Functional Analysis of the Transmembrane Domain and Activation Cleavage of Human Corin
2003; Elsevier BV; Volume: 278; Issue: 52 Linguagem: Inglês
10.1074/jbc.m309991200
ISSN1083-351X
AutoresSabine Knappe, Faye Wu, Mary Rose Masikat, John Morser, Qingyu Wu,
Tópico(s)Coagulation, Bradykinin, Polyphosphates, and Angioedema
ResumoCorin 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. 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. Corin is a mosaic serine protease that was recently identified from the human heart (1Yan W. Sheng N. Seto M. Morser J. Wu Q. J. Biol. Chem. 1999; 274: 14926-14935Abstract Full Text Full Text PDF PubMed Scopus (227) Google Scholar, 2Hooper J.D. Scarman A.L. Clarke B.E. Normyle J.F. Antalis T.M. Eur. J. Biochem. 2000; 267: 6931-6937Crossref PubMed Scopus (90) Google Scholar). 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 (3Hooper J.D. Clements J.A. Quigley J.P. Antalis T.M. J. Biol. Chem. 2001; 276: 857-860Abstract Full Text Full Text PDF PubMed Scopus (323) Google Scholar, 4Netzel-Arnett S. Hooper J.D. Szabo R. Madison E.L. Quigley J.P. Bugge T.H. Antalis T.M. Cancer Metastasis Rev. 2003; 22: 237-258Crossref PubMed Scopus (258) Google Scholar, 5Szabo R. Wu Q. Dickson R.B. Netzel-Arnett S. Antalis T.M. Bugge T.H. Thromb. Haemostasis. 2003; 90: 185-193Crossref PubMed Google Scholar, 6Wu Q. Curr. Top. Dev. 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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 cysteine-rich domains. enterokinase atrial natriuretic peptide human embryonic kidney high pressure liquid chromatography. Corin mRNA and protein are abundantly expressed in the heart (1Yan W. Sheng N. Seto M. Morser J. Wu Q. J. Biol. Chem. 1999; 274: 14926-14935Abstract Full Text Full Text PDF PubMed Scopus (227) Google Scholar, 2Hooper J.D. Scarman A.L. Clarke B.E. Normyle J.F. Antalis T.M. Eur. J. Biochem. 2000; 267: 6931-6937Crossref PubMed Scopus (90) Google Scholar), 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 (21Yan W. Wu F. Morser J. Wu Q. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 8525-8529Crossref PubMed Scopus (390) Google Scholar), both of which are cardiac hormones important in maintaining normal blood pressure and electrolyte homeostasis (22Levin E.R. Gardner D.G. Samson W.K. N. Engl. J. Med. 1998; 339: 321-328Crossref PubMed Scopus (2066) Google Scholar, 23Stein B.C. Levin R.I. Am. Heart J. 1998; 135: 914-923Crossref PubMed Scopus (248) Google Scholar, 24Wilkins M.R. Redondo J. Brown L.A. Lancet. 1997; 349: 1307-1310Abstract Full Text Full Text PDF PubMed Scopus (203) Google Scholar). Corin, however, does not convert pro-C-type natriuretic peptide to mature C-type natriuretic peptide (25Wu C. Wu F. Pan J. Morser J. Wu Q. J. Biol. Chem. 2003; 278: 25847-25852Abstract Full Text Full Text PDF PubMed Scopus (166) Google Scholar), the third member of the natriuretic peptide family (26Chen H.H. Burnett Jr., J.C. J. Cardiovasc. Pharmacol. 1998; 32: S22-S28Crossref PubMed Scopus (95) Google Scholar), which may play a role in angiogenesis and arterial restenosis. The results from these experiments suggest that corin is the pro-ANP/pro-brain natriuretic peptide convertase in the heart. This hypothesis is further supported by additional experiments in which overexpression of an active site mutant corin or transfection of small interfering RNA duplexes directed against the corin gene completely blocked pro-ANP processing in cultured cardiomyocytes (27Wu F. Yan W. Pan J. Morser J. Wu Q. J. Biol. Chem. 2002; 277: 16900-16905Abstract Full Text Full Text PDF PubMed Scopus (129) Google Scholar). To date, however, the reported studies of corin were performed in cell-based experiments, which do not allow elimination of the possibility that other proteins or enzymes might contribute to the observed pro-ANP processing activities. It is important, therefore, to