Abstract
Human Eosinophil Cationic Protein (hECP), also named as hRNase3, is the major component of eosinophil granule proteins and is used as a clinical bio-marker for asthma and allergic inflammatory disease. It is considered as a mediator of tissue damage based on the increased protein levels detected in serum and bronchoalveolar lavage fluid. It has been proposed that hRNase3 may interact with the target cells through other mediators such as some membrane proteins. In this study, a hRNase3-interacting membrane protein, carboxypeptidase E (CPE) was identified by yeast two-hybrid screening. Further in vitro binding assay demonstrated the residues 318–387 located in mature CPE were important for their interaction. Employing reinforced merging algorithms (RMA) we have identified a unique peptide motif 346-374, where residues E352, K355, E359, K362, N363, I366, E370 and R374 were apparently exposed to the surface. Site-directed mutagenesis was further carried out and the results showed that N363A and K362A possessed 4-fold and 2-fold decrease in interaction with hRNase3, respectively, indicating that these two residues were crucial for the molecular interaction. We also demonstrated that the interaction strength was reduced between mCPE E359A and hRNase3 using an MBP pull-down assay, surface plasmon resonance and molecular docking under in vitro conditions. In addition, we have further identified the epitope of a monoclonal antibody against hCPE employing RMA and expression of deletion mutants of CPE. Our data revealed that the epitope could be mapped to S180-D186 by both methods.