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 (cid:1) 40% activity, whereas corin mutants lacking single LDLR repeat 1, 2, 3, or 4 had (cid:1) 49, (cid:1) 12, (cid:1) 53, and (cid:1) 77% activity, respectively. We also made corin mutants with a single mutation at a conserved Asp residue that coordinates Ca

Human corin is a trypsin-like serine protease first cloned from the heart (1,2). Corin mRNA and protein are abundantly expressed in cardiomyocytes of the atrium and ventricle. In transfected human embryonic kidney (HEK) 1 293 cells, recombinant human corin converted pro-atrial natriuretic peptide (pro-ANP) to biologically active ANP (3), a cardiac hormone essential in controlling blood pressure and maintaining electrolyte and body fluid homeostasis (4 -6). In cultured cardiomyocytes, overexpression of an active site mutant corin or transfection of small interfering RNAs directed against the corin gene completely blocked the processing of pro-ANP (7).
These data indicate that corin is the pro-ANP convertase that had remained elusive for many years.
Human corin is a polypeptide of 1042 amino acids that contains a short cytoplasmic tail at the N terminus followed by an integral transmembrane domain (1,2). In the extracellular region, there are two frizzled-like cysteine-rich domains, eight LDLR class A repeats, a macrophage scavenger receptor-like domain, and a trypsin-like protease domain at the C terminus (see Fig. 1A). Topologically, corin belongs to a newly defined type II transmembrane serine protease family, which includes enterokinase (EK), hepsin, matriptases, TMPRSS2-5, human airway trypsin-like protease, and polyserase-I (8 -12). The combination of the domain structures in corin is unusual among this family of serine proteases. Corin, for example, contains two frizzled-like cysteine-rich domains that are common in Wntinteracting proteins but not in trypsin-like proteases. Most recently, we expressed and purified a soluble corin that consisted of only the extracellular fragment (13). The soluble corin converted human pro-ANP to biologically active ANP in a highly sequence-specific manner, indicating that the transmembrane domain of corin is not necessary for pro-ANP processing. It remained unknown, however, whether the other domains such as frizzled-like cysteine-rich domains and LDLR repeats contribute to the pro-ANP processing activity of corin.
In this study, we assessed the functional importance of the domain structures in the propeptide of corin for pro-ANP processing. We showed that a soluble corin that consisted of only the serine protease domain retained the catalytic activity toward small peptide substrates but was inactive in processing pro-ANP. We further identified a region in the propeptide of corin comprising frizzled 1 domain and LDLR repeats 1-4 that is critical for pro-ANP processing.

EXPERIMENTAL PROCEDURES
Materials-Cell culture medium, G418, anti-V5 and anti-Xpress antibodies, transfection reagent LipofectAMINE 2000, expression vectors pSecTag/FRT/V5-His-TOPO, and pcDNA4/HisMaxC were from Invitrogen. Fetal bovine serum was from SeraCare Life Sciences, Inc. (Oceanside, CA). HEK 293 cells were from the American Type Culture Collection (Manassas, VA). Oligonucleotide primers were synthesized by BIOSOURCE International Inc. (Camarillo, CA). Restriction enzymes and DNA polymerases were from New England Biolabs Inc. (Beverly, MA). Recombinant bovine light chain EK and EK capture beads (EKapture) were from Novagen Inc. (Madison, WI). Chromogenic substrates were from DiaPharma (West Chester, OH). Phenylmethylsulfonyl fluoride was from Bachem Bioscience Inc. (King of Prussia, PA). Protease inhibitors pepstatin, leupeptin, and EDTA-free protease inhibitor mixture tablets were from Roche Diagnostics. All other chemical reagents were from Sigma.
