Role of Glycosylation in Corin Zymogen Activation
2007; Elsevier BV; Volume: 282; Issue: 38 Linguagem: Inglês
10.1074/jbc.m703687200
ISSN1083-351X
AutoresXudong Liao, Wei Wang, Shenghan Chen, Qingyu Wu,
Tópico(s)Protease and Inhibitor Mechanisms
ResumoThe cardiac serine protease corin is the pro-atrial natriuretic peptide convertase. Corin is made as a zymogen, which is activated by proteolytic cleavage. Previous studies showed that recombinant human corin expressed in HEK 293 cells was biologically active, but activated corin fragments were not detectable, making it difficult to study corin activation. In this study, we showed that recombinant rat corin was activated in HEK 293 cells, murine HL-1 cardiomyocytes, and rat neonatal cardiomyocytes. In these cells, activated corin represented a small fraction of the total corin molecules. The activation of recombinant rat corin was inhibited by small molecule trypsin inhibitors but not inhibitors for matrix metalloproteinases or cysteine proteases, suggesting that a trypsin-like protease activated corin in these cells. Glycosidase digestion showed that rat and human corin proteins contained substantial N-glycans but little O-glycans. Treatment of HEK 293 cells expressing rat corin with tunicamycin prevented corin activation and inhibited its pro-atrial natriuretic peptide processing activity. Similar effects of tunicamycin on endogenous corin activity were found in HL-1 cells. Mutations altering the two N-glycosylation sites in the protease domain of rat corin prevented its activation in HEK 293 and HL-1 cells. Our results indicate that N-linked oligosaccharides play an important role in corin activation. The cardiac serine protease corin is the pro-atrial natriuretic peptide convertase. Corin is made as a zymogen, which is activated by proteolytic cleavage. Previous studies showed that recombinant human corin expressed in HEK 293 cells was biologically active, but activated corin fragments were not detectable, making it difficult to study corin activation. In this study, we showed that recombinant rat corin was activated in HEK 293 cells, murine HL-1 cardiomyocytes, and rat neonatal cardiomyocytes. In these cells, activated corin represented a small fraction of the total corin molecules. The activation of recombinant rat corin was inhibited by small molecule trypsin inhibitors but not inhibitors for matrix metalloproteinases or cysteine proteases, suggesting that a trypsin-like protease activated corin in these cells. Glycosidase digestion showed that rat and human corin proteins contained substantial N-glycans but little O-glycans. Treatment of HEK 293 cells expressing rat corin with tunicamycin prevented corin activation and inhibited its pro-atrial natriuretic peptide processing activity. Similar effects of tunicamycin on endogenous corin activity were found in HL-1 cells. Mutations altering the two N-glycosylation sites in the protease domain of rat corin prevented its activation in HEK 293 and HL-1 cells. Our results indicate that N-linked oligosaccharides play an important role in corin activation. Corin is a serine protease made primarily in atrial and ventricular cardiomyocytes (1Hooper 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, 2Wu Q. Front. Biosci. 2007; 12: 4179-4190Crossref PubMed Scopus (54) Google Scholar, 3Yan 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). Corin mRNA expression also has been detected in scar myofibroblasts in a rat model of myocardial infarction (4Calderone A. Bel-Hadj S. Drapeau J. El-Helou V. Gosselin H. Clement R. Villeneuve L. J. Cell. Physiol. 2006; 207: 165-173Crossref PubMed Scopus (54) Google Scholar). In cardiomyocytes, corin converts proatrial natriuretic peptide (pro-ANP) 2The abbreviations used are: ANPatrial natriuretic peptideDMEMDulbecco's modified Eagle's mediumFBSfetal bovine serumGAPDHglyceraldehyde-3-phosphate dehydrogenaseHEKhuman embryonic kidneyPBSphosphate-buffered salinePNGasepeptide-N-glycosidaseHRPhorseradish peroxidase. to active ANP (5Wu 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 hormone that regulates blood pressure by promoting natriuresis, diuresis, and vasodilatation (6Levin E.R. Gardner D.G. Samson W.K. N. Engl. J. Med. 1998; 339: 321-328Crossref PubMed Scopus (2066) Google Scholar, 7McGrath M.F. de Bold M.L. de Bold A.J. Trends Endocrinol. Metab. 