A Galectin-9-Driven CD11chigh Decidual Macrophage Subset Suppresses Uterine Vascular Remodeling in Preeclampsia.

BACKGROUND: Preeclampsia is a serious disease of pregnancy that lacks early diagnosis methods or effective treatment, except delivery. Dysregulated uterine immune cells and spiral arteries are implicated in preeclampsia, but the mechanistic link remains unclear. METHODS: Single-cell RNA sequencing and spatial transcriptomics were used to identify immune cell subsets associated with preeclampsia. Cell-based studies and animal models including conditional knockout mice and a new preeclampsia mouse model induced by recombinant mouse galectin-9 were applied to validate the pathogenic role of a CD11chigh subpopulation of decidual macrophages (dMφ) and to determine its underlying regulatory mechanisms in preeclampsia. A retrospective preeclampsia cohort study was performed to determine the value of circulating galectin-9 in predicting preeclampsia. RESULTS: We discovered a distinct CD11chigh dMφ subset that inhibits spiral artery remodeling in preeclampsia. The proinflammatory CD11chigh dMφ exhibits perivascular enrichment in the decidua from patients with preeclampsia. We also showed that trophoblast-derived galectin-9 activates CD11chigh dMφ by means of CD44 binding to suppress spiral artery remodeling. In 3 independent preeclampsia mouse models, placental and plasma galectin-9 levels were elevated. Galectin-9 administration in mice induces preeclampsia-like phenotypes with increased CD11chigh dMφ and defective spiral arteries, whereas galectin-9 blockade or macrophage-specific CD44 deletion prevents such phenotypes. In pregnant women, increased circulating galectin-9 levels in the first trimester and at 16 to 20 gestational weeks can predict subsequent preeclampsia onset. CONCLUSIONS: These findings highlight a key role of a distinct perivascular inflammatory CD11chigh dMφ subpopulation in the pathogenesis of preeclampsia. CD11chigh dMφ activated by increased galectin-9 from trophoblasts suppresses uterine spiral artery remodeling, contributing to preeclampsia. Increased circulating galectin-9 may be a biomarker for preeclampsia prediction and intervention.

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.

The protease corin regulates electrolyte homeostasis in eccrine sweat glands.

Sweating is a basic skin function in body temperature control. In sweat glands, salt excretion and reabsorption are regulated to avoid electrolyte imbalance. To date, the mechanism underlying such regulation is not fully understood. Corin is a transmembrane protease that activates atrial natriuretic peptide (ANP), a cardiac hormone essential for normal blood volume and pressure. Here, we report an unexpected role of corin in sweat glands to promote sweat and salt excretion in regulating electrolyte homeostasis. In human and mouse eccrine sweat glands, corin and ANP are expressed in the luminal epithelial cells. In corin-deficient mice on normal- and high-salt diets, sweat and salt excretion is reduced. This phenotype is associated with enhanced epithelial sodium channel (ENaC) activity that mediates Na+ and water reabsorption. Treatment of amiloride, an ENaC inhibitor, normalizes sweat and salt excretion in corin-deficient mice. Moreover, treatment of aldosterone decreases sweat and salt excretion in wild-type (WT), but not corin-deficient, mice. These results reveal an important regulatory function of corin in eccrine sweat glands to promote sweat and salt excretion.

Transmembrane serine protease TMPRSS2 implicated in SARS-CoV-2 infection is autoactivated intracellularly and requires N-glycosylation for regulation.

Transmembrane protease serine 2 (TMPRSS2) is a membrane-bound protease expressed in many human epithelial tissues, including the airway and lung. TMPRSS2-mediated cleavage of viral spike protein is a key mechanism in severe acute respiratory syndrome coronavirus 2 activation and host cell entry. To date, the cellular mechanisms that regulate TMPRSS2 activity and cell surface expression are not fully characterized. In this study, we examined two major post-translational events, zymogen activation and N-glycosylation, in human TMPRSS2. In experiments with human embryonic kidney 293, bronchial epithelial 16HBE, and lung alveolar epithelial A549 cells, we found that TMPRSS2 was activated via intracellular autocatalysis and that this process was blocked in the presence of hepatocyte growth factor activator inhibitors 1 and 2. By glycosidase digestion and site-directed mutagenesis, we showed that human TMPRSS2 was N-glycosylated. N-glycosylation at an evolutionarily conserved site in the scavenger receptor cysteine-rich domain was required for calnexin-assisted protein folding in the endoplasmic reticulum and subsequent intracellular trafficking, zymogen activation, and cell surface expression. Moreover, we showed that TMPRSS2 cleaved severe acute respiratory syndrome coronavirus 2 spike protein intracellularly in human embryonic kidney 293 cells. These results provide new insights into the cellular mechanism in regulating TMPRSS2 biosynthesis and function. Our findings should help to understand the role of TMPRSS2 in major respiratory viral diseases.

