Biosynthesis and Intracellular Fate of Two Mutant Factor VIII

• Main Authors: Liu, Yi-lin, 劉怡霖
• Other Authors: Lin, Shu-wha
• Format: Others
• Language: en_US
• Published: 1998
• Online Access: http://ndltd.ncl.edu.tw/handle/10509657823705830546

碩士 === 國立臺灣大學 === 醫事技術學系 === 86 === A型血友病是由於凝血蛋白第八因子異常所造成，是一種 極為顯見的性聯遺傳疾病。 在血液中，與其相類似的蛋白如第 五因子比較，第八因子蛋白的含量比第五因子蛋白低了100倍。 對於在表現量上這樣的差異，目前沒有完整的分析，主要是因 為第八因子的蛋白非常大，而且目前並沒有任何的細胞株可自 然表現此蛋白。 在我們實驗室中，已針對中國人A型血友病 病人的基因突變位置作一完整分析。 為了了解突變型基因的致 病機轉，與研究第八因子在細胞內的生合成，首先構築野生型 及兩個突變型的表達質體，利用細胞轉染的技術，在人類腎臟 細胞株293及肝癌細胞株HepG2中表現野生型及突變型第八因 子。 當測定病人血漿及細胞培養上清液得知，突變型蛋白P146S 及I386S在病人血漿的含量分別是0.87 ng/ml及0.63 ng/ml，而 在細胞培養上清液中，則是測不到P146S及測出0.01 ng/ml的 I386S突變型蛋白。 由於第八因子在細胞培養技術下的表現量 過低，進而造成許多實驗上的限制。 本篇論文試圖利用共軛焦 顯微鏡技術來追蹤第八因子在細胞內的路徑。 由觀察得知，轉 染293細胞時，在轉染後1.5小時，兩個突變型第八因子都可 發現堆積於細胞內，且螢光顯現位置可與內質網標記訊號重疊。 在轉染後3小時，突變型第八因子的螢光強度下降許多，推測 可能在這段時間中發生突變型蛋白的崩解 (degradation) 而造 成。 表現於HepG2細胞時，最短可在30分鐘內看到蛋白的表 現。 在轉染HepG2細胞後1.5、3及6小時，也同樣可在共軛 焦顯微鏡下追蹤野生型及兩個突變型第八因子蛋白的表現。 Factor VIII functions as a cofactor of factor IXa in the blood coagulation. The disorder of factor VIII deficiency causes hemophilia A, which is X-linked and is the most commonly- inherited bleeding disease. The intracellular biosynthesis and processing of factor VIII are not well known as well as the fact why it is 100 folds less in plasma concentration than its very similar homologue, factor V. To understand the biosynthesis of factor VIII and the mechanism of defects caused by some mutant factor VIIIs in hemophilia A patients, this study was decided to express factor VIII and two mutants with single base substitutions at position P146 (proline at amino acid residue 146) and I386, respectively, in human kidney 293 cells. Both mutants were replaced by serine. The two mutants caused low levels of factor VIII protein in patients' plasmas. Analysis of their plasma levels and the expressed protein levels in the cultured supernatant revealed that the plasma levels of P146S and I386S were 0.87 ng/ml and 0.63 ng/ml, respectively, and the levels in the supernatant were undetectable for P146S and 0.01 ng/ml for I386S. Since the biochemical experiments were limited due to the low expression of factor VIII in tissue culture system, an alternative way was taken to investigate the factor VIII biosynthesis in the cells by confocal microscopy. The results from confocal microscopy analysis demonstrated that both wildtype and mutant factor VIIIs were successfully expressed in 293 cells and a hepatoblastoma cell line, HepG2 cells. After plasmid electroporated into 293 cells for 1.5 hours, mutant proteins appeared to accumulate in the cells compared to the wildtype protein. At 3 hours after transfection, the fluorescence intensity of mutant proteins was decreased indicating the degradation of the two mutant factor VIIIs. The wildtype or mutant factor VIIIs seemed to be colocalized with BiP or calnexin since the fluorescence of those were in the same location when combined the fluorescence together. The situation of wildtype and mutant factor VIIIs' expression using a liposome-mediated transfection method were also examined at 1.5, 3, and 6 hours after overlaid the DNA-liposome mixture. Weak signals could be detected at 30 minutes after the beginning of transfection into HepG2 cells, and increasing intensity of signals were also observed until 6 hours.