Expression Vectors-The expression vector, pSECEKsolCorin, encoding a soluble corin (EKsolCorin) that consists of an Ig signal peptide at the N terminus followed by a 919-amino acid sequence from the extracellular region of corin (residues 124 -1042) and a viral V5 tag at the C terminus, was described previously (13). In EKsolCorin, the conserved activation cleavage sequence (RMNKR) was replaced by an EK recognition sequence (DDDDK), which allows for the activation of corin by EK (see Fig. 1A). The plasmid pSECEKsolCorin was used as a template to construct the expression vector pSECEKshortCorin, encoding a protein (EKshortCorin) that consists of an Ig signal peptide at the N terminus followed by the catalytic domain of human corin (residues 787-1042) and a viral V5 tag at the C terminus (see Fig. 1A). Like EKsolCorin, EKshortCorin contains the EK recognition sequence at the conserved activation cleavage site. Oligonucleotide primers 5Ј-GTT TAG GAG AAA GGT CTG GAT GTA AAT CTG-3Ј and 5Ј-AAA CAA GAC TGT GGG CGC CG-3Ј and Pfu polymerase were used in PCR to amplify a human corin cDNA fragment (2452-3222 bp) (1). After 30 cycles, 1 unit of Taq polymerase was added to the reaction and incubated at 72°C for 10 min. This step allows adding an extra base of adenine at the 3Ј-end of PCR products, facilitating the cloning of DNA fragments into the pSecTag/FRT/V5-His-TOPO vector.
To construct expression vectors encoding corin deletion mutants, a two-step PCR strategy was used. The full-length human corin cDNA was used as a template. The first step was to amplify a cDNA fragment flanked by an EcoRI site at the 5Ј-end and an XhoI site at the 3Ј end. The PCR products were digested with restriction enzymes EcoRI and XhoI and cloned into the vector pcDNA4/HisMaxC (Invitrogen). The next step was to amplify a second cDNA fragment flanked by an XhoI site at the 5Ј and an ApaI site at the 3Ј-end. The PCR products were digested with restriction enzymes XhoI and ApaI and cloned into the pcDNA4/HisMaxC vector that contained the first cDNA fragment. The segments derived from PCR in each expression plasmid were verified by DNA sequencing. Table I summarizes the corin cDNA sequences that were generated by PCR and the amino acids that were deleted in each mutant. All corin mutants in this series contain an Xpress tag at their N termini.
To construct plasmids expressing corin mutants D300Y, D336Y, D373Y, and D410Y, site-directed mutagenesis was performed using the QuikChange II site-directed mutagenesis kit (Stratagene) and a fulllength corin cDNA as template. The following oligonucleotide primers were used, 5Ј-C TGT GAC GAC TGG AGT TAC GAG GCT CAT TGC AAC-3Ј for mutant D300Y, 5Ј-GAC TGT GGG GAT TTG AGT TAT GAG CAA AAC TGT GAT TGC-3Ј for mutant D336Y, 5Ј-GAC TGT GTG GAT AAG TCC TAC GAG GTC AAC TGC TCC-3Ј for mutant D373Y, and 5Ј-GAC TGC AAG GAT GGG AGT TAT GAG GAG AAC TGC AG-3Ј for mutant D410Y. Like the deletion mutants described above, these corin mutants also contain an Xpress tag at their N termini.
Expression, Purification, and Activation of EKshortCorin-HEK 293 cells were co-transfected with the expression vector pSECEKshortCorin and a plasmid expressing the neomycin resistance gene using Lipo-fectAMINE 2000. Stable clones expressing EKshortCorin were selected in ␣-minimum essential medium containing 500 g/ml G418. Positive clones were identified by Western blotting and adapted for growth in serum-free Opti-MEM I medium. To purify EKshortCorin, the conditioned medium was collected, passed through a 0.2-m filter, and dialyzed against Buffer A (50 mM Tris-HCl, pH 7.5, 300 mM NaCl) using a Dialyze Direct L module (Qiagen, Valencia, CA). The medium was loaded onto a 23-ml nickel-nitrilotriacetic acid Superflow column (Qiagen) that was subsequently washed with Buffer A containing 10 mM imidazole and eluted with a 10 -250 mM imidazole linear gradient in Buffer A. Fractions containing the soluble corin were combined. The protein had a purity of Ͼ95%, as determined by SDS/PAGE followed by silver staining and Western blotting. To activate EKshortCorin, ϳ50 g of the purified protein was incubated with 15 units of recombinant EK in 5 ml of activation buffer (100 mM Tris-HCl, pH 7.5, 10 mM CaCl 2 ) at 25°C for 2 h. EK was then removed using EKapture beads by centrifugation (1000 rpm, 10 min). As a control, an assay buffer without corin protein underwent the same EK activation and removal procedures.
