Golgi α-mannosidases regulate cell surface N-glycan type and ectodomain shedding of the transmembrane protease corin.

Corin is a transmembrane protease that activates natriuretic peptides on the cell membrane. Reduced cell surface targeting or increased ectodomain shedding disrupts cell membrane homeostasis of corin, thereby impairing its cell surface expression and enzyme activity. N-glycans are essential in corin ectodomain shedding. Lack of N-glycans promotes corin ectodomain shedding in the juxtamembrane and frizzled-1 domains. The nascent N-glycans, transferred onto the polypeptide of corin, undergo multistep N-glycan processing in the endoplasmic reticulum and Golgi. It remains unclear how trimming by Golgi α-mannosidases, the critical N-glycan processing steps in N-glycan maturation, may regulate corin biosynthesis. In this study, we examined the effects of kifunensine and swainsonine, the inhibitors for α-mannosidases I and II, on corin expression and function. Western analysis of corin proteins in cell lysates and conditioned media from the inhibitor-treated corin-stable HEK293 cells and AC16 cells showed that both α-mannosidases I and II were required to maintain complex N-glycans on cell surface corin and protect corin from ectodomain shedding in the juxtamembrane and frizzled-1 domains. Cell viability analysis revealed that inhibition of α-mannosidase I or II sensitized cardiomyocytes to hydrogen peroxide-induced injury via regulating corin. Moreover, either one of the two coding genes was sufficient to perform Golgi α-mannosidase I trimming of N-glycans on corin. Similarly, this sufficiency was observed in Golgi α-mannosidase II-coding genes. Inhibition of ectodomain shedding restored corin zymogen activation from kifunensine- or swainsonine-induced reduction. Together, our results show the important roles of Golgi α-mannosidases in maintaining cell membrane homeostasis and biological activities of corin.

Krüppel-like factor 17 upregulates uterine corin expression and promotes spiral artery remodeling in pregnancy.

Spiral artery remodeling is an important physiological process in the pregnant uterus which increases blood flow to the fetus. Impaired spiral artery remodeling contributes to preeclampsia, a major disease in pregnancy. Corin, a transmembrane serine protease, is up-regulated in the pregnant uterus to promote spiral artery remodeling. To date, the mechanism underlying uterine corin up-regulation remains unknown. Here we show that Krüppel-like factor (KLF) 17 is a key transcription factor for uterine corin expression in pregnancy. In cultured human uterine endometrial cells, KLF17 binds to the CORIN promoter and enhances the promoter activity. Disruption of the KLF17 gene in the endometrial cells abolishes CORIN expression. In mice, Klf17 is up-regulated in the pregnant uterus. Klf17 deficiency prevents uterine Corin expression in pregnancy. Moreover, Klf17-deficient mice have poorly remodeled uterine spiral arteries and develop gestational hypertension and proteinuria. Together, our results reveal an important function of KLF17 in regulating Corin expression and uterine physiology in pregnancy.

Natriuretic Peptide Signaling in Uterine Biology and Preeclampsia.

Endometrial decidualization is a uterine process essential for spiral artery remodeling, embryo implantation, and trophoblast invasion. Defects in endometrial decidualization and spiral artery remodeling are important contributing factors in preeclampsia, a major disorder in pregnancy. Atrial natriuretic peptide (ANP) is a cardiac hormone that regulates blood volume and pressure. ANP is also generated in non-cardiac tissues, such as the uterus and placenta. In recent human genome-wide association studies, multiple loci with genes involved in natriuretic peptide signaling are associated with gestational hypertension and preeclampsia. In cellular experiments and mouse models, uterine ANP has been shown to stimulate endometrial decidualization, increase TNF-related apoptosis-inducing ligand expression and secretion, and enhance apoptosis in arterial smooth muscle cells and endothelial cells. In placental trophoblasts, ANP stimulates adenosine 5'-monophosphate-activated protein kinase and the mammalian target of rapamycin complex 1 signaling, leading to autophagy inhibition and protein kinase N3 upregulation, thereby increasing trophoblast invasiveness. ANP deficiency impairs endometrial decidualization and spiral artery remodeling, causing a preeclampsia-like phenotype in mice. These findings indicate the importance of natriuretic peptide signaling in pregnancy. This review discusses the role of ANP in uterine biology and potential implications of impaired ANP signaling in preeclampsia.

Investigating the Effects of Atrial Natriuretic Peptide on the Maternal Endothelium to Determine Potential Implications for Preeclampsia.

