STRUCTURAL DETERMINANTS OF BIG ENDOTHELIN 1 PROCESSING

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Epidemiologic studies indicate that death from cardiovascular disease is the number one cause of natural deaths in this country. It is well established that the endothelium is very important in the control of cardiovascular homeostasis in health and disease. Factors released from endothelial cells modulate the vascular smooth muscle contractility and proliferation, which are altered in some disease states such as hypertension and atherosclerosis. One of these factors, endothelin-1 (ET-1), has been shown to be involved in systemic and pulmonary hypertension, advanced atherosclerosis, and acute myocardial infarction. The elevated plasma ET-1 levels in these patients can be regulated at the biosynthesis level by blocking the conversion of big ET-1 to bioactive ET-1 with endothelin converting enzyme (ECE) inhibitors and/or at the receptor level using receptor antagonists. Although much work has been done on endothelin receptor antagonists and on the purification and cloning of ECE, the structural determinants required for the high specificity of ECE-1 for big ET-1 is not known. One approach is to study the contribution of conserved amino acid residues or specific domains to the structure of big ET-1 and ECE-1. In this research proposal, highly conserved amino acid residues in big ET-1 will be replaced by the corresponding residues in big ET-2 and big ET-3 using site-directed mutagenesis of preproendothelin-1 (PPET-1) cDNA. The mutant or wild-type cDNAs and ECE-1 cDNA will then be co-transfected into Chinese hamster ovary (CHO) cells. The effects of these mutations on the conversion of big ET-1 to ET-1 by ECE-1 will be tested by analyzing the transfection media for immunoreactive big ET-1 and ET-1, and the results will be compared to that of wild-type big ET- 1. In addition, a truncated ECE- 1 cDNA will be constructed by replacing the sequence of the ECE- 1 cDNA encoding the N-terminal region of ECE- 1, including the membrane-spanning region (amino acid residues 1-77 in ECE-1), by the secretion signal sequence of PPET-1 and an affinity tag of six consecutive histidine residues using polymerase chain reaction. The wild-type and truncated ECE-1 proteins will be expressed in CHO cells. The His-tagged soluble enzyme will be purified using nickel-nitrilotriacetate (Ni-NAT) columns and soluble membrane fractions containing the native enzyme will be prepared. The recombinant native membrane-bound and soluble ECE-1 will then be characterized by analyzing the conversion of synthetic big ET-1 to ET-1 in vitro. The proposed studies using current techniques in molecular biology will provide information on the structure of ECE-1, as well as developing new approaches to prepare increased amounts of enzyme for structural studies and antibody production. The results of this study have the potential to lead to the development of new ECE inhibitors which could be used as therapeutic agents.