2005; 16: 469-477Abstract Full Text Full Text PDF PubMed Scopus (212) Google Scholar, 8Potter L.R. Abbey-Hosch S. Dickey D.M. Endocr. Rev. 2006; 27: 47-72Crossref PubMed Scopus (784) Google Scholar). ANP also suppresses renin and endothelin release (9Burnett J.C. Am. J. Physiol. 1984; 247: F863PubMed Google Scholar, 10Granger J.P. Meadows E. Sooudi S. Stacy D.L. Am. J. Physiol. 1989; 257: H502PubMed Google Scholar, 11Kohno M. Yasunari K. Yokokawa K. Murakawa K. Horio T. Takeda T. J. Clin. Investig. 1991; 87: 1999-2004Crossref PubMed Scopus (142) Google Scholar), which represents an additional mechanism regulating vascular tone. In corin null mice, pro-ANP to ANP conversion was abolished, demonstrating that corin is the long sought physiological pro-ANP convertase (12Chan J.C. Knudson O. Wu F. Morser J. Dole W.P. Wu Q. Proc. Natl. Acad. Sci. U. S. A. 2005; 102: 785-790Crossref PubMed Scopus (211) Google Scholar). Corin null mice develop hypertension, indicating the importance of corin in maintaining normal blood pressure in vivo (12Chan J.C. Knudson O. Wu F. Morser J. Dole W.P. Wu Q. Proc. Natl. Acad. Sci. U. S. A. 2005; 102: 785-790Crossref PubMed Scopus (211) Google Scholar). Most recently, nonsynonymous single nucleotide polymorphisms in a corin gene allele were found to be associated with an increased risk for hypertension in African-Americans (13Dries D.L. Victor R.G. Rame J.E. Cooper R.S. Wu X. Zhu X. Leonard D. Ho S.I. Wu Q. Post W. Drazner M.H. Circulation. 2005; 112: 2403-2410Crossref PubMed Scopus (177) Google Scholar). Patients with this gene allele also exhibited an enhanced cardiac hypertrophic response to high blood pressure, as indicated by a greater left ventricular mass that is disproportional to their systolic blood pressure (14Burnett J.C. Hypertension. 2007; 49: 765-766Crossref PubMed Scopus (6) Google Scholar, 15Rame J.E. Drazner M.H. Post W. Peshock R. Lima J. Cooper R.S. Dries D.L. Hypertension. 2007; 49: 857-864Crossref PubMed Scopus (115) Google Scholar). These data suggest that corin might be involved in hypertensive disease in humans. atrial natriuretic peptide Dulbecco's modified Eagle's medium fetal bovine serum glyceraldehyde-3-phosphate dehydrogenase human embryonic kidney phosphate-buffered saline peptide-N-glycosidase horseradish peroxidase. Structurally, corin belongs to the type II transmembrane serine protease family (16Hooper 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, 17Szabo R. Wu Q. Dickson R.B. Netzel-Arnett S. Antalis T.M. Bugge T.H. Thromb. Haemostasis. 2003; 90: 185-193Crossref PubMed Google Scholar, 18Wu Q. Curr. Top. Dev. Biol. 2003; 54: 167-206Crossref PubMed Google Scholar). Corin has a short N-terminal cytoplasmic tail and an integral transmembrane domain. In its extracellular region, there are two frizzled-like cysteine-rich domains, eight low density lipoprotein receptor repeats, a scavenger receptor-like cysteine-rich domain, and a trypsin-like protease domain at the C terminus (1Hooper 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, 3Yan 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). Like most trypsin-like proteases, corin is made as a single chain zymogen, as shown by Western blots under reducing conditions (1Hooper 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, 19Knappe S. Wu F. Masikat M.R. Morser J. Wu Q. J. Biol. Chem. 2003; 278: 52363-52370Abstract Full Text Full Text PDF PubMed Scopus (100) Google Scholar, 20Yan W. Wu F. Morser J. Wu Q. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 8525-8529Crossref PubMed Scopus (390) Google Scholar). Human corin contains a conserved activation cleavage sequence Arg ↓ Ile-Leu-Gly-Gly at residues 801–805 (3Yan 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). Mutant corin R801A, in which Arg-801 was replace by an Ala, had no detectable activity in functional assays, indicating that proteolytic cleavage at Arg-801 is required to convert corin zymogen to an active enzyme (19Knappe S. Wu F. Masikat M.R. Morser J. Wu Q. J. Biol. Chem. 2003; 278: 52363-52370Abstract Full Text Full Text PDF PubMed Scopus (100) Google Scholar). For many proteolytic enzymes, zymogen activation is one of the most important steps in regulating their biological activities. To date, however, the physiological corin activator has not been identified. Little is known about how corin activity is controlled in vivo. The calculated mass for human corin protein is ∼116 kDa (3Yan 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). In Western blotting analysis, recombinant human corin from human embryonic kidney (HEK) 293 cells appeared as a band of ∼150 kDa (19Knappe S. Wu F. Masikat M.R. Morser J. Wu Q. J. Biol. Chem. 2003; 278: 52363-52370Abstract Full Text Full Text PDF PubMed Scopus (100) Google Scholar, 20Yan W. Wu F. Morser J. Wu Q. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 8525-8529Crossref PubMed Scopus (390) Google Scholar). Similar molecular mass also was observed in Western blotting of native corin from human hearts (1Hooper 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). The difference between the calculated and actually observed values in molecular mass suggests that corin protein may be extensively glycosylated. This is consistent with 19 predicted N-linked glycosylation sites in the extracellular region of human corin (3Yan 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). Most of these N-glycosylation sites are conserved in rat and mouse corin proteins (21Langenickel T.H. Pagel I. Buttgereit J. Tenner K. Lindner M. Dietz R. Willenbrock R. Bader M. Am. J. Physiol. 2004; 287: H1516Crossref PubMed Scopus (40) Google Scholar, 22Tomita Y. Kim D.H. Magoori K. Fujino T. Yamamoto T.T. J. Biochem. (Tokyo). 1998; 124: 784-789Crossref PubMed Scopus (45) Google Scholar). To date, the extent of N-linked oligosaccharides on corin protein and their functional importance have not been examined. Studies of other type II transmembrane serine proteases such as matriptase (23Oberst M.D. Williams C.A. Dickson R.B. Johnson M.D. Lin C.Y. J. Biol. Chem. 2003; 278: 26773-26779Abstract Full Text Full Text PDF PubMed Scopus (161) Google Scholar) and enteropeptidase (24Zheng X. Lu D. Sadler J.E. J. Biol. Chem. 1999; 274: 1596-1605Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar, 25Zheng X. Sadler J.E. J. Biol. Chem. 2002; 277: 6858-6863Abstract Full Text Full Text PDF PubMed Scopus (26) Google Scholar) have shown that N-glycosylation is critical for zymogen activation and subcellular targeting. It is possible that carbohydrate moieties may play a similar role in corin. In this study, we tested this hypothesis by examining corin zymogen activation in HEK 293 cells, mouse atrial cardiomyocyte HL-1 cells (26Claycomb W.C. Lanson N.A. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 2979-2984Crossref PubMed Scopus (1250) Google Scholar), and primary rat neonatal cardiomyocytes. Our results showed that a fraction of rat corin zymogen was converted to biologically active enzyme in these cells and that N-glycosylation was required for this activation process. Treatment of HEK 293 and HL-1 cells with tunicamycin or mutations altering the N-glycosylation sites in the protease domain prevented rat corin zymogen activation. Our data indicate that N-glycans are important in regulating corin biosynthesis and activity. Cell Culture-HEK 293 and its derived cell lines were cultured in Dulbecco's modified Eagle's medium (DMEM) with 10% fetal bovine serum (FBS). The murine atrial cardiomyocyte cell line HL-1, a generous gift from Dr. William C. Claycomb (Louisiana State University Medical Center, New Orleans), was cultured in Claycomb medium with 10% FBS, 100 μm norepinephrine, and 4 mm l-glutamine in gelatin/fibronectin-coated flasks or plates. Primary neonatal cardiomyocytes were isolated from hearts of 1-day-old rats and cultured in DMEM/F-12 medium containing 10% FBS, 100 units/ml penicillin, and 100 μg/ml streptomycin, as described previously (27Liao