Functional analysis of the transmembrane domain and activation cleavage of human corin: design and characterization of a soluble corin.
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 EK-solCorin 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.
Knappe
Sabine
S
Department of Cardiovascular Research, Berlex Biosciences, Richmond, California 94804, USA.
Wu
Faye
F
Masikat
Mary Rose
MR
Morser
John
J
Wu
Qingyu
Q
D009320:Atrial Natriuretic Factor / Q000737:chemistry; D001665:Binding Sites; D015153:Blotting, Western; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism*; D006152:Cyclic GMP / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D004591:Electrophoresis, Polyacrylamide Gel; D004765:Enteropeptidase / Q000378:metabolism; D004791:Enzyme Inhibitors / Q000494:pharmacology; D015951:Factor Xa / Q000737:chemistry; D005822:Genetic Vectors; D006801:Humans; D007610:Kallikreins / Q000737:chemistry; D007700:Kinetics; D009154:Mutation; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D012697:Serine Endopeptidases / Q000737:chemistry*; D013917:Thrombin / Q000737:chemistry; D014162:Transfection; D014357:Trypsin / Q000737:chemistry
D004791:Enzyme Inhibitors; D011994:Recombinant Proteins; D009320:Atrial Natriuretic Factor; C119812:CORIN protein, human; D007610:Kallikreins; D012697:Serine Endopeptidases; D014357:Trypsin; D013917:Thrombin; D015951:Factor Xa; D004765:Enteropeptidase; D006152:Cyclic GMP