Abstract
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.