Identification of domain structures in the propeptide of corin essential for the processing of proatrial natriuretic peptide

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 similar to 40% activity, whereas corin mutants lacking single LDLR repeat 1, 2, 3, or 4 had similar to 49, similar to 12, similar to 53, and similar to 77% activity, respectively. We also made corin mutants with a single mutation at a conserved Asp residue that coordinates Ca2+-binding in LDLR repeats 1, 2, 3, or 4 (D300Y, D336Y, D373Y, and D410Y) and showed that these mutants had similar to 25, similar to 11, similar to 16, and similar to 82% 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.