X. Liu J.M. Du L. Tang A. Shang Y. Wang S.Q. Chen L.Y. Chen Q. FASEB J. 2006; 20: 1883-1885Crossref PubMed Scopus (35) Google Scholar). The use of new born rats was approved by the local Institutional Animal Care and Use Committee and in compliance with the Guide for the Care and Use of Laboratory Animals, National Institutes of Health. All cells were cultured at 37 °C in humidified incubators with 5% CO2 and 95% air. Corin Expression Vectors-The full-length rat corin cDNA was cloned from a heart library. First, total RNA was isolated from adult Sprague-Dawley rat hearts using TRIzol reagents (Invitrogen). First strand cDNAs were synthesized using SuperScript III reverse transcriptase (Invitrogen). The full-length cDNA was amplified by PCR using Pfu polymerase (Stratagene, La Jolla, CA) and oligonucleotide primers based on the published rat corin cDNA sequence (21Langenickel T.H. Pagel I. Buttgereit J. Tenner K. Lindner M. Dietz R. Willenbrock R. Bader M. Am. J. Physiol. 2004; 287: H1516Crossref PubMed Scopus (40) Google Scholar). The cDNA was subjected to a second round PCR using Phusion High Fidelity polymerase (New England Biolabs, Inc., Ipswich, MA) with sense primer 5′-CAG TCA TGG GCA GGG TTT CTT TCA-3′ and antisense primer 5′-TCC TTG GGA TTT CTT TTG GAG AAA GGT C-3′ to delete the original stop codon. The PCR product was gel-purified and made an overhanging 3′-adenine by a 20-min incubation at 72 °C with Taq polymerase and a dNTP mixture (1 mmol/liter), and cloned into pcDNA3.1-V5-6xHis TOPO vector (Invitrogen). The resultant plasmid, pcDNAratCorin, encoded rat corin protein containing a viral V5 tag at its C terminus, which allowed the detection by an anti-V5 antibody (Invitrogen) in Western blotting. Plasmids expressing rat corin mutants R866A, N968S, and N1087S to replace residues Arg-866, Asn-968, and Asn-1087 with Ala, Ser, and Ser, respectively, were constructed by PCR-based site-directed mutagenesis using vector pcDNAratCorin as a template. The oligonucleotide primers used for mutagenesis (with mutation site underlined) were as follows: R866A-forward, 5′-A ATG AAC AAG GCC ATC CTT GGG GGT CGG AC-3′, and R866A-reverse, 5′-GT CCG ACC CCC AAG GAT GGC CTT GTT CAT T-3′; N968S-forward, 5′-AGT GAT GAT ATC ATC AGT GAG ACA AG-3′, and N968S-reverse, 5′-TGT CTC ACT GAT ATC ATC ACT C-3′; N1087S-forward, 5′-GTG TAC AGC AGT GTG TCT TAC TTT-3′, and N1087S-reverse, 5′-AAA GTA AGA CAC ACT GCT GTA CAC-3′. A human corin cDNA fragment containing the entire open reading frame was amplified by PCR from a human fetal heart library (BioChain, Hayward, CA) using Phusion High Fidelity polymerase with sense primer 5′-AGA GAA AAG CGA CCA AGA TAA A-3′ and antisense primer 5′-AGG CCA TTT TCT TTT AGT GTA GC-3′ based on the published human corin cDNA sequence (3Yan 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). The PCR products were cloned into pBS(II)-KS plasmid (Stratagene) and subjected to a second round PCR using sense primer 5′-AGA GAA AAG CGA CCA AGA TAA A-3′ and antisense primer 5′-GTT TAG GAG AAA GGT CTG GAT G-3′ to remove the original stop codon. The PCR fragment was cloned into pcDNA3.1-V5–6xHis TOPO vector, and the resultant plasmid, pcDNAhumanCorin, encoded human corin with a V5 tag at the C terminus. All expression plasmids used in this study were verified by restriction enzyme digestion and DNA sequencing. To express recombinant rat corin in primary cardiomyocytes, a lentiviral vector and packaging system, a generous gift from the Stark laboratory (Cleveland Clinic Foundation), was used. The plasmid pcDNAratCorin was digested with HindIII and PmeI to release the cDNA insert, which was blunted with Klenow enzyme and inserted into pLV-puro vector to yield vector pLVratCorin. Lentivirus was packaged in HEK 293T cells by co-transfection of pLVratCorin with packaging plasmid pCMVΔR8.74 and envelope plasmid pDM2G (28Jackson M.W. Agarwal M.K. Yang J. Bruss P. Uchiumi T. Agarwal M.L. Stark G.R. Taylor W.R. J. Cell Sci. 2005; 118: 1821-1832Crossref PubMed Scopus (81) Google Scholar). Primary rat neonatal cardiomyocytes were infected twice with the lentivirus