demonstrate directly that corin itself possesses the pro-ANP processing activity by using purified active corin. To test the hypothesis that purified active corin is able to process pro-ANP, we first examined the importance of the transmembrane domain and activation cleavage in corin for its biological activity. Our results showed that the transmembrane domain is not required for corin to process pro-ANP, but proteolytic cleavage of corin at its conserved activation site is essential. Based on these results, we designed, expressed, and purified a soluble form of human corin and studied its biochemical properties. Our results showed that activated soluble human corin hydrolyzed synthetic peptic substrates and activated human pro-ANP in a highly sequence-specific manner. Materials—Cell culture medium, G418, anti-V5 antibody, transfection reagent LipofectAMINE 2000, and expression vector pSecTag/FRT/V5-His-TOPO were purchased from Invitrogen. Fetal bovine serum was from SeraCare Life Sciences, Inc. (Oceanside, CA). Human embryonic kidney (HEK) 293 cells were obtained from the American Type Culture Collection and maintained at the Core Facility at Berlex Biosciences. Oligonucleotide primers were synthesized by BIOSOURCE International Inc. (Camarillo, CA). Restriction enzymes and DNA polymerases were obtained 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 purchased from DiaPharma (West Chester, OH). Phenylmethylsulfonyl fluoride and tosyl-Lys-chloromethylketone were from Bachem Bioscience Inc. (King of Prussia, PA). Protease inhibitors antipain, pepstatin, bestatin, chymostatin, phosphoramidon, leupeptin, and aprotinin were purchased from Roche Applied Science. Heparin was from U. S. Biochemical Corp. All other chemical reagents were obtained from Sigma. Expression Vectors—Expression plasmids encoding human wild-type corin (pcDNACorin), active site mutant corin S985A (pcDNACorinS985A), human wild-type pro-ANP (pcDNAproANP), and mutant pro-ANPs R98A (pcDNAproANPR98A), R101A (pcDNAproANPR101A), and R102A (pcDNAproANPR102A) were described previously (21Yan W. Wu F. Morser J. Wu Q. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 8525-8529Crossref PubMed Scopus (390) Google Scholar, 27Wu F. Yan W. Pan J. Morser J. Wu Q. J. Biol. Chem. 2002; 277: 16900-16905Abstract Full Text Full Text PDF PubMed Scopus (129) Google Scholar). A plasmid vector encoding corin activation cleavage site mutant R801A (pcDNACorinR801A) was constructed by a PCR-based mutagenesis method (QuikChange site-directed mutagenesis kit; Stratagene, La Jolla, CA) using pcDNACorin as a template and oligonucleotide primer 5′-CCG AAT GAA CAA AGC AAT CCT TGG AGG TCG-3′. To construct a plasmid expressing a soluble corin, a cDNA fragment containing nucleotides 463–3219 of human corin cDNA (1Yan W. Sheng N. Seto M. Morser J. Wu Q. J. Biol. Chem. 1999; 274: 14926-14935Abstract Full Text Full Text PDF PubMed Scopus (227) Google Scholar) was amplified by PCR and inserted into the expression vector pSEC to yield the plasmid pSECsolCorin. This plasmid encodes a protein, WTsolCorin, consisting of an Igκ 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 (see Fig. 1). To construct a plasmid encoding a soluble corin that can be activated by EK, PCR-based site-directed mutagenesis was performed using plasmid pSECsolCorin as a template. Nucleotides 2482–2496 (CGA ATG AAC AAA AGG) of human corin cDNA were replaced by nucleotides GAC GAT GAC GAT AAG. The resulting plasmid, pSECEKsolCorin, encodes a soluble corin, EKsolCorin, with the amino acid sequence DDDDK replacing the original sequence RMNKR at the conserved activation cleavage site (see Fig. 1). All plasmid constructs were verified by restriction enzyme digestion and direct DNA sequencing. Cell Culture, Transfection, and Western Analysis of Pro-ANP Processing—HEK 293 cells were cultured in α-minimum essential medium