Membrane-bound carboxypeptidase E facilitates the entry of eosinophil cationic protein into neuroendocrine cells.
ECP (eosinophil cationic protein) is a major component of eosinophil granule proteins, and is used as a clinical biomarker 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 the present study, mECP-eGFP-6H, a recombinant fusion protein containing mature ECP (mECP), enhanced green fluorescence protein (eGFP) and a His(6) tag (6H), 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 an IC(50) of 0.8 microM. By yeast two-hybrid screening and immunoprecipitation, we have identified a specific protein-protein interaction between mECP and CPE (carboxypeptidase E), a well characterized metalloprotease. Further in vivo yeast two-hybrid screening has also revealed that residues 318-387 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 pre-pro-HA-CPE(S471A,E472A) in GH3 cells, suggesting that the entry of mECP-eGFP-6H is associated with the recycling of CPE in GH3 cells. Taken together, we have demonstrated that CPE possesses a novel function to facilitate the entry of ECP to neuroendocrine cells, and such an endocytotic process allows the cytotoxic ECP to inhibit growth of the target cells.
Wu
Chia-Mao
CM
Department of Life Science, Institute of Molecular and Cellular Biology, National Tsing Hua University, Hsinchu, Taiwan 30013, Republic of China.
Chang
Hao-Teng
HT
Chang
Margaret Dah-Tsyr
MD
D000595:Amino Acid Sequence; D000818:Animals; D043423:Carboxypeptidase H / Q000378:metabolism*; D002460:Cell Line; D002462:Cell Membrane / Q000201:enzymology; D049109:Cell Proliferation; D004705:Endocytosis / Q000502:physiology; D047150:Eosinophil Cationic Protein / Q000378:metabolism* / Q000502:physiology; D049452:Green Fluorescent Proteins; D008969:Molecular Sequence Data; D009490:Neurosecretory Systems / Q000166:cytology / Q000378:metabolism*; D017354:Point Mutation; D011485:Protein Binding; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D022142:Secretory Vesicles / Q000378:metabolism
D011993:Recombinant Fusion Proteins; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins; D047150:Eosinophil Cationic Protein; C479220:RNASE3 protein, human; D043423:Carboxypeptidase H