in the conditioned medium from transfected 293T packaging cells. Transfection and Western Blotting-HEK 293 and HL-1 cells were transfected with expression plasmids using Lipofectamine 2000 (Invitrogen) according to manufacturer's instruction. Expressed proteins were detected by Western blotting. Cells were lysed in a buffer containing 50 mm Tris-HCl, pH 8.0, 150 mm NaCl, 1% Triton X-100 (v/v), 10% glycerol (v/v), and a protease inhibitor mixture (1:100 dilution; Sigma). Protein samples were mixed with a loading buffer with (reducing) or without (nonreducing) 2% β-mercaptoethanol and boiled at 100 °C for 5 min before being loaded onto an SDS-polyacrylamide gel. Western blotting of recombinant corin and pro-ANP was done using a horseradish peroxidase conjugated anti-V5 (anti-V5-HRP) antibody, as described previously (5Wu 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, 20Yan W. Wu F. Morser J. Wu Q. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 8525-8529Crossref PubMed Scopus (390) Google Scholar). For stable transfection, transfected cells were selected in DMEM containing G418 (500 μg/ml, Sigma). Cell clones expressing corin were identified by Western blotting. To quantify activated corin fragments, x-ray films from Western blotting were scanned by a densitometer (The PharosFx System, Bio-Rad). The optical density of the bands representing corin zymogen and the protease domain was measured, and the percentage of the activated molecules was calculated using the Quantity One one-dimensional analysis software (Bio-Rad). Corin-mediated Pro-ANP Processing-A cell-based assay was used to examine corin-mediated pro-ANP processing, as described previously (20Yan W. Wu F. Morser J. Wu Q. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 8525-8529Crossref PubMed Scopus (390) Google Scholar, 29Wu 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). HEK 293 cells in 6-well plates were co-transfected with plasmids expressing human pro-ANP (pcDNAproANP) (20Yan W. Wu F. Morser J. Wu Q. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 8525-8529Crossref PubMed Scopus (390) Google Scholar) and rat or human corin (pcDNAratCorin or pcDNAhumanCorin) and incubated for 16 h. Conditioned medium was collected and centrifuged at 13,000 rpm for 10 min to remove cell debris. Pro-ANP and its derivatives in the conditioned medium were analyzed by immunoprecipitation using an anti-V5 antibody against the tag attached to the C terminus of recombinant pro-ANP/ANP followed by Western blotting using the anti-V5-HRP antibody. On Western blots, recombinant human pro-ANP and ANP with the V5 tag appeared as bands of ∼23 and ∼11 kDa, respectively, as described previously (20Yan W. Wu F. Morser J. Wu Q. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 8525-8529Crossref PubMed Scopus (390) Google Scholar, 29Wu 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). In some experiments, conditioned medium containing pro-ANP was first prepared in transfected HEK 293 cells, then added (2 ml/well) to HL-1 cells or HEK 293 cells expressing wild-type or mutant corin in 6-well plates, and incubated at 37 °C for 1 h. Pro-ANP and its derivatives in the conditioned medium were analyzed by immunoprecipitation and Western blotting, as described above. Effects of Small Molecule Inhibitors-Transfected HEK 293 and HL-1 cells expressing recombinant rat corin were grown in DMEM. Small molecule inhibitors, including trypsin-like protease inhibitors (10 mm benzamidine and 100 μm leupeptin), a metalloproteinase inhibitor (20 μm GM 6001) (30Park H.I. Turk B.E. Gerkema F.E. Cantley L.C. Sang Q.X. J. Biol. Chem. 2002; 277: 35168-35175Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar), and a cysteine protease inhibitor (100 μm N-acetyl-leucyl-leucyl-methionine (ALLM)) (31Inoue S. Bar-Nun S. Roitelman J. Simoni R.D. J. Biol. Chem. 1991; 266: 13311-13317Abstract Full Text PDF PubMed Google Scholar) were added to the cells in separate wells and incubated for 16 h. Recombinant rat corin and its derived fragments were analyzed by Western blotting, as described above. Glycosidase Digestion-To