supplemented with 10% fetal bovine serum. Transient transfection was performed using LipofectAMINE 2000 according to the manufacturer's instructions. The conditioned medium was collected 12–16 h after transfection and subjected to centrifugation at 15,000 rpm to remove cell debris. The cells were lysed in a buffer containing 100 mm Tris-HCl, pH 7.5, and 1% Triton X-100. To analyze pro-ANP processing, the conditioned medium containing recombinant wild-type or mutant pro-ANPs was incubated with purified soluble corin (1.8 μg/ml) at 37 °C for 4 h. Recombinant human pro-ANP and its derivatives in the conditioned medium were immunoprecipitated by an anti-V5 antibody. The protein samples were separated by SDS-PAGE and analyzed by Western blotting using a horseradish peroxidase-conjugated anti-V5 antibody. On Western blots under reducing conditions, recombinant human corin zymogen, the corin protease domain, pro-ANP, and ANP migrate as bands of ∼150, ∼35, ∼24, and ∼7 kDa, respectively. Expression and Purification of EKsolCorin—HEK 293 cells were co-transfected with the expression vector pSECEKsolCorin and a plasmid expressing the neomycin resistance gene using LipofectAMINE 2000. Stable clones were selected in α-minimum essential medium containing 10% fetal bovine serum and 500 μg/ml G418 and screened by Western blotting using an anti-V5 antibody for corin protein expression. Positive clones were adapted for growth in serum-free OPTI-MEM I medium containing 500 μg/ml G418. The conditioned medium was collected, passed through a 0.2-μm filter, and dialyzed against Buffer A (50 mm Tris-HCl, pH 7.5, 300 mm NaCl) using a Dialyze Direct L Module (Qiagen). 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. The fractions containing soluble corin were identified by Western blotting and combined. The pooled fractions were then diluted 1:3 in Buffer B (20 mm Tris-HCl, pH 8.0) and loaded onto a 5-ml Hi Trap Q Sepharose column (Amersham Biosciences). The column was equilibrated with Buffer B containing 100 mm NaCl, washed with Buffer B containing 200 mm NaCl, and eluted with a 200–750 mm NaCl linear gradient in Buffer B. The fractions containing the soluble corin, EKsolCorin, were identified by Western blotting and pooled. The purified protein was further characterized by Coomassie Blue staining, analytical size exclusion chromatography, and N-terminal protein sequencing. Activation of EKsolCorin by Recombinant EK—To activate the recombinant soluble corin, EKsolCorin, 2.5 μg of purified EKsolCorin protein was incubated with increasing concentrations of recombinant EK (1–10 units/ml) in 100 μl of Activation buffer (100 mm Tris-HCl, pH 7.5, 10 mm CaCl2) at 25 °C for 2 h. The samples (2 μl) were taken and analyzed by SDS-PAGE under reducing and nonreducing conditions followed by Western blotting using an anti-V5 antibody. For activation of large batches, 1 mg of purified EKsolCorin in 40 ml of activation buffer was incubated with 300 units of recombinant EK at 25 °C for 3 h. To remove the recombinant EK, 11 ml of EKapture beads were added to the solution and incubated at room temperature for 15 min. EKapture beads were removed by centrifugation (1,000 rpm, 10 min), and the supernatant was collected and stored at -20 °C until further use. EK was removed from the EKsolCorin preparation to below the limit of detection when analyzed by both SDS-PAGE followed by silver staining and by HPLC-based analytical size exclusion chromatography. As another control, an assay buffer without corin protein underwent the same EK activation and removal procedures. Enzyme Kinetics—Kinetic constants were determined using a panel of selected synthetic chromogenic substrates. For each assay, which was carried out in 96-well plates, 50 μl of substrates (final concentrations ranging from 0.2 to 2 mm in 100 mm Tris-HCl, pH 7.5, 10 mm CaCl2) were mixed with 50 μl of