Preeclampsia is associated with an increased lifelong risk of cardiovascular disease (CVD). It is not clear whether this is induced by persistent systemic organ and vascular damage following preeclampsia or due to a predisposition to both conditions that share cardiovascular pathophysiology. Common to both CVD and preeclampsia is the dysregulation of corin and its proteolytic product, atrial natriuretic peptide (ANP). ANP, a hypotensive hormone converted from pro-ANP by corin, is involved in blood pressure homeostasis. While corin is predominantly a cardiac enzyme, both corin and pro-ANP are significantly upregulated in the gravid uterus and dysregulated in preeclampsia. Relatively little is known about ANP function in the endothelium during a pregnancy complicated by preeclampsia. Here, we investigated the effect of ANP on endothelial cell proliferation and migration, markers of endothelial dysfunction, and receptor expression in omental arteries exposed to circulating preeclamptic toxins. ANP receptor expression is significantly upregulated in preeclamptic vasculature but not because of exposure to preeclampsia toxins tumour necrosis factor α or soluble fms-like tyrosine kinase-1. The supplementation of endothelial cells with ANP did not promote proliferation or migration, nor did ANP improve markers of endothelial dysfunction. The role of ANP in preeclampsia is unlikely to be via endothelial pathways.

Golden cats: A never-ending story!

In the British feline breed a golden coat modification, called light-gold, akita or copper, was reported by breeders during the 2010s. This modification restricted eumelanin to the tip of the tail and hairs showed a wideband modification. Pedigree analyses revealed an autosomal recessive inheritance pattern. A single candidate region was identified using a genome-wide association study. Within that region, we identified CORIN (Corin, serine peptidase) as the strongest candidate gene, since two CORIN variants have previously been identified in Siberian cats with a golden phenotype. A homozygous CORIN:c.2425C>T nonsense variant was identified in copper British cats. Segregation of the variant was consistent with recessive inheritance. This nonsense CORIN:c.2425C>T variant, located in CORIN exon 19, was predicted to produce a truncated CORIN protein - CORIN:p.(Arg809Ter) - that would lack part of the scavenger receptor domain and the trypsine-like serine protease catalytic domain. All 30 copper cats were T/T homozygous for the variant, which was also found in 20 C/T heterozygous British control cats but was absent in 340 cats from the 99 Lives dataset. Finally, genotyping of 218 cats from 12 breeds failed to identify carriers in cats from other breeds. We propose that this third CORIN:c.2425C>T variant represents the wbBSH (British recessive wideband) allele in the domestic cat.

Renal Corin Is Essential for Normal Blood Pressure and Sodium Homeostasis.

Atrial natriuretic peptide (ANP)-mediated natriuresis is known as a cardiac endocrine function in sodium and body fluid homeostasis. Corin is a protease essential for ANP activation. Here, we studied the role of renal corin in regulating salt excretion and blood pressure. We created corin conditional knockout (cKO), in which the Corin gene was selectively disrupted in the kidney (kcKO) or heart (hcKO). We examined the blood pressure, urinary Na+ and Cl- excretion, and cardiac hypertrophy in wild-type, corin global KO, kcKO, and hcKO mice fed normal- and high-salt diets. We found that on a normal-salt diet (0.3% NaCl), corin kcKO and hcKO mice had increased blood pressure, indicating that both renal and cardiac corin is necessary for normal blood pressure in mice. On a high-salt diet (4% NaCl), reduced urinary Na+ and Cl- excretion, increased body weight, salt-exacerbated hypertension, and cardiac hypertrophy were observed in corin kcKO mice. In contrast, impaired urinary Na+ and Cl- excretion and salt-exacerbated hypertension were not observed in corin hcKO mice. These results indicated that renal corin function is important in enhancing natriuresis upon high salt intakes and that this function cannot be compensated by the cardiac corin function in mice.

Proprotein Convertase Subtilisin/Kexin 6 in Cardiovascular Biology and Disease.

Proprotein convertase subtilisin/kexin 6 (PCSK6) is a secreted serine protease expressed in most major organs, where it cleaves a wide range of growth factors, signaling molecules, peptide hormones, proteolytic enzymes, and adhesion proteins. Studies in Pcsk6-deficient mice have demonstrated the importance of Pcsk6 in embryonic development, body axis specification, ovarian function, and extracellular matrix remodeling in articular cartilage. In the cardiovascular system, PCSK6 acts as a key modulator in heart formation, lipoprotein metabolism, body fluid homeostasis, cardiac repair, and vascular remodeling. To date, dysregulated PCSK6 expression or function has been implicated in major cardiovascular diseases, including atrial septal defects, hypertension, atherosclerosis, myocardial infarction, and cardiac aging. In this review, we describe biochemical characteristics and posttranslational modifications of PCSK6. Moreover, we discuss the role of PCSK6 and related molecular mechanisms in cardiovascular biology and disease.