analyze the carbohydrate contents in rat and human corin proteins, glycosidase digestion was done using a deglycosylation kit from Prozyme (San Leandro, CA). Briefly, HEK 293 cells expressing rat or human corin in 10-cm dishes were washed twice with phosphate-buffered saline (PBS), collected, and lysed in 100 μl of a buffer containing 50 mm Tris-HCl, pH 8.0, 150 mm NaCl, and 1% Nonidet P-40. Cell lysate containing ∼100 μg of protein was incubated at 100 °C for 5 min in 42.5 μl of a buffer containing 50 mm sodium phosphate, pH 7.0, 0.1% SDS, and 50 mm β-mercaptoethanol. Denatured and reduced proteins were digested without (control) or with 5 × 10–3 units of N-glycanase, O-glycanase, and sialidase A, either individually or in combination, at 37 °C for 16 h. Protein samples were analyzed by Western blotting using an anti-V5 antibody. Effects of Tunicamycin-Effects of tunicamycin on corin zymogen activation and pro-ANP processing activity were examined in HEK 293 cells expressing rat corin. Confluent (∼95%) cells in 6-well plates were cultured in DMEM with 10% FBS with or without tunicamycin (1 μg/ml; Sigma). After 16 h, the medium was removed, and the cells were washed with serum-free medium. Conditioned medium containing recombinant pro-ANP was added to the cells and incubated at 37 °C for 1 h. As a control for potential direct effect of tunicamycin on corin or pro-ANP, tunicamycin (1 μg/ml) was added to pro-ANP containing medium and incubated for 1 h with HEK 293 that were not pretreated with the antibiotic. Pro-ANP and its derivatives in the conditioned medium were analyzed by immunoprecipitation and Western blotting. Recombinant corin expression in HEK 293 cells with or without tunicamycin treatment was verified by Western blotting of cell lysate. Similar experiments were done with HL-1 cells, which appeared to be more resistant to tunicamycin inhibition. As a result, a higher concentration (10 μg/ml) of tunicamycin was used during the 16-h incubation period. Pro-ANP processing was examined as described above. Analysis of Biotin-labeled Cell Surface Protein Expression-HEK 293 cells expressing recombinant rat corin in 60-mm dishes were washed three times with PBS and incubated with 3 ml of PBS, pH 8.0, containing 1 mm sulfo-NHS-biotin (Pierce) at room temperature for 30 min. Reactions were quenched by a 10-min incubation in 3 ml of PBS with 100 mm glycine. The cells were washed with PBS and lysed on ice for 30 min in 300 μl of a lysis buffer containing 50 mm Tris-HCl, pH 8.0, 150 mm NaCl, 1% Nonidet P-40 (v/v), and a protease inhibitor mixture (1:100 dilution). Cell lysate was centrifuged at 4 °C at 13,000 rpm for 10 min, and protein concentration was adjusted to 1 μg/μl using the lysis buffer. Streptavidin-Sepharose beads (50 μl in a 50:50 (beads/buffer volume) slurry) were added to 300 μl of cell lysate, and the mixture was rocked at 4 °C for 2 h. The beads were washed three times with the lysis buffer and boiled in 50 μl of sample loading buffer. Protein eluates were analyzed by Western blotting. Expression of Rat Corin in HEK 293 Cells-The full-length rat corin cDNA was cloned from a Sprague-Dawley rat heart library by PCR based on the published sequence (21Langenickel T.H. Pagel I. Buttgereit J. Tenner K. Lindner M. Dietz R. Willenbrock R. Bader M. Am. J. Physiol. 2004; 287: H1516Crossref PubMed Scopus (40) Google Scholar). A plasmid vector was made to express recombinant rat corin with a V5 tag at the C terminus (Fig. 1A). To test the activity of recombinant rat corin, HEK 293 cells were co-transfected with a pro-ANP expression vector and the rat corin expression vector. Western blotting of the conditioned medium indicated that pro-ANP was converted to ANP in cells expressing rat corin (data not shown), confirming that recombinant rat corin was biologically active. N-Linked Glycosylation in Rat and Human Corin-HEK 293 cell lysates containing recombinant rat or human corin were digested with N-glycanase, O-glycanase, and sialidase A and analyzed by Western blotting using an antibody against the V5 tag at the C termini of recombinant rat and human corin. Under reducing conditions, rat corin appeared as a major band of ∼170 kDa (Fig. 1B, lane 1), whereas human corin appeared as two bands of ∼150 and ∼130 kDa, respectively (Fig. 1B, lane 7). The result was consistent with rat corin being 69 amino acids longer than human corin (1111 versus 1042 amino acids) (3Yan 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, 21Langenickel T.H. Pagel I. Buttgereit J. Tenner K. Lindner M. Dietz R. Willenbrock R. Bader M. Am. J. Physiol. 