Ectodomain Shedding and Autocleavage of the Cardiac Membrane Protease Corin

Abstract

Corin is a cardiac membrane protease that activates natriuretic peptides. It is unknown how corin function is regulated. Recently, soluble corin was detected in human plasma, suggesting that corin may be shed from cardiomyocytes. Here we examined soluble corin production and activity and determined the proteolytic enzymes responsible for corin cleavage. We expressed human corin in HEK 293 cells and detected three soluble fragments of ϳ180, ϳ160, and ϳ100 kDa, respectively, in the cultured medium by Western blot analysis. All three fragments were derived from activated corin molecules. Similar results were obtained in HL-1 cardiomyocytes. Using protease inhibitors, ionomycin and phorbol myristate acetate stimulation, small interfering RNA knockdown, and site-directed mutagenesis, we found that ADAM10 was primarily responsible for shedding corin in its juxtamembrane region to release the ϳ180-kDa fragment, corresponding to the near-entire extracellular region. In contrast, the ϳ160-and ϳ100-kDa fragments were from corin autocleavage at Arg-164 in frizzled 1 domain and Arg-427 in LDL receptor 5 domain, respectively. In functional studies, the ϳ180-kDa fragment activated atrial natriuretic peptide, whereas the ϳ160-and ϳ100-kDa fragments did not. Our data indicate that ADAM-mediated shedding and corin autocleavage are important mechanisms regulating corin function and preventing excessive, potentially hazardous, proteolytic activities in the heart. Natriuretic peptides act as a cardiac endocrine mechanism to regulate blood volume and pressure (1, 2). Corin is a serine protease that activates atrial natriuretic peptides (ANP) 2 and B-type natriuretic peptides in the heart (3, 4). The physiological importance of corin function has been shown in mouse models, in which disruption of the corin gene causes hypertension and cardiac hypertrophy (5, 6). In humans, population studies have identified polymorphisms (T555I/Q568P) in the corin gene in African Americans who had high blood pressure and cardiac hypertrophy (7-9). The polymorphisms were shown to alter corin protein structure and impair its biological activity in functional studies (10). In patients with heart failure (HF), the corin variants were associated with poor natriuretic peptide processing and worse clinical outcomes (11).Corin belongs to the type II transmembrane serine protease family, a subclass of trypsin-like enzymes defined by an N-terminal transmembrane domain and a C-terminal protease domain (12, 13). The transmembrane domain anchors the proteases on the cell surface, localizing the biological activities at specific sites. Under physiological and/or pathological conditions, type II transmembrane serine proteases can be shed from the cell surface. Soluble forms of enteropeptidase, hepsin and matriptases, for example, have been reported (14 -17). Recently, soluble corin was detected in human blood, indicating that corin is shed from the heart (18 -20). Interestingly, plasma corin levels were lower in patients with severe HF compared with those of normal...

Corin Mutation R539C from Hypertensive Patients Impairs Zymogen Activation and Generates an Inactive Alternative Ectodomain Fragment

“…As shown in Fig. 5A, GM6001 and TAPI-1 reduced the level of the ϳ180-kDa fragment, consistent with a previous report that the fragment was cleaved by an ADAM protease (32). The results also indicated that the production of the ϳ75-kDa fragment may not depend on the ϳ180-kDa fragment.…”

“…4B, upper panel). The sample from the inactive corin mutant R801A had only the ϳ180-kDa band, consistent with previous findings that the ϳ160-and ϳ100-kDa bands are products of corin autocleavage (32). In the sample from the R539C mutant, a prominent extra band of ϳ75 kDa was detected (Fig.…”

“…4A) (32). To examine if the R539C mutation affects corin shedding, we analyzed corin fragments in the conditioned medium from transfected HEK293 cells.…”

“…Plasmids-Plasmids expressing human WT corin, the S985A mutant (in which the catalytic Ser-985 was replaced with Ala), and the R801A mutant (in which the activation cleavage site Arg-801 was replaced with Ala) were described previously (32,33). Plasmids expressing corin mutants R539C, R539A, R539C/ R480A, R530A/R539C, R539C/R588A, R539C/R597A/R598A, R539C/R619A, R539C/R670A, R539C/S985A, C540A, and R539C/C540A were made by PCR-based site-directed mutagenesis.…”

“…Plasmids expressing corin mutants R539C, R539A, R539C/ R480A, R530A/R539C, R539C/R588A, R539C/R597A/R598A, R539C/R619A, R539C/R670A, R539C/S985A, C540A, and R539C/C540A were made by PCR-based site-directed mutagenesis. Recombinant corin proteins encoded by these plasmids contained a C-terminal V5 tag to facilitate protein detection by Western blotting (32,33).…”