activated EKsolCorin (final concentration of 58 nm). The plates were incubated at 37 °C and read at 405-nm wavelength over 15 min at 20-s intervals in a Spectra MAX 250 plate reader (Molecular Devices Corp., Sunnyvale, CA). In these experiments, controls included purified EKsolCorin that was not activated by EK and an assay buffer that underwent the same EK treatment and removal procedures. Readings from the controls, which were minimal, were subtracted as the background. In addition, plasmin (for S-2222, S-2251, S-2302, S-2366, S-2403, and S-2444), kallikrein (for S-2266 and S-2288), thrombin (for S-2238), trypsin (for S-2765), and elastase (for S-2484) were used as positive controls under the conditions recommended by the manufacturer. The Km and Vmax values were determined by Line-weaver-Burk double-reciprocal plot. Each enzymatic assay was carried out in triplicate and repeated at least twice. Effects of Protease Inhibitors—Effects of protease inhibitors on EK-solCorin were tested in an assay using the chromogenic substrate S-2403. In each experiment, 45 μl of activated EKsolCorin (final concentration of 104 nm) was mixed with 5 μl of an inhibitor (final concentrations ranging from 0.1 μm to 20 mm) and incubated at 37 °C for 30 min. To measure the remaining hydrolytic activity of EKsolCorin, 50 μl of S-2403 (final concentration of 500 μm) was added to the mixture, and the absorbance was measured at 405 nm after 2 h. Each experiment was performed in triplicate and repeated at least twice. cGMP Assay—To examine the biological activity of corin-processed recombinant ANP, a cGMP assay was performed using an enzyme immunoassay kit (Biotrak; Amersham Biosciences), as described previously (27Wu F. Yan W. Pan J. Morser J. Wu Q. J. Biol. Chem. 2002; 277: 16900-16905Abstract Full Text Full Text PDF PubMed Scopus (129) Google Scholar). In these experiments, synthetic human ANP (Peninsula Laboratories Inc., San Carlos, CA) was used as a standard. Each experimental condition was assayed in triplicate. Processing of Pro-ANP by Wild Type and a Soluble Corin, WTsolCorin—To examine the importance of the transmembrane domain of corin for its pro-ANP processing activity, we constructed a plasmid that encodes a soluble form of human corin containing all of the extracellular domains (Fig. 1). Recombinant wild-type corin, active site mutant corin S985A, and the soluble corin, WTsolCorin, were expressed transiently in HEK 293 cells. Wild-type corin and active site mutant corin S985A were detected by Western analysis in the cell lysate but not in the conditioned medium (Fig. 2A), consistent with corin being a transmembrane protein. In contrast, WTsolCorin was detected in both the cell lysate and conditioned medium (Fig. 2A), confirming that the soluble corin was secreted from the cells. We determined the activity of these recombinant corins in pro-ANP processing in co-transfection experiments using a plasmid expressing human pro-ANP together with plasmids expressing either wild-type corin, mutant corin S985A, or WT-solCorin. Pro-ANP and its derivatives in the conditioned medium were analyzed by Western blotting. As shown in Fig. 2B, pro-ANP, but not ANP, was detected in the cell lysate. In the conditioned medium, conversion of pro-ANP to ANP was observed when cells were transfected with the pro-ANP expressing plasmid together with plasmids expressing wild-type corin or WTsolCorin. As controls, the cells were co-transfected with the pro-ANP expressing construct and a plasmid expressing either active site mutant corin S985A or a control vector. Without the presence of a plasmid expressing an active corin, no pro-ANP processing was detected, showing that the cells do not contain any detectable endogenous pro-ANP processing activity. The results indicate that the transmembrane domain of corin is not necessary for the pro-ANP processing activity