AE1蛋白表現在紅血球和腎臟遠端小管細胞之細胞膜,其功能為維持細胞形狀及進行細胞內外之Cl-與HCO3- 交換。AE1突變則被證明為人類遺傳性球狀紅血球症及遠端腎小管酸血症形成原因之一。但是人類遺傳性球狀紅血球症和遠端腎小管酸血症幾乎不會同時存在於同一個體。唯一人類的病例為V488M的單一突變而導致AE1蛋白質功能完全喪失所造成遺傳性球狀紅血球症和遠端腎小管酸血症。本研究中為目前所知全世界第二個同時具人類遺傳性球狀紅血球症和遠端腎小管酸血症之病例。基因定序分析結果得知,此病人之AE1為一個新突變E522K合併一個已知的G70D的突變。我們利用分子生物學方法建構了eAE1及kAE1和AE1突變蛋白質之cDNA,並將之嵌入數種可在哺乳類細胞表現之載體中。以轉殖方法將這些eAE1及kAE1和AE1突變蛋白質表現於狗的腎臟細胞 (MDCK)內,以觀察eAE1和kAE1和AE1突變蛋白質的運送,蛋白質表現的位置,與其穩定度。結果發現無論是AE1 WT,E522K或G701D突變蛋白間皆可以形成同質性或異質性複合體 (homodimer or heterodimer)。在形成同質複合體的情況下,AE1 WT和E522K突變蛋白質皆可被運送到細胞膜,而AE1 G701D或E522K&G701D則不能被運送至細胞膜而停置在細胞質內。在形成異質複合體的情況下,結果顯示E522K或G701D和AE1 WT所組成的異質複合體,可被運送到細胞表面去,而E522K和G701D所組成的異質複合體,則大多數停置於細胞質內,並可能在溶小體的位置進行降解。由於E522K&G701D蛋白質大多停置於細胞質內,與溶小體有部分共位現象;且蛋白質的穩定度較WT顯著下降,因此我們推論AE1 E522K/G701D運送至細胞膜的阻斷及蛋白質穩定度降低是造成球型血球症與遠端腎小管酸血症的主要原因。

The function of AE1 was to help maintain the cell shape and to exchang Cl- and HCO3-. AE1 is mainly expressed in the erythrocyte and in the acid-secreting, type A intercalated cells of the kidney. It has been demonstrated that nonsense and frameshift mutations of AE1 gene are associated with hereditary spherocytosis (HS) and inherited distal renal tubular acidosis (dRTA). HS and dRTA, however, are almost always mutually exclusive. The first human exception is a single instance of the total absence of AE1 secondary to the mutation Coimbra (V488M) in the homozygous state. We report the second human exception with nearly total absence of AE1 protein due to a novel heterozygous E522K mutation in combination with a heterozygous G701D mutation that show the phenotypes of severe HS and complete dRTA. The protein trafficking and subcellular localization of the kAE1 and kAE1 mutants in transfected MDCK cells were examined. Our results show that AE1 WT, AE1 E522K, or AE1 G701D can form homodimer or heterodimer to each other. For homodimer, kAE1 WT or E522K mutation can traffic to the plasma membrane (44.50% and 33.50%, respectively). Whereas kAE1 G701D or kAE1 E522K&G701D was largely retained in cytoplasm (99.99% and 99.99%, respectively). For heterodimer, our results show that kAE1 E522K/WT or kAE1 G701D/WT traffic to the plasma membrane (40.25% and 43.50%, respectively). Whereas, E522K/G701D also mistargeted to the plasma membrane (0.04%) and retained in the cytoplasm (99.96%, respectively). The protein stability of kAE1 E522K&G701D decreases significantly when compared with that of kAE1 WT. Together with that the kAE1 E522K/G701D largely colocalized with losozyme, these data suggest that the defect of the E522K/G701D trafficking to the plasma membrane and the decrease of protein stability cause the hereditary spherocytosis (HS) and distal renal tubular acidosis (dRTA).

中文摘要.............................................3
Abstract..............................................5
Abbreviation......................................7
Introduction.......................................8
Subjects .........................................18
Materials and Methods.................21
Results............................................31
Discussion.....................................41
References.....................................45
Table and figure.............................57
Appendix..........................................71

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