本論文係探討人類嗜酸性白血球神經毒性蛋白（核醣核酸水解酵素2）與人類嗜酸性白血球陽離子蛋白（核醣核酸水解酵素3）之不同生物功能，研究的重點為二者啟動子活性及訊號胜肽毒性的差異。人類嗜酸性白血球神經毒性蛋白與人類嗜酸性白血球陽離子蛋白為核醣核酸水解酵素家族的成員，二者的基因均位於第14號染色體長臂第2區第4條帶和長臂第3區第4條帶之間。序列比對其基因上游一千個鹼基對的啟動子區域顯示二者具92%的相似度，但在嗜酸性白血球神經毒性蛋白啟動子的-81/-48位置發現一段含34個鹼基對的區域，而人類嗜酸性白血球陽離子蛋白則無。由啟動子分析的實驗得知，在人類肝癌細胞株、胚胎腎臟細胞、慢性原髓白血病細胞株以及急性原髓白血病細胞株中這34個鹼基對區域能加強人類嗜酸性白血球神經毒性蛋白之轉錄能力。膠體電泳流動轉移試驗(EMSA)以及去氧核醣核酸親和力沈澱試驗(DAPA)則發現轉錄因子MAZ跟Sp1會結合在此34個鹼基對的區域，繼而造成基因表現的組織專一性。
人類嗜酸性白血球陽離子蛋白是由人類嗜酸性白血球所分泌的的毒性蛋白，此毒性蛋白不只具備核醣核酸水解活性，還有抗寄生蟲、抗細菌以及神經毒素等特性。過往的研究中顯示表現嗜酸性白血球陽離子蛋白的訊號胜肽會抑制細菌的生長，進而造成細胞毒性。本研究表現出嗜酸性白血球陽離子蛋白訊號胜肽銜接強化綠色螢光蛋白的重組融合蛋白，並製備在訊號胜肽第三個胺基酸變異的突變株P3X，分別轉殖到大腸桿菌及嗜甲醇酵母菌中。細胞生長實驗顯示P3位置對標的蛋白質表現及細胞毒性均有影響，製備嗜酸性白血球神經毒性蛋白訊號胜肽的突變株P3X，其結果不同於嗜酸性白血球陽離子蛋白訊號胜肽的突變株，結果顯示出二者在轉譯上之不同生物功能。

Human eosinophil-derived neurotoxin (edn, RNase2) and human eosinophil cationic protein (ecp, RNase3) belong to the ribonuclease A (RNase A) superfamily, and the genes of edn and ecp are located in the q24-q31 region of chromosome 14. This thesis aims to investigate the differential function of edn and ecp in terms of promoter activity and function of signal peptides. Meanwhile, multiple sequences alignment of upstream 1 kb region of human edn and ecp showed 92% identity, but a major difference was a 34-nucleotide element (-81/-48) only appeared in the edn promoter. Using leuciferase as a reporter, the 34-nucleotide (34-nt) element was found to be essential for transactivation of edn promoter in transiently transfected human HepG2, HEK293, K562 and HL60-C15 cell lines. In order to investigate transcription factor binding sites governing the regulatory transactivation, electrophoretic mobility shift assay (EMSA) and DNA affinity precipitation assay (DAPA) were performed. The results revealed that two transcription factors, MAZ and Sp1, were involved in binding to the 34-nt region of edn promoter. Our data suggested that the 34-nt element played an important role in tissue specific expression of edn, whereas the expression level of ecp was much lower.
ECP is a small cationic toxin secreted by human eosinophil and it possesses not only ribonucleolytic activity, but also anti-parasitic, anti-bacterial and neurotoxic properties. In this study, we found that the expression of signal peptide of ECP (ECPsp) inhibited growth of bacterial and yeast cells. In order to investigate the cytotoxic effects, the third residue of ECPsp was mutated and fused with enhanced green fluorescent protein (eGFP), and transformed into E. coli BL21 (DE3) and P. pastoris GS115. Unlike the wild type ECPsp P3 clone, P3W and P3G survived in E. coli BL21 (DE3) cells. Further analysis revealed that upon induction with IPTG, the bacterial growth of P3W mutant ceased immediately, while the growth of P3G mutant was unaffected. In the P. pastoris, expression of P3W and P3G led to the cessation of growth upon the methanol induction. Therefore, ECPsp was not only a signal sequence, but also toxic to cells. The third amino acid proline of ECPsp played a key role for its toxicity. Interestingly, when the third residue of signal peptide of EDN (EDNsp) was mutated, none of the transformant survived, quite similar to what happened with the wild type EDNsp clones. Taken together, they revealed differential function between ECP and EDN at translation.

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