Enzyme Kinetics-Kinetic constants were determined using two selected chromogenic substrates, S-2366 (pyroGlu-Pro-Arg-p-nitroanilide-HCl) and S-2403 (pyroGlu-Phe-Lys-p-nitroanilide-HCl), which were used previously for a full-length soluble corin (EKsolCorin) (13). For each assay, 50 l of substrates (final concentrations from 0.2 to 2 mM in 100 mM Tris-HCl, pH 7.5, 10 mM CaCl 2 ) were mixed with 50 l of activated EKshortCorin (final concentration of 40 nM) in a 96-well plate. The plate was incubated at 37°C and read at 405 nm wavelength over 15 min at 20 s intervals in a plate reader (Spectra MAX 250, Molecular Devices Corp., Sunnyvale, CA). In these experiments, controls included purified EKshortCorin that was not activated by EK and an assay buffer that underwent the same EK treatment and removal procedures. Readings from these controls, which were minimal, were subtracted as the background. The K m and V max values were determined by a Lineweaver-Burk double-reciprocal plot. Each assay was carried out in triplicate and repeated at least three times.
Effects of Protease Inhibitors-Effects of protease inhibitors on EK-shortCorin and EKsolCorin were tested in an assay using the chromogenic substrate S-2403. In each experiment, 45 l of activated EKshort-Corin or EKsolCorin (final concentration of 50 nM) was mixed with 5 l of an inhibitor (final concentrations ranging from 50 to 20 mM) and incubated at 37°C for 30 min. The substrate S-2403 (final concentration of 500 M) was added to the mixture, and the absorbance was measured at 405 nm after 2 h. Each assay was performed in triplicate and repeated at least twice.
Cell Culture, Transfection, and Pro-ANP Processing-HEK 293 cells were transfected with plasmids expressing either human pro-ANP or corin using LipofectAMINE 2000 according to the manufacturer's instructions. Conditioned medium containing recombinant human pro-ANP was collected after 16 h, added to cells expressing either the full-length or mutant corin, and incubated at 37°C for 4 h. Recombinant corin proteins in cell lysate and recombinant pro-ANP and its derivatives in the conditioned medium were analyzed by Western blotting using an anti-Xpress-or anti-V5 antibody. To examine the pro-ANP processing activity of the soluble corins, EKshortCorin or EKsolCorin was first activated by EK and then added to the conditioned medium containing pro-ANP. Pro-ANP processing was analyzed by Western blotting. To quantify the conversion of pro-ANP to ANP, protein bands were analyzed using the ChemiImager TM 4400 instrument (Alpha Innotech Corp., San Leandro, CA). The optical density of the bands representing pro-ANP and ANP was measured, and the percentage of pro-ANP conversion was calculated. For each corin mutant, at least three independent experiments were performed.
Detection of Corin Protein on the Cell Surface-HEK 293 cells in 100-mm dishes were transiently transfected with plasmids expressing either the full-length or mutant corin. As controls, a parental plasmid and plasmids for soluble corins were included. Cell surface proteins were biotinylated using the ECL protein biotinylation module (Amersham Biosciences) according to the manufacturer's instructions. The cells were washed with saline and lysed in 1 ml of a lysis buffer (250 mM NaCl, 25 mM Tris-HCl, pH 7.5, 5 mM EDTA, 0.5% Nonidet P-40, and one tablet of protease inhibitors/15 ml). After the removal of cell debris by centrifugation at 13,000 rpm for 10 min, recombinant corin proteins were immunoprecipitated with an anti-Xpress-or anti-V5 antibody. To detect total corin proteins, cell lysate was analyzed by SDS-PAGE and Western blotting using a horseradish peroxidase-conjugated anti-Xpress or anti-V5 antibody. The membranes were then stripped with a glycine solution (100 mM, pH 3) and reprobed with horseradish peroxidase-labeled streptavidin, which detects cell surface proteins.