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.

Probing the functional consequence and clinical relevance of CD320 p.E88del, a variant in the transcobalamin receptor gene.

The biological and clinical significance of the p.E88del variant in the transcobalamin receptor, CD320, is unknown. This allele is annotated in ClinVar as likely benign, pathogenic, and of uncertain significance. To determine functional consequence and clinical relevance of this allele, we employed cell culture and genetic association studies. Fibroblasts from 16 CD320 p.E88del homozygotes exhibited reduced binding and uptake of cobalamin. Complete ascertainment of newborns with transiently elevated C3 (propionylcarnitine) in New York State demonstrated that homozygosity for CD320 p.E88del was over-represented (7/348, p < 6 × 10-5 ). Using population data, we estimate that ~85% of the p.E88del homozygotes born in the same period did not have elevated C3, suggesting that cobalamin metabolism in the majority of these infants with this genotype is unaffected. Clinical follow-up of 4/9 homozygous individuals uncovered neuropsychological findings, mostly in speech and language development. None of these nine individuals exhibited perturbation of cobalamin metabolism beyond the newborn stage even during periods of acute illness. Newborns homozygous for this allele in the absence of other factors are at low risk of requiring clinical intervention, although more studies are required to clarify the natural history of various CD320 variants across patient populations.

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.

Intracellular autoactivation of TMPRSS11A, an airway epithelial transmembrane serine protease.

Type II transmembrane serine proteases (TTSPs) are a group of enzymes participating in diverse biological processes. Some members of the TTSP family are implicated in viral infection. TMPRSS11A is a TTSP expressed on the surface of airway epithelial cells, which has been shown to cleave and activate spike proteins of the severe acute respiratory syndrome (SARS) and the Middle East respiratory syndrome coronaviruses (CoVs). In this study, we examined the mechanism underlying the activation cleavage of TMPRSS11A that converts the one-chain zymogen to a two-chain enzyme. By expression in human embryonic kidney 293, esophageal EC9706, and lung epithelial A549 and 16HBE cells, Western blotting, and site-directed mutagenesis, we found that the activation cleavage of human TMPRSS11A was mediated by autocatalysis. Moreover, we found that TMPRSS11A activation cleavage occurred before the protein reached the cell surface, as indicated by studies with trypsin digestion to remove cell surface proteins, treatment with cell organelle-disturbing agents to block intracellular protein trafficking, and analysis of a soluble form of TMPRSS11A without the transmembrane domain. We also showed that TMPRSS11A was able to cleave the SARS-CoV-2 spike protein. These results reveal an intracellular autocleavage mechanism in TMPRSS11A zymogen activation, which differs from the extracellular zymogen activation reported in other TTSPs. These findings provide new insights into the diverse mechanisms in regulating TTSP activation.

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.

Autoactivation and calpain-1-mediated shedding of hepsin in human hepatoma cells.

Hepsin is a transmembrane serine protease implicated in many biological processes, including hepatocyte growth, urinary protein secretion, auditory nerve development, and cancer metastasis. Zymogen activation is critical for hepsin function. To date, how hepsin is activated and regulated in cells remains an enigma. In this study, we conducted site-directed mutagenesis, cell expression, plasma membrane protein labeling, trypsin digestion, Western blotting, and flow cytometry experiments in human hepatoma HepG2 cells, where hepsin was originally discovered, and SMMC-7721 cells. Our results show that hepsin is activated by autocatalysis on the cell surface but not intracellularly. Moreover, we show that hepsin undergoes ectodomain shedding. In the conditioned medium from HepG2 and SMMC-7721 cells, we detected a soluble fragment comprising nearly the entire extracellular region of hepsin. By testing protease inhibitors, gene knockdown, and site-directed mutagenesis, we identified calpain-1 as a primary protease that acted extracellularly to cleave Tyr52 in the juxtamembrane space of hepsin. These results provide new insights into the biochemical and cellular mechanisms that regulate hepsin expression and activity.

High-Mobility Group Box 1 From Hypoxic Trophoblasts Promotes Endothelial Microparticle Production and Thrombophilia in Preeclampsia.