Corin plays a protective role via upregulating MAPK and downregulating eNOS in diabetic nephropathy endothelial dysfunction.

Diabetic nephropathy (DN) is one of the leading causes of mortality in diabetic patients, but its pathogenesis is unclear. We aimed to study the role of the pro-ANP convertase Corin in the pathogenesis of DN. Corin and ANP expression in DN rat kidneys and high-glucose-treated HK-2 cells was analyzed by real-time PCR, western blotting, and immunohistochemical staining. The effect of Corin-siRNA or ANP-siRNA HK-2 cells on EA.hy926 cell migration was determined by scratch-wound healing assay. The expression of mitogen-activated protein kinase (MAPK) and endothelial NO synthase (eNOS) in EA.hy926 cells treated with conditioned medium from Corin-siRNA- or ANP-siRNA-transfected HK-2 cells was determined by western blotting. We found a significant reduction in Corin and ANP expression in DN rat kidneys. These results were recapitulated in HK-2 cells treated with high glucose. EA.hy926 cells treated with conditioned medium from Corin-deficient HK-2 cells had inhibited migration, increased MAPK activity, and decreased eNOS activity. Similar effects were observed with ANP-siRNA transfection. Finally, adding ANP to the Corin-deficient HK-2 conditioned medium rescued the above defects, indicating that Corin mediates its effects through ANP. In conclusion, Corin plays a renoprotective role through pro-ANP processing, and defects in Corin cause endothelial dysfunction through MAPK and eNOS signaling in DN.

Siberian cats help in solving part of the mystery surrounding golden cats.

Golden cats have been appreciated since the beginning of the cat fancy. Golden is a modification of the tabby coat. In the Siberian breed, a specific golden phenotype, named sunshine, has been described. Sunshine tabby cats exhibit a warm tone of tabby, a pink nose lacking the black lining and a large light cream area around the nose. Pedigree analyses revealed an autosomal recessive inheritance pattern. A single candidate region was identified by genome-wide association study (GWAS) and homozygosity mapping. Within that region, we identified CORIN (Corin, serine peptidase) as a strong candidate gene, since CORIN variants have been identified in mice and tigers with a golden phenotype and CORIN has been described as a modifier of the ASIP (Agouti Signaling Protein) pathway. A homozygous CORIN:c.2383C>T missense variant was identified in sunshine tabby cats. Segregation of the variant was consistent with recessive inheritance. The variant was also found in three Kurilian bobtail cats and in two ToyBob cats from the 99 Lives dataset but genotyping of 106 cats from 13 breeds failed to identify carriers in cats from other breeds. The CORIN:c.2383C>T variant was predicted to change an arginine to a cysteine at position 795 in the protein: CORIN:p.(Arg795Cys). Finally, hair observation in Siberian cats was consistent with elongated ASIP signaling as golden hair showed a large yellow band instead of the short subapical one usually observed in agouti hair. These results support an association of the Siberian sunshine modification with the CORIN:c.2383C>T variant. The Siberian cat has helped us to decipher one of the golden phenotypes observed in cats and we propose that the CORIN:c.2383C>T variant represents the wbSIB (Siberian recessive wideband) allele in the domestic cat.

A common CORIN variant in hypertension reduces corin intracellular trafficking by exposing an inhibitory N-terminus.

Corin is a transmembrane serine protease that activates atrial natriuretic peptide, a cardiac hormone essential for normal blood pressure. Corin is synthesized as a zymogen and activated on the cell surface. In previous studies, we identified a CORIN variant allele with an adenine insertion in the 5'-end of the coding region in ∼5% of hypertensive individuals in a Chinese population. The protein, named insA, encoded by the CORIN variant allele has a shortened cytoplasmic tail and reduced atrial natriuretic peptide processing activity. It remains unknown how a shortened cytoplasmic tail impairs corin function. In this study, we expressed a series of corin mutants with different N-terminal sequences and analyzed them by Western blotting, flow cytometry, protein chase, and immunostaining. Our results revealed that a Gly-Asn sequence after the initiating Met at the newly generated N-terminus was responsible for delaying corin trafficking in the Golgi. Deletion of the N-terminal Gly and Asn residues increased the intracellular trafficking, cell surface expression, and activation cleavage of the insA variant. These results help to explain how the CORIN variant allele impairs corin structure and function as an underlying mechanism in hypertension.

