Abstract
Eosinophil cationic protein (ECP) is a major component of eosinophil granule proteins and is used as a clinical bio-marker for asthma and allergic inflammatory disease. ECP has been implicated in damage to the cell membrane of many tissue types, but the mechanism is not well known. In this study, mECP-eGFP-6H, a recombinant fusion protein containing mature ECP (mECP), green fluorescence protein (eGFP) and Histag, has been expressed, purified and added to GH3 neuroendocrine cells to study the internalization ability of ECP. We found that mECP-eGFP-6H entered into GH3 neuroendocrine cells and inhibited the growth of the cells with the IC50 of 0.8 □M. By yeast two-hybrid screening and immunoprecipitation, we have identified a specific protein-protein interaction between mature mECP and carboxypeptidase E (CPE), a well characterized metalloprotease. Further in vivo yeast two-hybrid screening has also revealed that residues 318 to 387 located in a region of unknown function in mature CPE are indispensable for association with mECP. In addition, the uptake of mECP-eGFP-6H is suppressed by dominant-negative expression of the recycling defect mutant preproHA-CPES471A,E472A in GH3 cells, suggesting that the entrance of mECP-eGFP-6H is associated with the recycling of CPE in GH3 cells. On the other hand, the properties of mature ECP have been well studied but that of the signal peptide of ECP (ECPsp) are not clear. In this thesis, several chimeric proteins containing N-terminal fusion of ECPsp were generated, and introduced into Escherichia coli, Pichia pastoris and human epidermoid carcinoma cell line A431 to study the function of ECPsp. We found that expression of ECPsp chimeric proteins inhibited the growth of E. coli and P. pastoris but not A431 cells. Primary sequence analysis and in vitro transcription/translation of ECPsp have revealed that it is a potential substrate for human signal peptide peptidase (hSPP), an intramembrane protease located in endoplasmic reticulum. In addition, knockdown of the hSPP mRNA expression in ECPsp-eGFP/A431 cells caused the growth inhibitory effect, whereas complementary expression of hSPP in Pichia pastoris system rescued the cell growth. Taken together, we have demonstrated that CPE possesses a novel function to facilitate the entry of ECP to neuroendocrine cell, and such endocytosis process allows the cytotoxic ECP to inhibit growth of the target cells. In addition, we also demonstrated that ECPsp is a toxic signal peptide, and expression of hSPP protects the cells from growth inhibition.