OBJECTIVE: Thrombophilia is a major complication in preeclampsia, a disease associated with placental hypoxia and trophoblast inflammation. Preeclampsia women are known to have increased circulating microparticles that are procoagulant, but the underlying mechanisms remain unclear. In this study, we sought to understand the mechanism connecting placental hypoxia, circulating microparticles, and thrombophilia. APPROACH AND RESULTS: We analyzed protein markers on plasma microparticles from preeclampsia women and found that the increased circulating microparticles were mostly from endothelial cells. In proteomic studies, we identified HMGB1 (high-mobility group box 1), a proinflammatory protein, as a key factor from hypoxic trophoblasts in stimulating microparticle production in human umbilical vein endothelial cells. Immunodepletion or inhibition of HMGB1 in the conditioned medium from hypoxic human trophoblasts abolished the endothelial microparticle-stimulating activity. Conversely, recombinant HMGB1 stimulated microparticle production in cultured human umbilical vein endothelial cells. The microparticles from recombinant HMGB1-stimulated human umbilical vein endothelial cells promoted blood coagulation and neutrophil activation in vitro. Injection of recombinant HMGB1 in pregnant mice increased plasma endothelial microparticles and promoted blood coagulation. In preeclampsia women, elevated placental HMGB1 expression was detected and high levels of plasma HMGB1 correlated with increased plasma endothelial microparticles. CONCLUSIONS: Our results indicate that placental hypoxia-induced HMGB1 expression and release from trophoblasts are important mechanism underlying increased circulating endothelial microparticles and thrombophilia in preeclampsia.

Role of corin in trophoblast invasion and uterine spiral artery remodelling in pregnancy.

In pregnancy, trophoblast invasion and uterine spiral artery remodelling are important for lowering maternal vascular resistance and increasing uteroplacental blood flow. Impaired spiral artery remodelling has been implicated in pre-eclampsia, a major complication of pregnancy, for a long time but the underlying mechanisms remain unclear. Corin (also known as atrial natriuretic peptide-converting enzyme) is a cardiac protease that activates atrial natriuretic peptide (ANP), a cardiac hormone that is important in regulating blood pressure. Unexpectedly, corin expression was detected in the pregnant uterus. Here we identify a new function of corin and ANP in promoting trophoblast invasion and spiral artery remodelling. We show that pregnant corin- or ANP-deficient mice developed high blood pressure and proteinuria, characteristics of pre-eclampsia. In these mice, trophoblast invasion and uterine spiral artery remodelling were markedly impaired. Consistent with this, the ANP potently stimulated human trophoblasts in invading Matrigels. In patients with pre-eclampsia, uterine Corin messenger RNA and protein levels were significantly lower than that in normal pregnancies. Moreover, we have identified Corin gene mutations in pre-eclamptic patients, which decreased corin activity in processing pro-ANP. These results indicate that corin and ANP are essential for physiological changes at the maternal-fetal interface, suggesting that defects in corin and ANP function may contribute to pre-eclampsia.

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.

Transplantation of human villous trophoblasts preserves cardiac function in mice with acute myocardial infarction.

Over the past decade, cell therapies have provided promising strategies for the treatment of ischaemic cardiomyopathy. Particularly, the beneficial effects of stem cells, including bone marrow stem cells (BMSCs), endothelial progenitor cells (EPCs), mesenchymal stem cells (MSCs), embryonic stem cells (ESCs), and induced pluripotent stem cells (iPSCs), have been demonstrated by substantial preclinical and clinical studies. Nevertheless stem cell therapy is not always safe and effective. Hence, there is an urgent need for alternative sources of cells to promote cardiac regeneration. Human villous trophoblasts (HVTs) play key roles in embryonic implantation and placentation. In this study, we show that HVTs can promote tube formation of human umbilical vein endothelial cells (HUVECs) on Matrigel and enhance the resistance of neonatal rat cardiomyocytes (NRCMs) to oxidative stress in vitro. Delivery of HVTs to ischaemic area of heart preserved cardiac function and reduced fibrosis in a mouse model of acute myocardial infarction (AMI). Histological analysis revealed that transplantation of HVTs promoted angiogenesis in AMI mouse hearts. In addition, our data indicate that HVTs exert their therapeutic benefit through paracrine mechanisms. Meanwhile, injection of HVTs to mouse hearts did not elicit severe immune response. Taken together, our study demonstrates HVT may be used as a source for cell therapy or a tool to study cell-derived soluble factors for AMI treatment.

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.

Localization of corin and atrial natriuretic peptide expression in human renal segments.