RESULTS
Processing Pro-ANP by EKshortCorin-To assess the importance of the propeptide of corin in pro-ANP processing, we expressed and purified a soluble form of corin, EKshortCorin, that consists of only the catalytic domain and contains an EK recognition sequence at the conserved activation cleavage site (Fig. 1A). EKshortCorin and a control full-length soluble corin, EKsolCorin (13), were activated by recombinant EK and tested for their pro-ANP processing activity by Western analysis. As shown in Fig. 1B, the zymogen forms of EKsolCorin (left panel) and EKshortCorin (right panel) migrated on SDS-PAGE as single bands of ϳ150 and ϳ38 kDa, respectively. The observed size of EKshortCorin was consistent with its calculated mass of ϳ33 kDa. EKshortCorin also contains one putative N-linked glycosylation site (1). When treated with increasing concentrations of EK, corin zymogens were converted to two-chain proteins in a dose-dependent manner. Because the V5 tag is located at the C termini of the proteins, the antibody detected only the C-terminal protease domain, which appeared as a band of ϳ35 kDa under reducing conditions (Fig. 1B). When activated EKsolCorin and EKshortCorin were added to the conditioned medium containing recombinant pro-ANP, only EKsolCorin, but not EKshortCorin, converted pro-ANP to ANP (Fig. 1B).
Catalytic Activity of EKshortCorin-One possibility for the failure of EKshortCorin to cleave pro-ANP could be that the protein was not properly folded in the absence of the propeptide, making it catalytically inactive. We then compared enzyme kinetics of EKshortCorin and EKsolCorin, which is biologically active (13) (13) and show that EKshortCorin is catalytically active toward small peptide substrates.
Effects of Protease Inhibitors-We next examined the effects of protease inhibitors on the catalytic activity of EKshortCorin and EKsolCorin in a chromogenic substrate-based assay. As summarized in Table II, the catalytic activity of EKshortCorin and EKsolCorin was inhibited similarly by trypsin-like serine  protease inhibitors including benzamidine, phenylmethylsulfonyl fluoride, leupeptin, and soybean trypsin inhibitor but not by metallo-and cysteine-protease inhibitors such as EDTA and pepstatin. The data show that the active center of EKshort-Corin is properly formed consistent with the results from the kinetic studies described above. Together, these data indicate that the propeptide of corin is not necessary for its interactions with small peptide substrates and inhibitors but is required for its interaction with the physiological substrate pro-ANP.
Membrane-bound Corin with Internal Domain Deletions-To understand how different extracellular domains of corin contribute to its activity in pro-ANP processing, we constructed a series of mutants by deleting increasing numbers of domains at the N terminus of the extracellular region ( Fig. 2A). All of these mutants were designed to contain the transmembrane domain, allowing us to examine pro-ANP processing in cell-based assays. The plasmids encoding these mutants were transfected in HEK 293 cells. Expression of recombinant corin proteins was confirmed by Western blotting (Fig. 2B, upper panel). Conditioned medium containing recombinant pro-ANP was incubated with the transfected cells, and pro-ANP processing was examined by Western blotting (Fig. 2B, lower panel). Compared with the full-length corin, mutant corin ⌬Fz1, which lacks the frizzled 1 domain, exhibited a reduced (ϳ40%) activity in processing pro-ANP, whereas corin mutants lacking either frizzled 1 domain and LDLR repeats 1-5 (⌬Fz1R1-5) or only LDLR repeats 1-5 (⌬R1-5) had no detectable activity of pro-ANP processing. Corin mutants with additional deletions that extend to include frizzled 2 domain, LDLR repeats 6 -8, and the scavenger receptor repeat also lost their activities in pro-ANP processing (data not shown). These results indicate that the fragment containing frizzled 1 domain and LDLR repeats 1-5 is critical for the function of corin in pro-ANP processing.
Expression of Corin Mutants on the Cell Surface-To exclude the possibility that the corin deletion mutants lost their activity because of the lack of cell surface expression, we examined the cell surface expression using a biotin-labeling method. HEK 293 cells were transiently transfected with plasmids encoding the full-length corin or corin mutants ⌬Fz1, ⌬Fz1R1-5, and ⌬R1-5. Cell surface proteins were then labeled with biotin. Cells transfected with plasmids encoding soluble EKsolCorin and EKshortCorin or a parental vector were included as controls. Cells transfected with a plasmid expressing the fulllength corin but that were not biotinylated were used as an additional control. By SDS-PAGE and Western blotting, we showed that the full-length corin and corin mutants ⌬Fz1, ⌬Fz1R1-5, ⌬R1-5, EKsolCorin, and EKshortCorin proteins were present in cell lysate (Fig. 2C, upper panels). When the same blots were reprobed with horseradish peroxidase-conjugated streptavidin, which binds to biotin-labeled cell surface proteins, the full-length corin and mutant corin ⌬Fz1, ⌬Fz1R1-5, and ⌬R1-5 proteins, but not control soluble EKsol-

FIG. 2. Expression and activity of corin mutants lacking frizzled 1 domain and LDLR repeats 1-5.
