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 approximately 40% activity, whereas corin mutants lacking single LDLR repeat 1, 2, 3, or 4 had approximately 49, approximately 12, approximately 53, and approximately 77% 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 approximately 25, approximately 11, approximately 16, and approximately 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.
Knappe
Sabine
S
Department of Cardiovascular Research, Berlex Biosciences, 2600 Hilltop Drive, Richmond, CA 94804, USA.
Wu
Faye
F
Madlansacay
Mary Rose
MR
Wu
Qingyu
Q
D000595:Amino Acid Sequence; D009320:Atrial Natriuretic Factor / Q000737:chemistry*; D001665:Binding Sites; D002118:Calcium / Q000378:metabolism; D002384:Catalysis; D020134:Catalytic Domain; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D017353:Gene Deletion; D005822:Genetic Vectors; D006801:Humans; D007700:Kinetics; D008954:Models, Biological; D008957:Models, Genetic; D008969:Molecular Sequence Data; D009154:Mutation; D010455:Peptides / Q000737:chemistry; D010957:Plasmids / Q000378:metabolism; D017354:Point Mutation; D016133:Polymerase Chain Reaction; D011480:Protease Inhibitors / Q000494:pharmacology; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D017386:Sequence Homology, Amino Acid; D012697:Serine Endopeptidases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D014162:Transfection
D010455:Peptides; D011480:Protease Inhibitors; D009320:Atrial Natriuretic Factor; C119812:CORIN protein, human; D012697:Serine Endopeptidases; D002118:Calcium