Atrial natriuretic peptide (ANP)-mediated natriuretic response is a well-established cardiac endocrine function. Corin is a transmembrane protease that activates ANP in the heart. Corin expression has been detected in non-cardiac tissues including the kidney. Here we examined corin, pro-ANP/ANP and natriuretic peptide receptor-A (NPR-A) expression in human renal segments. By immunostaining and in situ hybridization, we found similar corin, pro-ANP/ANP and NPR-A protein and mRNA expression in human renal segments. The expression was most abundant in the proximal convoluted tubules and the medullary connecting ducts. In the proximal tubules, corin protein was present in the apical membrane region underneath the brush border where the ANP-degrading protease neprilysin was abundant. These results suggest that corin-mediated pro-ANP activation may occur in renal segments and that locally produced ANP may act in an autocrine manner to regulate sodium and water reabsorption in situ Our results also point to the proximal convoluted tubules as a major site for local ANP action. Such a renal corin/ANP autocrine mechanism may differ from the cardiac corin/ANP endocrine mechanism in regulating sodium homoeostasis under physiological and pathological conditions.

N-glycosylation is required for matriptase-2 autoactivation and ectodomain shedding.

Matriptase-2 is a hepatic membrane serine protease that regulates iron homeostasis. Defects in matriptase-2 cause iron deficiency anemia. In cells, matriptase-2 is synthesized as a zymogen. To date, how matriptase-2 expression and activation are regulated remains poorly understood. Here we expressed human matriptase-2 in HEK293 and hepatic BEL-7402, SMMC-7721, and QGY-7703 cells. By labeling cell surface proteins and Western analysis, we examined matriptase-2 cell surface expression, zymogen activation, and ectodomain shedding. Our results show that matriptase-2 was activated on the cell surface but not intracellularly. Activated matriptase-2 underwent ectodomain shedding, producing soluble fragments in the conditioned medium. By testing inactive mutants, R576A and S762A, we found that matriptase-2 activation and shedding were mediated by its own catalytic activity and that the one-chain form of matriptase-2 had little activity in ectodomain shedding. We made additional matriptase-2 mutants, N136Q, N184Q, N216Q, N338Q, N433Q, N453Q, and N518Q, in which each of the predicted N-glycosylation sites was mutated. All of these mutants were expressed on the cell surface. However, mutants N216Q, N453Q, and N518Q, but not the other mutants, had impaired zymogen activation and ectodomain shedding. Our results indicate that N-glycans at specific sites are critical for matriptase-2 activation. Together, these data provide new insights into the cell surface expression, zymogen activation, and ectodomain shedding of matriptase-2.

Corin mutations K317E and S472G from preeclamptic patients alter zymogen activation and cell surface targeting. [Corrected].

Corin is a membrane-bound serine protease that acts as the atrial natriuretic peptide (ANP) convertase in the heart. Recent studies show that corin also activates ANP in the pregnant uterus to promote spiral artery remodeling and prevent pregnancy-induced hypertension. Two CORIN gene mutations, K317E and S472G, were identified in preeclamptic patients and shown to have reduced activity in vitro. In this study, we carried out molecular modeling and biochemical experiments to understand how these mutations impair corin function. By molecular modeling, the mutation K317E was predicted to alter corin LDL receptor-2 module conformation. Western blot analysis of K317E mutant in HEK293 cells showed that the mutation did not block corin expression on the cell surface but inhibited corin zymogen activation. In contrast, the mutation S472G was predicted to abolish a ß-sheet critical for corin frizzled-2 module structure. In Western blot analysis and flow cytometry, S472G mutant was not detected on the cell surface in transfected HEK293 cells. By immunostaining, the S472G mutant was found in the ER, indicating that the mutation S472G disrupted the ß-sheet, causing corin misfolding and ER retention. Thus, these results show that mutations in the CORIN gene may impair corin function by entirely different mechanisms. Together, our data provide important insights into the molecular basis underlying corin mutations that may contribute to preeclampsia in patients.

A novel cytoplasmic tail motif regulates mouse corin expression on the cell surface.

Type II transmembrane serine proteases (TTSPs) are important in many biological processes. Cell surface expression is critical for TTSP activation and function. To date, the mechanism underlying TTSP cell surface expression is poorly understood. Corin is a TTSP and acts as the pro-atrial natriuretic peptide convertase that is essential for sodium homeostasis and normal blood pressure. In this study, we investigated how cytoplasmic tail sequences may regulate corin expression and activation on the cell surface. By site-directed mutagenesis, we made mouse corin proteins with truncations or point-mutations in the cytoplasmic tail. We expressed the mutants in transfected HEK293 cells and analyzed corin cell surface expression and activation by Western blotting and flow cytometry. We found that corin truncation mutants lacking a Lys-Phe-Gln sequence at residues 71-73 had higher levels of cell surface expression and activation compared with that in wild-type corin. When Lys-71, Phe-72 and Gln-73 residues were mutated together, but not individually, in corin with the full-length cytoplasmic tail, increased levels of cell surface expression and zymogen activation were also observed. These results indicate that residues Lys-71, Phe-72 and Gln-73 serve as a novel retention motif in the intracellular pathway to regulate corin cell surface expression and activation.