemostasis 1
i、 Primary hemostasis 1
ii、 Secondary hemostasis 2
(2)、 Human coagulation factor V protein 4
iii、 Structure of coagulation factor V 4
iv、 The role of FV protein in the coagulation cascade 4
(3)、 Human FV gene (F5) 5
(4)、 Congenital FV deficiency 6
(5)、 FV deficiency related mutations 6
(6)、 Identification of mutations in Chinese population with FV deficiency 7
(7)、 Identification of mutations in Taiwan population with FV deficiency 8
(8)、 Potential roles of the identified missense mutations in FV protein expression 9
i、 The importance on the characterization of missense mutations 9
ii、 Hot spots of the missense mutations on FV protein 9
iii、 Missense mutations on the C2 domain of FV protein 10
iv、 Missence mutations on the A2 domain of FV protein 10
v、 Missence mutations on the A1 domain of FV protein 10
(9)、 The possible functions of FV A1 domain 11
(10)、 Post-modification and secretion of FV protein 12
II. Specific aims 14
III. Materials and methods 16
(1)、 Subjects and blood collection 16
(2)、 FV mutant plasmids construct 16
(3)、 DNA sequencing 17
(4)、 Sequence alignment and Molecular modeling 17
(5)、 Cell cultures and transient transfections 17
(6)、 mRNA degradation assay by quantitative RT- PCR 18
(7)、 FV antigen and activity measurements in conditioned media and cell lysates 19
(8)、 Molecular dynamics and mutation modeling 19
(9)、 Western blotting protein degradation studies 20
(10)、 N-linked glycosylation and Western blotting 21
(11)、 Immunofluorescence staining 21
(12)、 Immunoprecipitation and thrombin activation 22
(13)、 Determination of the stability of the FV variants 22
(14)、 Statistical analysis 23
IV. Results 24
(1)、 Pedigree of FV deficient patients with novel mutations 24
(2)、 Sequence alignment and Molecular modeling 24
(3)、 Biosynthesis of wild type and mutant FV mRNA in COS-1 cells 25
(4)、 Expression of mutant recombinant FV in COS-1 cells 25
(5)、 Molecular modeling of Asp68His mutation on FV protein 26
(6)、 Impaired secretion of FV-Asp68His mutant protein 26
(7)、 Effect of protein degradation inhibitors on FV protein biosynthesis 27
(8)、 Defects in post-translational processing of the FV-Asp68His mutant protein 28
(9)、 Subcellular localisation of FV-wt or FV-Asp68His mutant proteins 29
(10)、 Expression of His147Arg mutant recombinant FV in COS-1 cells 30
(11)、 Molecular modeling 31
(12)、 Assessment of the activity of wild-type and mutant FV molecules 32
(13)、 Enhanced dissociation of heavy and light chains of activated FV-His147Arg protein 33
V. Discussion 35
VI. Reference 59
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