Function and regulation of corin in physiology and disease.

Atrial natriuretic peptide (ANP) is of major importance in the maintenance of electrolyte balance and normal blood pressure. Reduced plasma ANP levels are associated with the increased risk of cardiovascular disease. Corin is a type II transmembrane serine protease that converts the ANP precursor to mature ANP. Corin deficiency prevents ANP generation and alters electrolyte and body fluid homeostasis. Corin is synthesized as a zymogen that is proteolytically activated on the cell surface. Factors that disrupt corin folding, intracellular trafficking, cell surface expression, and zymogen activation are expected to impair corin function. To date, CORIN variants that reduce corin activity have been identified in hypertensive patients. In addition to the heart, corin expression has been detected in non-cardiac tissues, where corin and ANP participate in diverse physiological processes. In this review, we summarize the current knowledge in corin biosynthesis and post-translational modifications. We also discuss tissue-specific corin expression and function in physiology and disease.

Corin Overexpression Reduces Myocardial Infarct Size and Modulates Cardiomyocyte Apoptotic Cell Death.

Altered expression of corin, a cardiac transmembrane serine protease, has been linked to dilated and ischemic cardiomyopathy. However, the potential role of corin in myocardial infarction (MI) is lacking. This study examined the outcomes of MI in wild-type vs. cardiac-specific overexpressed corin transgenic (Corin-Tg) mice during pre-MI, early phase (3, 24, 72 h), and late phase (1, 4 weeks) post-MI. Corin overexpression significantly reduced cardiac cell apoptosis (p < 0.001), infarct size (p < 0.001), and inhibited cleavage of procaspases 3, 9, and 8 (p < 0.05 to p < 0.01), as well as altered the expression of Bcl2 family proteins, Bcl-xl, Bcl2 and Bak (p < 0.05 to p < 0.001) at 24 h post-MI. Overexpressed cardiac corin also significantly modulated heart function (ejection fraction, p < 0.0001), lung congestion (lung weight to body weight ratio, p < 0.0001), and systemic extracellular water (edema, p < 0.05) during late phase post-MI. Overall, cardiac corin overexpression significantly reduced apoptosis, infarct size, and modulated cardiac expression of key members of the apoptotic pathway in early phase post-MI; and led to significant improvement in heart function and reduced congestion in late phase post-MI. These findings suggest that corin may be a useful target to protect the heart from ischemic injury and subsequent post-infarction remodeling.

Associations between potentially functional CORIN SNPs and serum corin levels in the Chinese Han population.

BACKGROUND: Corin is an important convertase involved in the natriuretic peptide system and may indirectly regulate blood pressure. Genetic factors relate to corin remain unclear. The purpose of the current study was to comprehensively examine the associations among CORIN SNPs, methylations, serum corin levels and hypertension. RESULTS: We genotyped 9 tag-SNPs in the CORIN gene and measured serum corin levels in 731 new-onset hypertensive cases and 731 age- and sex-matched controls. DNA methylations were tested in 43 individuals. Mendelian randomization was used to investigate the causal associations. Under additive models, we observed associations of rs2289433 (p.Cys13Tyr), rs6823184, rs10517195, rs2271037 and rs12509275 with serum corin levels after adjustment for covariates (P = 0.0399, 0.0238, 0.0016, 0.0148 and 0.0038, respectively). The tag-SNP rs6823184 and SNPs that are in strong linkage disequilibrium with it, i.e., rs10049713, rs6823698 and rs1866689, were associated with CORIN gene expression (P = 2.38 × 10- 24, 5.94 × 10- 27, 6.31 × 10- 27 and 6.30 × 10- 27, respectively). Neither SNPs nor corin levels was found to be associated with hypertension. SNP rs6823184, which is located in a DNase hypersensitivity cluster, a CpG island and transcription factor binding sites, was significantly associated with cg02955940 methylation levels (P = 1.54 × 10- 7). A putative causal association between cg02955940 methylation and corin levels was detected (P = 0.0011). CONCLUSION: This study identified potentially functional CORIN SNPs that were associated with serum corin level in the Chinese Han population. The effect of CORIN SNPs on corin level may be mediated by DNA methylation.

Cardiac-Specific Overexpression of Catalytically Inactive Corin Reduces Edema, Contractile Dysfunction, and Death in Mice with Dilated Cardiomyopathy.