A, schematic diagrams of the full-length (FL) and mutant corins ⌬Fz1, ⌬Fz1R1-5, and ⌬R1-5. Dotted lines represent deleted domains. The domains structures C-terminal to frizzled 2 domain are omitted. B, recombinant corin proteins in HEK 293 cells transfected with plasmids encoding FL corin and mutants ⌬Fz1, ⌬Fz1R1-5, and ⌬R1-5, or a control vector (vector) were detected by Western blotting using an anti-Xpress antibody (upper panel). Pro-ANP and its derivatives in the conditioned medium were analyzed by immunoprecipitation and Western blotting using an anti-V5 antibody (lower panel). C, cell-surface proteins in transfected HEK 293 cells were biotinylated. Total recombinant corin proteins in cell lysate were detected by immunoprecipitation and Western blotting (upper panels). The same blots were reprobed with horseradish peroxidase-conjugated streptavidin to detect the corin proteins that were present on the cell surface. Cells transfected with the plasmid expressing the full-length corin but not biotinylated (FLØ) or transfected with plasmids expressing soluble EKsolCorin and EKshortCorin or a control vector and biotinylated were included as controls (lower panels).
Corin and EKshortCorin, were detected (Fig. 2C, lower panels). No specific bands were detected in cells transfected with the plasmid encoding the full-length corin but not biotinylated or cells transfected with a control vector and biotinylated (Fig. 2C,  lower panels). The results show that mutant corin ⌬Fz1, ⌬Fz1R1-5, and ⌬R1-5 proteins were indeed present on the cell surface.
Functional Importance of Individual LDLR Repeats-To determine the functional importance of each individual repeat within the fragment containing LDLR repeats 1-5, we first generated a series of plasmids expressing corin mutants lacking frizzled 1 domain and increasing numbers of LDLR repeats (⌬Fz1R1, ⌬Fz1R12, ⌬Fz1R1-3, ⌬Fz1R1-4) (Fig. 3A). The plasmids were transfected into HEK 293 cells, and the expression of mutant corin proteins were confirmed by Western analysis of cell lysate (Fig. 3B, upper panel). Conditioned medium containing recombinant pro-ANP was incubated with these cells and pro-ANP processing was analyzed by Western blotting. Con-sistent with data shown in Fig. 2B, mutant corin ⌬Fz1 had a reduced activity in pro-ANP processing (Fig. 3B, lower panel). No pro-ANP processing activity was detected in cells expressing corin mutants ⌬Fz1R1, ⌬Fz1R12, ⌬Fz1R1-3, and ⌬Fz1R1-4 (Fig. 3B, lower panel). These data show that in the absence of frizzled 1 domain deletion of LDLR repeat 1 alone or together with LDLR repeats 2-4 abolished the pro-ANP processing activity of corin.
To examine the contribution of individual LDLR repeats, we constructed a third series of corin mutants lacking each LDLR repeat (⌬R2, ⌬R3, and ⌬R4) (Fig. 5A) and expressed them in HEK 293 cells (Fig. 5B, upper panel). In pro-ANP processing assays, corin mutants ⌬R2, ⌬R3, and ⌬R4 had ϳ12, ϳ52, and ϳ77% activity, respectively, when compared with that of the full-length corin (Fig. 5B, lower panel). Together with the data from mutant corin ⌬R1 (ϳ49% activity) (Fig. 4B, lower panel), these results show that LDLR repeats 1-4 each contributes to the function of pro-ANP processing with LDLR repeat 2 being the most important.
