Corin, a transmembrane cardiac serine protease, acts as a pro-atrial natriuretic peptide-converting enzyme

Abstract

Atrial natriuretic peptide (ANP) is a cardiac hormone essential for the regulation of blood pressure. In cardiac myocytes, ANP is synthesized as a precursor, pro-ANP, that is converted to biologically active ANP by an unknown membrane-associated protease. Recently, we cloned a transmembrane serine protease, corin, that is highly expressed in the heart. In this study, we examine effects of corin on pro-ANP processing. Our results show that recombinant human corin converts pro-ANP to ANP and that the cleavage in pro-ANP by corin is highly sequence specific. Our findings suggest that corin is the long-sought pro-ANP-converting enzyme and that the corin-mediated pro-ANP activation may play a role in regulating blood pressure.

Figure 1

Cleavage of pro-ANP by corin. (a) Western analysis of recombinant corin. Human embryonic kidney 293 cells were stably transfected with corin expression vector pcDNACorin/V5 or a control vector pcDNA. Conditioned medium, total cell lysate, and membrane fractions were prepared based on methods described previously (27). Expression of corin was detected by SDS/PAGE and Western blotting using an anti-V5 antibody. (b) Cotransfection of pro-ANP and corin expression vectors. Pro-ANP expression vector pcDNAproANP was cotransfected into 293 cells with vectors expressing corin (pcDNACorin, lane 2), prothrombin (pProthrombin, lane 3), hepsin (pHepsin, lane 4), or a control vector (pcDNA, lane 1). Conditioned medium was collected and processing of pro-ANP was analyzed by Western blotting using an anti-V5 antibody. (c) Incubation of conditioned medium containing pro-ANP with transfected cells. Conditioned medium from pcDNAproANP transfected cells was collected and incubated with 293 cells transfected with vectors expressing corin (pcDNACorin, lane 2), prothrombin (pProthrombin, lane 3), hepsin (pHepsin, lane 4), or a control vector (pcDNA, lane 1). Processing of pro-ANP was analyzed by Western blotting.

Figure 2

Cleavage of pro-ANP depended on the catalytic activity of corin. (a) Pro-ANP expression vector pcDNAproANP was cotransfected into 293 cells with vectors expressing wild-type corin (pcDNACorin, lane 2) and mutant corin S985A (pcDNACorin S985A, lane 1) or a control vector (pcDNA, lane 3). Pro-ANP and its derivatives were detected by Western blotting using an anti-V5 antibody. (b) Expression of recombinant corin in transfected 293 cells. To verify expression of recombinant corin in transfected cells, cell lysate was prepared and analyzed by SDS/PAGE and Western blotting. Expression of recombinant corin was detected in pcDNACorinV5 and pcDNACorin S985A but not pcDNA, transfected cells. A nonspecific (ns) band was present in samples from pcDNACorinV5, pcDNACorinS985A, and pcDNA transfected 293 cells.

Figure 3

Determination of the corin cleavage site in pro-ANP. Expression vectors for wild-type pro-ANP or mutant pro-ANPs R98G, R101A, and R102A were cotransfected into 293 cells with corin expression vector, pcDNACorin. Pro-ANP and its derivatives in conditioned medium were analyzed by Western blotting using an anti-V5 antibody. At high resolution, two specific bands of pro-ANP were detected on the Western blot possibly caused by differences in glycosylation in transiently transfected 293 cells.

Figure 4

Cleavage of pro-BNP by corin. Expression vectors for human pro-ANP or pro-BNP were cotransfected into 293 cells with corin expression vector, pcDNACorin, or a control vector pcDNA. Pro-ANP, pro-BNP, and their derivatives in conditioned medium were analyzed by Western blotting using an anti-V5 antibody.

Figure 5

Corin regulates the ANP-mediated pathway by activating pro-ANP. Deficiency in either ANP or its receptor causes hypertension (–13), suggesting that defects in corin might also lead to hypertension and preeclampsia.