2004; 287: H1516Crossref PubMed Scopus (40) Google Scholar). Peptide-N-glycosidase (PNGase) F digestion reduced the apparent mass of rat corin to ∼130 kDa (Fig. 1B, lane 2) and that of human corin to ∼110 kDa (Fig. 1B, lane 8). It appeared that the ∼150- and ∼130-kDa species of human corin were differentially N-glycosylated products. Digestion with O-glycanase and sialidase A, either individually or in combination, did not yield noticeable reduction in the apparent mass of rat and human corin (Fig. 1B, lanes 3–6, and 9). The results indicate that the majority, if not all, of carbohydrate moieties on corin are N-linked oligosaccharides. Interestingly, a minor band of ∼40 kDa was detected in cell lysate with rat, but not human, corin (Fig. 1B, lane 1). Upon PNGase F, but not O-glycanase or sialidase A, digestion, this band was reduced to ∼35 kDa (Fig. 1B, lanes 2–6). Based on its size, this band appeared to be the corin protease fragment produced by activation cleavage (Fig. 1A). Consistently, this domain of rat corin contains two potential N-glycosylation sites (Asn-968 and Asn-1087) (21Langenickel T.H. Pagel I. Buttgereit J. Tenner K. Lindner M. Dietz R. Willenbrock R. Bader M. Am. J. Physiol. 2004; 287: H1516Crossref PubMed Scopus (40) Google Scholar). Because the V5 tag was attached to the C terminus of recombinant corin proteins, the anti-V5 antibody would not detect the cleaved N-terminal propeptide in our experiments. Detection of Cleaved Rat Corin Protease Fragments in HEK 293 and HL-1 Cells, and Neonatal Cardiomyocytes-If the observed ∼40-kDa band in lysate from HEK cells expressing rat corin was the activated corin protease fragment, it is expected to be attached to the propeptide region by a disulfide bond connecting Cys residues 855 and 977 (Fig. 1A) (3Yan 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, 21Langenickel T.H. Pagel I. Buttgereit J. Tenner K. Lindner M. Dietz R. Willenbrock R. Bader M. Am. J. Physiol. 2004; 287: H1516Crossref PubMed Scopus (40) Google Scholar). On Western blots, the propeptide and the activated protease domain should appear as a single band under nonreducing conditions. This was confirmed upon further studies. The ∼40-kDa band was detected by Western blotting under reducing but not nonreducing conditions in HEK 293 cells expressing rat corin (Fig. 2A). To examine if the activation cleavage occurs in physiologically relevant cardiomyocytes, recombinant rat corin was expressed in murine atrial cell line HL-1 and rat primary neonatal cardiomyocytes. Western blotting analysis of cell lysate showed a similar ∼40-kDa band in HL-1 cells and rat neonatal cardiomyocytes under reducing but not nonreducing conditions (Fig. 2A). This band represented a small fraction of overall corin protein in these cells, as estimated by densitometry. In lysate from HEK 293 cells expressing human corin, this ∼40-kDa band was barely visible (Fig. 2B). The results were consistent with our previous observation that only a very low percentage of human corin molecules was activated in HEK 293 cells. Lack of Activation Cleavage in Mutant Rat Corin R866A-To confirm that the observed ∼40-kDa band indeed was generated by cleavage at the conserved activation site, a plasmid construct was made to express mutant rat corin R866A, in which Arg-866 was replaced by an Ala to abolish the activation site (21Langenickel T.H. Pagel I. Buttgereit J. Tenner K. Lindner M. Dietz R. Willenbrock R. Bader M. Am. J. Physiol. 2004; 287: H1516Crossref PubMed Scopus (40) Google Scholar). HEK 293 cells were transfected with plasmids expre
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