Effect of Point Mutations in LDLR Repeats 1-4 -To further confirm the importance of each repeat in the LDLR1-4 fragment, we constructed a new series of corin mutants by replacing a highly conserved Asp residue with a Tyr residue in LDLR repeats 1, 2, 3, or 4 (D300Y, D336Y, D373Y, and D410Y) (Fig.  6, A and B). In the LDLR, the corresponding Asp residue has been shown to coordinate Ca 2ϩ binding (14, 15) (Fig. 6A), which   FIG. 6. Processing of pro-ANP by corin mutants with a point mutation in LDLR repeats 1-4. A, alignment of amino acid sequences of the LDLR repeats 1-5 of corin with the consensus sequence from the human LDLR. Numbers for the first amino acid residues in each LDLR repeat of corin are indicated. Black dots below the sequences indicate the acidic residues contributing to Ca 2ϩ coordination. The star indicates the conserved Asp residue that was mutated to Tyr residue in corin mutants. B, schematic diagrams of the full-length corin (FL) and corin mutants D300Y, D336Y, D373Y, and D410Y. Stars indicate the LDLR repeats in which a conserved Asp residue was mutated to Tyr. C, recombinant corin proteins in HEK 293 cells transfected with plasmids encoding the full-length corin and corin mutants D300Y, D336Y, D373Y, and D410Y, or a control vector (vector) were detected by Western blotting using an anti-Xpress antibody (upper panel). Pro-ANP and its derivatives in the conditioned medium were analyzed by immunoprecipitation and Western blotting using an anti-V5 antibody (lower panel). is required to maintain the structural integrity of the protein.
A point mutation at the Asp residue is expected to alter the conformation of individual LDLR repeats without causing structural perturbation to its neighboring repeats (16). The mutant corin proteins were expressed in HEK 293 cells (Fig.  6C, upper panel), and their pro-ANP processing activities were analyzed by Western blotting (Fig. 6C, lower panel) and quantified by densitometry. Corin mutants D300Y, D336Y, D373Y, and D410Y had ϳ25, ϳ11, ϳ16, and ϳ82% of pro-ANP processing activities, respectively, compared with that of the fulllength corin. These results are consistent with data from corin mutants with single LDLR repeat deletions (Figs. 4 and 5) demonstrating the individual contribution of LDLR repeats 1-4 in pro-ANP processing. DISCUSSION In this study, we examined the functional importance of the domain structures in the propeptide of corin for pro-ANP processing. We expressed a soluble corin, EKshortCorin, that consists of only the serine protease domain and contains an EK recognition sequence at the conserved activation cleavage site. We showed that EKshortCorin was activated by EK and retained the catalytic activity when examined in chromogenic substrate-based assays. Unlike the full-length soluble corin, EKsolCorin, which is biologically active (13), EKshortCorin failed to cleave pro-ANP indicating that certain domain structures in the propeptide are required for this activity of corin. We constructed a series of corin mutants by deleting increasing numbers of domain structures starting at the N terminus of the extracellular region. We showed that a deletion of frizzled 1 domain reduced the pro-ANP processing activity, whereas a deletion of LDLR repeats 1-5 abolished the activity. By analyzing additional corin mutants that either lacked individual LDLR repeats or contained point mutations at a conserved Ca 2ϩ binding site in the LDLR repeats, we showed that a region spanning from frizzled 1 domain to LDLR repeat 4 is critical for the activity of corin in processing pro-ANP. Fig. 7 summarizes the corin mutants described in this study and their activities in pro-ANP processing.
Corin is a mosaic protease that contains a transmembrane domain near the N terminus and several distinct domain structures in its C-terminal extracellular region. In a recent study, we showed that the transmembrane domain is not required for the activity of corin in pro-ANP processing (13), but the functional importance of other extracellular noncatalytic domains of corin was not determined. Previous studies have shown that noncatalytic domains in the extracellular region of other type II transmembrane serine proteases are important for their biological functions. For example, Lu et al. (17) have reported that a soluble bovine EK consisting of only the protease domain is catalytically active toward small peptide substrates but fails to activate trypsinogen indicating that the propeptide of EK plays a role in recognizing trypsinogen. In this study, we showed that frizzled 1 domain and LDLR repeats 1-4 in the propeptide of corin are required for the processing of pro-ANP. To our knowledge, EK and corin are the only two members from the type II transmembrane serine protease family for which physiological substrates have been identified. These data suggest that the propeptide in other type II transmembrane serine proteases may also have an important function in interacting with their physiological substrates. It will be important to test this concept in future studies.