in this cell-based assay. Processing of Pro-ANP by Wild-type Corin and Activation Cleavage Site Mutant Corin R801A—The human corin protein contains a conserved activation cleavage sequence Arg-Ile-Leu-Gly-Gly at residues 801–805 (1Yan W. Sheng N. Seto M. Morser J. Wu Q. J. Biol. Chem. 1999; 274: 14926-14935Abstract Full Text Full Text PDF PubMed Scopus (227) Google Scholar). To examine the functional importance of the activation cleavage of human corin, we constructed a plasmid expressing a mutant that would have impaired activation (activation cleavage site mutant corin R801A; Fig. 1). Co-transfection experiments were performed using a plasmid expressing pro-ANP together with plasmids expressing either wild-type corin, active site mutant corin S985A, or activation cleavage site mutant corin R801A. Recombinant corin proteins were present in the cell lysate but not in the conditioned medium (Fig. 3A). Recombinant pro-ANP was detected in the cell lysate after transfection with the pro-ANP expressing plasmid (Fig. 3B, left panel). In the conditioned medium, processing of pro-ANP to ANP was observed when cells were co-transfected with a plasmid encoding wild-type corin but not those encoding mutant corins S985A and R801A or a control vector (Fig. 3B, right panel). The results demonstrate that proteolytic cleavage at Arg801 is required for the pro-ANP processing activity of corin. The fact that wild-type corin was capable of processing pro-ANP in cell-based transfection experiments indicates that some corin molecules must be activated. In the Western analysis (Figs. 2 and 3), however, we were unable to detect the cleaved protease fragment from wild-type corin or mutant corin S985A, which is expected to migrate as a band at ∼35 kDa under reducing conditions. This would suggest that the number of activated corin molecules is a low fraction of the overall amount of corin. Effects of Thrombin, Factor Xa, and Kallikrein on Corin Activation—To examine whether plasma-derived serine proteases could activate corin, we examined the effects of thrombin, blood clotting factor Xa, and kallikrein on corin activation. Recombinant human wild-type corin was stably expressed in HEK 293 cells. Purified human plasma thrombin, factor Xa, or kallikrein was added to the cell culture and incubated at 37 °C for 1 h. The cell lysates were prepared and analyzed by Western blotting under reducing conditions. The results showed that recombinant human corin was not cleaved in cells treated with either thrombin, factor Xa, or kallikrein (data not shown). We also added thrombin, factor Xa, or kallikrein directly to 293 cell lysates containing recombinant corin and analyzed the corin protein by SDS-PAGE and Western blotting. Again, no activation cleavage of corin was detected in these experiments (data not shown). Expression and Purification of Soluble EK-activable Corin, EKsolCorin—To produce an active soluble corin for further biochemical studies, we designed a mutant corin, EKsolCorin, in which an EK recognition sequence (DDDDK) (7Kitamoto Y. Yuan X. Wu Q. McCourt D.W. Sadler J.E. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 7588-7592Crossref PubMed Scopus (149) Google Scholar) was used to replace the activation cleavage sequence of human corin (RMNKR) (1Yan W. Sheng N. Seto M. Morser J. Wu Q. J. Biol. Chem. 1999; 274: 14926-14935Abstract Full Text Full Text PDF PubMed Scopus (227) Google Scholar). Stable cell lines expressing EKsolCorin were established. The conditioned medium from these cells was collected, and EKsolCorin was purified by nickel affinity and ion exchange chromatography. SDS-PAGE followed by Coomassie Blue staining and Western blotting using an anti-V5 antibody showed that EKsolCorin migrated as a single band at ∼150 kDa under reducing conditions and at ∼145 kDa under nonreducing conditions (Fig. 4). The results were consistent with