Functional Analysis of the Transmembrane Domain and Activation Cleavage of Human Corin

Abstract

Corin is a cardiac transmembrane serine protease. In cell-based studies, corin converted pro-atrial natriuretic peptide (pro-ANP) to mature ANP, suggesting that corin is potentially the pro-ANP convertase. In this study, we evaluated the importance of the transmembrane domain and activation cleavage in human corin. We showed that a soluble corin that consists of only the extracellular domain was capable of processing recombinant human pro-ANP in cell-based assays. In contrast, a mutation at the conserved activation cleavage site, R801A, abolished the function of corin, demonstrating that the activation cleavage is essential for corin activity. These results allowed us to design, express, and purify a mutant soluble corin, EKsolCorin, that contains an enterokinase recognition sequence at the activation cleavage site. Purified EKsolCorin was activated by enterokinase in a dose-dependent manner. Activated EKsolCorin had hydrolytic activity toward peptide substrates with a preference for Arg and Lys residues in the P-1 position. This activity of EKsolCorin was inhibited by trypsin-like serine protease inhibitors but not inhibitors of chymotrypsin-like, cysteine-, or metallo-proteases. In pro-ANP processing assays, purified active EKsolCorin converted recombinant human pro-ANP to biologically active ANP in a highly sequence-specific manner. The pro-ANP processing activity of EKsolCorin was not inhibited by human plasma. Together, our data indicate that the transmembrane domain is not necessary for the biological activity of corin but may be a mechanism to localize corin at specific sites, whereas the proteolytic cleavage at the activation site is an essential step in controlling the activity of corin.Corin is a mosaic serine protease that was recently identified from the human heart (1, 2). It consists of 1,042 amino acids and contains an integral transmembrane domain near the N terminus. In the extracellular region of corin, there are two frizzled-like cysteine-rich domains, eight low density lipoprotein receptor type A repeats, a scavenger receptor-like cysteinerich domain, and a C-terminal trypsin-like protease domain. Topologically, corin belongs to the newly defined type II transmembrane serine protease family (3-6), which includes enterokinase (EK) 1 (7), hepsin (8), matriptases (9 -12), TMPRSS2-5 (13-16), human airway trypsin-like protease (17), MSPL (18), DESC1 (19), and polyserase-I (20). The combination of domains present in corin, however, is unique among the trypsin-like serine proteases, because corin is the only serine protease identified so far that contains frizzled-like cysteinerich domains. Corin mRNA and protein are abundantly expressed in the heart (1, 2), suggesting that corin might have a role in the cardiovascular system. In cell-based experiments, we showed that recombinant human corin mediated the conversion of proatrial natriuretic peptide (pro-ANP) and pro-brain natriuretic peptide to mature ANP and brain natriuretic peptide (21), both of which are cardiac hormones important in maintaining ...

Matriptase-2 (TMPRSS6): a proteolytic regulator of iron homeostasis

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IL-1β increases urinary corin in patients with primary proteinuric kidney diseases and in 293 cells

“…293 cells (26) were obtained from ATCC. 293-corin cells were stably transfected with pcDNA3.1-h corin (plasmid containing full-length human corin; Invitrogen, Carlsbad, USA) (27), which was constructed by the Cyrus Tang Medical Institute of Soochow University (Soochow, China) (9,22,28). In the present study, 293 corin cells were cultured in Dulbecco's modified Eagle's medium with 500 ng/ml G418, at 37˚C with 95% air and 5% CO 2 .…”

New issues on measurement of B-type natriuretic peptides

“…NT-proBNP is an inactive peptide produced in an equimolar ratio together with BNP by action of some cellular or circulating proteolytic enzymes (such as corin and furin) on the precursor pro-hormone (proBNP) (Figure 2) [1,14,[24][25][26][27][28]. As a result, NT-proBNP assay is not a reliable index of the biological (natriuretic) activity of cardiac endocrine system in severe HF because the concentration of less active peptides in patients with severe HF (such as NT-proBNP and proBNP) is greatly higher than that of the active peptide BNP (Figure 1) [1,[29][30][31][32][33].…”

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