Abstract-Corin is a cardiac serine protease that acts as the pro-atrial natriuretic peptide (ANP) convertase. Recently, 2 single-nucleotide polymorphisms (SNPs) (T555I and Q568P) in the human corin gene have been identified in genetic epidemiological studies. The minor I555/P568 allele, which is more common in African Americans, is associated with hypertension and cardiac hypertrophy. In this study, we examined the effect of T555I and Q568P amino acid substitutions on corin function. We found that corin frizzled-like domain 2, where T555I/Q568P substitutions occur, was required for efficient pro-ANP processing in functional assays. Mutant corin lacking this domain had 30Ϯ5% (PϽ0.01) activity compared to that of wild type. Similarly, corin variant T555I/Q568P had a reduced (38Ϯ7%, PϽ0.01) pro-ANP processing activity compared to that of wild type. The variant also exhibited a low activity (44Ϯ15%, PϽ0.05) in processing pro-brain natriuretic peptide (BNP). We next examined the biochemical basis for the loss of activity in T555I/Q568P variant and found that the zymogen activation of the corin variant was impaired significantly, as indicated by the absence of the activated protease domain fragment. This finding was confirmed in human embryonic kidney (HEK)293 cells and murine HL-1 cardiomyocytes. Thus, our results show that the corin gene SNPs associated with hypertension and cardiac hypertrophy impair corin zymogen activation and natriuretic peptide processing activity. Our data suggest that corin deficiency may be an important mechanism in hypertensive and heart diseases. (Circ Res. 2008;103:502-508.)Key Words: natriuretic peptides Ⅲ genetic variants Ⅲ protease A trial natriuretic peptide (ANP) is a cardiac hormone that regulates blood pressure and salt-water balance. 1-3 The ANP pathway also has an antihypertrophic function in the heart, which is independent of its systemic action on blood pressure. 4 -7 Corin is a cardiac enzyme of the type II transmembrane serine protease family. 8 -10 It has a cytoplasmic tail and a transmembrane domain near the N terminus. In its extracellular region, there are 2 frizzled-like domains, 8 LDL receptor (LDLR) repeats, a scavenger receptor-like domain, and a C-terminal trypsin-like protease domain. Corin converts pro-ANP into active ANP. 11,12 In mice, corin deficiency prevented pro-ANP activation, 13 demonstrating that corin is the physiological pro-ANP convertase. Corin-null mice developed hypertension and cardiac hypertrophy. 13 Corin also cleaved pro-brain natriuretic peptide (BNP) in vitro, although the cleavage was less efficient than that for pro-ANP. 12 Hypertension is the most common cardiovascular disease, and its prevalence is even higher in African Americans, but the underlying mechanism is unclear. 14,15 Recently, 2 nonsynonymous and nonconservative single-nucleotide polymorphisms (SNPs) (T555I and Q568P) are found in the human corin gene. These 2 SNPs are in complete linkage disequilibrium in the population and, as a result, are colocalized in a minor allele (I555...

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 ...

Corin is a type II transmembrane serine protease and functions as the proatrial natriuretic peptide (pro-ANP) convertase in the heart. In the extracellular region of corin, there are two frizzled-like cysteine-rich domains, eight low density lipoprotein receptor (LDLR) repeats, a macrophage scavenger receptor-like domain, and a trypsin-like protease domain at the C terminus. To examine the functional importance of the domain structures in the propeptide of corin for pro-ANP processing, we constructed a soluble corin, EKshortCorin, that consists of only the protease domain and contains an enterokinase (EK) recognition sequence at the conserved activation cleavage site. After being activated by EK, EKshortCorin exhibited catalytic activity toward chromogenic substrates but failed to cleave pro-ANP, indicating that certain domain structures in the propeptide are required for pro-ANP processing. We then constructed a series of corin deletion mutants and studied their functions in pro-ANP processing. Compared with that of the full-length corin, a corin mutant lacking frizzled 1 domain exhibited ϳ40% activity, whereas corin mutants lacking single LDLR repeat 1, 2, 3, or 4 had ϳ49, ϳ12, ϳ53, and ϳ77% activity, respectively. We also made corin mutants with a single mutation at a conserved Asp residue that coordinates Ca 2؉ -binding in LDLR repeats 1, 2, 3, or 4 (D300Y, D336Y, D373Y, and D410Y) and showed that these mutants had ϳ25, ϳ11, ϳ16, and ϳ82% pro-ANP processing activity, respectively. Our results indicate that frizzled 1 domain and LDLR repeats 1-4 are important structural elements for corin to recognize its physiological substrate, pro-ANP.

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The labeling of biomolecules has become standard practice in molecular biosciences. Modifications are used for detection, sorting and isolation of small molecules, complexes and entire cells. We have recently reported a method for introducing internal chemical and structural modifications into kbp-sized DNA target substrates that are frequently used in single-molecule experiments. It makes use of nicking enzymes that create single-stranded DNA gaps, which can be subsequently filled with labeled oligonucleotides. Here we provide a detailed protocol and further expand this method. We show that modifications can be introduced at distant loci within one molecule in a simple one-pot reaction. In addition, we achieve labeling on both strands at a specific locus, as demonstrated by Förster resonance energy transfer (FRET) experiments. The protocol requires an initial cloning of the target substrate (3-5 d), whereas the labeling itself takes 4-6 h. More elaborate purification and verification of label incorporation requires 2 h for each method.