The frizzled-like cysteine-rich domain, which is ϳ120 amino acids in length and contains 10 conserved cysteine residues, was first discovered in members of the Frizzled family of seventransmembrane receptors for Wnt signaling proteins (18,19). Subsequently, the frizzled-like cysteine-rich domain also has been found in soluble Frizzled-related proteins that act as antagonists of Wnt signaling (20 -22). Studies have shown that the frizzled-like cysteine-rich domain interacts directly with Wnt proteins (23,24). In addition to Wnt receptors and inhibitors, other proteins such as human carboxypeptidase Z (25), mouse collagen (XVIII) ␣1 chain (26), and several receptor tyrosine kinases including muscle-specific kinase and Smoothened (27,28) also contain frizzled-like cysteine-rich domains. A recent study (29) has indicated that the frizzled-like cysteinerich domain in chick carboxypeptidase Z binds to Wnt proteins and plays a role in the formation of skeleton. At this time, the functional significance of the frizzled-like domain in collagen (XVIII) ␣1 chain, muscle-specific kinase, and Smoothened is not known. In this study, we showed that the deletion of frizzled 1 domain reduced the activity of corin in pro-ANP processing by 60%, indicating that the frizzled 1 domain is involved in the interaction of corin with pro-ANP. Thus, our data suggest that frizzled-like cysteine-rich domains have a much broader role in protein-protein interactions.
In the extracellular region of corin, there are eight LDLR class A repeats in two separate clusters. This type of repeat, which is ϳ40 amino acids in length and contains six conserved cysteine residues, was first identified in the LDLR as lipoprotein binding sites (30,31). Naturally occurring mutations in the LDLR repeats alter the function of the receptor and cause familial hypercholesterolemia in patients (32). Site-directed mutagenesis studies have shown that an individual repeat in the LDLR binds differentially to lipoproteins and has an additive effect on ligand binding (33,34). In corin, however, the LDLR repeats are unlikely to interact with lipoproteins, because no binding of lipoproteins to mouse corin was detected in cell-based assays (35). In contrast, we showed that LDLR repeats 1-4 are important for corin to recognize pro-ANP. Corin mutants lacking single LDLR repeats 1-4 (⌬R1, ⌬R2, ⌬R3, and ⌬R4) exhibited ϳ49, ϳ12, ϳ53, and ϳ77% of pro-ANP processing activities, respectively, compared with that of the fulllength corin. We also made corin mutants with a single mutation at a conserved Ca 2ϩ -binding Asp residue in LDLR repeats 1, 2, 3, or 4 (D300Y, D336Y, D373Y, and D410Y). The mutation is expected to affect only the function of the individual LDLR repeat in which it resides but not the overall protein structure. Unlike the deletion mutants, the mutants with a single amino acid substitution shall also maintain the distance between the cell membrane and individual LDLR repeats, which may be important for the binding of macromolecular substrates. In pro-ANP processing assays, mutants D300Y, D336Y, D373Y, and D410Y had ϳ25, ϳ11, ϳ16, and ϳ82% activities, respectively. The overall data are consistent with the results from corin mutants ⌬R1, ⌬R2, ⌬R3, and ⌬R4, showing that LDLR repeats 1-4 all contribute to the binding of pro-ANP and that among these LDLR repeats repeat 2 appears to be most critical, whereas repeat 4 is least important. Based on these results, we propose a model in which pro-ANP binds to corin by interacting with frizzled 1 domain and LDLR repeats 1-4 (Fig. 8). The binding of pro-ANP to this region of corin may allow the protease domain to cleave the substrate more efficiently. It is equally possible that the binding induces conformational changes in pro-ANP, making its activation cleavage site accessible to the protease domain of corin. This model may explain why pro-ANP is resistant to the cleavage by EKshortCorin, which contains only the catalytic domain. At this time, the specific amino acid residues in frizzled 1 domain and LDLR repeats 1-4 of corin that make contact with pro-ANP are not known. Further mutagenesis experiments shall help to verify and refine this model.