Humans with dilated cardiomyopathy (DCM) and heart failure (HF) develop low levels of corin, a multi-domain, cardiac-selective serine protease involved in natriuretic peptide cleavage and sodium and water regulation. However, experimental restoration of corin levels markedly attenuates HF progression. To determine whether the beneficial effects of corin in HF require catalytic activity, we engineered cardiac overexpression of an enzymatically inactive corin transgene (corin-Tg(i)). On a wild-type (WT) background, corin-Tg(i) had no evident phenotypic effects. However, in a well-established genetic model of DCM, corin-Tg(i)/DCM mice had increased survival (p < 0.01 to 0.001) vs. littermate corin-WT/DCM controls. Pleural effusion (p < 0.01), lung edema (p < 0.05), systemic extracellular free water (p < 0.01), and heart weight were decreased (p < 0.01) in corin-Tg(i)/DCM vs. corin-WT/DCM mice. Cardiac ejection fraction and fractional shortening improved (p < 0.01), while ventricular dilation decreased (p < 0.0001) in corin-Tg(i)/DCM mice. Plasma atrial natriuretic peptide, cyclic guanosine monophosphate, and neprilysin were significantly decreased. Cardiac phosphorylated glycogen synthase kinase-3ß (pSer9-GSK3ß) levels were increased in corin(i)-Tg/DCM mice (p < 0.01). In summary, catalytically inactive corin-Tg(i) decreased fluid retention, improved contractile function, decreased HF biomarkers, and diminished cardiac GSK3ß activity. Thus, the protective effects of cardiac corin on HF progression and survival in experimental DCM do not require the serine protease activity of the molecule.

Natriuretic Peptide Processing in Patients with and Without Left Ventricular Dysfunction.

This study aimed to examine the relationship between corin expression and circulating brain natriuretic peptide in patients with left ventricular (LV) dysfunction.Circulating levels of B-type natriuretic peptide (BNP) can be an indicator of LV dysfunction. The 32-amino-acid BNP is cleaved by corin, a cardiac serine protease, from its108-amino-acid pro-brain natriuretic peptide (proBNP) precursor.This study included 25 patients with idiopathic dilated cardiomyopathy (DCMP) and LV dysfunction and 44 heart transplant recipients with normal LV function who underwent diagnostic left and right heart catheterization. Blood samples were used to determine the ratio of plasma proBNP/BNP levels, and LV endomyocardial biopsies were used to determine the expression of NPPB, which encode BNP and corin, respectively, by quantitative reverse transcription-polymerase chain reaction.Patients with DCMP revealed worse hemodynamic profiles and higher plasma proBNP and BNP levels than those of the transplant recipients. Myocardial NPPB expression was higher and CORIN expression was lower in the DCMP patients than in the transplant recipients. CORIN expression significantly correlated with NPPB expression (r = -0.585; P < 0.001), ejection fraction (EF; r = 0.694; P < 0.01), LV end-diastolic pressure (r = -0.373; P < 0.05), and indexed end-diastolic LV volume (r = -0.452; P < 0.001). In addition, the plasma proBNP/BNP levels inversely correlated with the CORIN expression (r = -0.362; P < 0.005).Decreased myocardial CORIN expression and the corresponding higher levels of circulating unprocessed proBNP in DCMP may partly account for the relative BNP resistance observed in patients with LV dysfunction.

Corin is highly expressed in atherosclerosis models.

Corin is a serine protease mainly expressed in the heart, where it regulates blood pressure and cardiac function through activating pro-atrial natriuretic peptide (pro-ANP) to ANP. Its expression has also been detected in non-cardiac tissues. However, there is no report so far about the distribution and function of corin in aorta and in related diseases such as atherosclerosis. This study was the first to explore corin expression in aorta both under normal conditions and in atherosclerosis models. In vivo, we found corin had a basal level of expression in aortas, mainly in intimal endothelial cells and was significantly elevated in mouse atherosclerosis model. Moreover, we observed pro-ANP, the specific substrate of corin, was also expressed in mice aortas and increased in mouse atherosclerosis model. In vitro, we further demonstrated corin expression in cultured vascular endothelial cells and its induced expression after ox-LDL stimulation. Our results suggested that corin may play important roles in aorta physiology and in the pathophysiological process of atherosclerosis in an autocrine manner and has potential clinical value for the treatment of atherosclerosis.

Identification and functional analysis of CORIN variants in hypertensive patients.