the calculated mass of ∼108 kDa for EKsolCorin, which also contained 19 potential N-linked glycosylation sites (1Yan W. Sheng N. Seto M. Morser J. Wu Q. J. Biol. Chem. 1999; 274: 14926-14935Abstract Full Text Full Text PDF PubMed Scopus (227) Google Scholar). HPLC-based gel filtration chromatography showed that the purified EKsolCorin had a purity of >98% (data not shown). The N-terminal sequence of EKsolCorin was confirmed by protein sequencing. The purified protein was quantified by UV spectrometry at 280 nm using an extinction coefficient (1 mg/ml) of 1.45 calculated from the protein sequence. Activation of EKsolCorin by EK—Purified EKsolCorin protein was activated with increasing concentrations of recombinant EK at 25 °C for 2 h. The protein samples were analyzed by SDS-PAGE under reducing and nonreducing conditions followed by Western analysis using an anti-V5 antibody. As shown in Fig. 5, EKsolCorin was activated by recombinant EK in a dose-dependent manner. Under nonreducing conditions, EKsolCorin appeared as a single band of ∼145 kDa. Under reducing conditions, activated EKsolCorin migrated as two fragments: an N-terminal propeptide (∼115 kDa) and a C-terminal protease domain (∼35 kDa) (Fig. 1). Because the V5 tag is located at the C terminus, the anti-V5 antibody detected only the C-terminal protease domain once EKsolCorin was activated (Fig. 5). Enzymatic Properties of Soluble Corin—Substrate specificity and kinetic constants of EKsolCorin for a panel of selected peptide substrates were determined using purified and activated EKsolCorin. As controls, EKsolCorin without EK activation and a buffer that was treated with EK were included. Initial experiments showed that activated EKsolCorin, but not the zymogen form of EKsolCorin or the buffer control, hydrolyzed some chromogenic substrates (S-2222, S-2302, S-2366, S-2403, and S-2444). No significant hydrolysis of substrates S-2238 (H-d-Phe-Pip-Arg-pNA·2HCl), S-2251 (H-d-Val-Leu-Lys-pNA·2HCl), S-2266 (H-d-Val-Leu-Arg-pNA·2HCl), S-2288 (H-d-Ile-Pro-Arg-pNA·2HCl), S-2484 (pyroGlu-Pro-Val-pNA), and S-2765 (N-α-Z-d-Arg-Gly-Arg-pNA·2HCl) was detected (Km values were >50 mm). This profile was very different from that of recombinant bovine light chain EK used in this study (data not shown), indicating that the observed activity was not derived from any potential contamination of EK. We further determined the kinetics of EKsolCorin-mediated hydrolysis of the substrates S-2403, S-2366, S-2302, S-2222, and S-2444 (Table I). The results showed that EKsolCorin cleaved peptide substrates with either Arg or Lys at the P-1 position. For example, the Km values were 1.28 ± 0.46 and 3.52 ± 1.07 mm for S-2403 and S-2366, respectively. Pro, Phe, and Gly residues appeared to be preferred at the P-2 position, and a pyro-Glu residue, an analog of small neutral amino acids, seemed to be preferred at the P-3 position. The overall results are consistent with the corin cleavage sequence (Thr-Ala-Pro-Arg ↓ Ser) in human pro-ANP (28Koller K.J. Goeddel D.V. Circulation. 1992; 86: 1081-1088Crossref PubMed Scopus (364) Google Scholar).Table IKinetic parameters of activated EKsolCorin for selected chromogenic substrates The kinetic constants were determined as described under “Experimental Procedures.” The data are presented as the means ± S.D. from at least three independent experiments.SubstrateKmkcatkcat/Kmmms-1m-1·s-1S-2403, pyroGlu-Phe-Lys-pNA·HCl1.28 ± 0.460.47 ± 0.10389.1 ± 74.2S-2366, pyroGlu-Pro-Arg-pNA·HCl3.52 ± 1.070.48 ± 0.21138.6 ± 56.7S-2302, H-d-Pro-Phe-Arg-pNA·2HCl2.95 ± 0.900.23 ± 0.0479.9 ± 14.0S-2222, Bz-Ile-Glu-(γ-OR)-Gly-Arg-pNA·HCl1.92 ± 0.530.09 ± 0.0150.8 ± 8.5S-2444, pyroGlu-Gly-Arg-pNA·HCl16.0 ± 3.770.40 ± 0.0825.0 ± 3.4 Open table in a new tab Effects of Protease Inhibitors—To examine the effects of protease inhibitors
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