Corin is a serine protease that activates atrial natriuretic peptide (ANP). CORIN gene variants have been reported in patients with hypertension. To date, however, the prevalence of CORIN variants in hypertensive patients remains unknown. To understand the prevalence and functional significance of CORIN variants in hypertension, we sequenced CORIN exons in 300 normal and 401 hypertensive individuals in a Chinese population and identified nine nonsynonymous variants, of which eight were not characterized previously. Among them, variants c.131A > G (p.Tyr13Cys), c.376G > T (p.Asp95Tyr), c.1094T > G (p.Leu334Trp), and c.1667G > A (p.Arg525His) occurred similarly in both normal and hypertensive individuals. Variants c1139G > A (p.Arg349His), c.2689C > T (p.Pro866Ser), and c.2864C > T (p.Thr924Met) were found once each in hypertensive individuals. Variant c.1683G > T (p.Arg530Ser) occurred preferentially in hypertensive individuals [10/401 (2.5%) vs. 1/300 (0.3%) in normal individuals; P = 0.023], which was confirmed in another independent cohort [9/368 (2.44%) in hypertensive and 2/377 (0.53%) in normal individuals; P = 0.033]. In biochemical and cell-based functional studies, variants p.Arg530Ser and p.Thr924Met, but not p.Tyr13Cys, p.Asp95Tyr, p.Leu334Trp, p.Arg349His, p.Arg525His, and p.Pro866Ser, exhibited reduced pro-ANP processing activity, which was caused by endoplasmic reticulum retention and poor zymogen activation, respectively. These results indicate that genetic variants impairing corin function are not uncommon in general populations and that such variants may be an important contributing factor in hypertension.

The Involvement of Corin in the Progression of Diabetic Erectile Dysfunction in a Rat Model by Down-Regulating ANP /NO/cGMP Signal Pathway.

This study was aimed to analyze the potential role of Corin in the procession of diabetic ED and to explore the underlying mechanism. Diabetic ED rat model was constructed and the characteristics of diabetic ED and control rats were recorded at 4, 8, 12, and 16 weeks. qRT-PCR and Western bloting were used to detected the mRNA and protein levels. Intracellular cGMP detection was accomplished using a commercial radioimmunoassay method. Vascular endothelial cell from rat corpus cavernosum spiral artery was isolated and transfected with si- Corin to analyzed the potential role of Corin. Cell viability was assessed using crystal violet. The results showed that diabetic ED rats showed significantly higher glucose level, and lower body weight, ICP level, and ICP/MAP ratio at 12 and 16 weeks in diabetic ED rats compared with control rats. The protein levels of Corin, atrial natriuretic peptide (ANP) and eNOS, and the level of cGMP were significantly down-regulated in corpus cavernosum in diabetic ED rats, revealing the potential role of Corin in NO-associated diabetic ED. Further, studies proved that defect of Corin not only inhibited the vascular endothelial cell viability in high-glucose condition, but also suppressed ANP, eNOS, and cGMP expression in vascular endothelial cells. To sum up, Corin contributes to the progression of diabetic ED and the underlying mechanism is associated with the down-regulation of ANP /NO/cGMP signal pathway. J. Cell. Biochem. 118: 2325-2332, 2017. © 2017 Wiley Periodicals, Inc.

Distinct roles of N-glycosylation at different sites of corin in cell membrane targeting and ectodomain shedding.

Corin is a membrane-bound protease essential for activating natriuretic peptides and regulating blood pressure. Human corin has 19 predicted N-glycosylation sites in its extracellular domains. It has been shown that N-glycans are required for corin cell surface expression and zymogen activation. It remains unknown, however, how N-glycans at different sites may regulate corin biosynthesis and processing. In this study, we examined corin mutants, in which each of the 19 predicted N-glycosylation sites was mutated individually. By Western analysis of corin proteins in cell lysate and conditioned medium from transfected HEK293 cells and HL-1 cardiomyocytes, we found that N-glycosylation at Asn-80 inhibited corin shedding in the juxtamembrane domain. Similarly, N-glycosylation at Asn-231 protected corin from autocleavage in the frizzled-1 domain. Moreover, N-glycosylation at Asn-697 in the scavenger receptor domain and at Asn-1022 in the protease domain is important for corin cell surface targeting and zymogen activation. We also found that the location of the N-glycosylation site in the protease domain was not critical. N-Glycosylation at Asn-1022 may be switched to different sites to promote corin zymogen activation. Together, our results show that N-glycans at different sites may play distinct roles in regulating the cell membrane targeting, zymogen activation, and ectodomain shedding of corin.