Identification And Functional Studies Of Corin Variants In Hypertensive Patients

Chapter One.Identification and Functional Analysis of Corin Variants in Hypertensive PatientsObjective:Genetic factors altering sodium homeostasis play a major role in the pathogenesis of hypertension.Atrial natriuretic peptide(ANP)is a cardiac hormone that plays an important role in maintaining sodium homeostasis.Corin is a serine protease critical for ANP generation.Corin converts the inactive ANP precursor,pro-ANP,to active ANP.This corin function is essential for salt-water balance and normal blood pressure.To date,corin variants and mutations have been reported in patients with hypertension and heart disease.In African Americans,for example,corin variant T555I/Q568P with reduced pro-ANP processing activity has been associated with hypertension and cardiac hypertrophy.More recently,low levels of plasma or serum corin have been reported in patients with coronary artery disease,myocardial infarction,heart failure and stroke,suggesting that corin deficiency may be a contributing factor in major cardiovascular diseases.The goal of this study is to understand the prevalence and functional significance of CORIN variants in hypertension.Methods:1.Venous blood was obtained from a cohort of 300 normal and 401 hypertensive individuals.Plasma corin,N-terminal(NT)-pro-ANP and NT-pro-B-type natriuretic peptide(NT-pro-BNP)levels were measured by ELISA.2.Genomic DNA was isolated from white blood cells and used in PCR to amplify CORIN exons and intron-exons boundaries.PCR fragments were sequenced.3.Plasmids expressing human wild-type(WT)and proprotein convertase subtilisin/kexin-6(PCSK6)were made in previous studies.Plasmids expressing corin variants were made by site-directed mutagenesis using PCR-based methods.4.Plasmids were transfected into human embryonic kidney 293(HEK293)cells.Conditioned medium containing human pro-ANP from stable cells was added to the cell culture at 37?.Pro-ANP,ANP and corin protein in the conditioned medium and cell lysates were analyzed by immunoprecipitation and Western blotting.5.Flow cytometry was performed to examine WT and variants corin expression on the HEK293 cell surface.6.Plasmids were transfected into HEK293 cells,and corin protein fragments in the conditioned medium and cell lysates were analyzed by ELISA,immunoprecipitation,Western blotting.7.The subcellular distribution of the corin variants in the transfected cells was examined by immunofluorescent staining and confocal microscopy.8.Molecular modeling analysis was performed to understand the impact of R530S and T924M changes on corin structure.Results:1 We sequenced CORIN exons in genomic DNAs from 301 patients and identified nine non-synonymous variants,of which eight were not characterized previously.The insA variant we identified previously was confirmed in hypertensive individuals.2.Y13C,D95Y,L334W and R525H variants occurred similarly in both normal and hypertensive groups.R349H,P866S and T924M variants were found once each in hypertensive individuals.The R530S variant occurred preferentially in hypertensive individuals,which was confirmed in another independent cohort of 377 normal and 368 hypertensive individuals.3.In biochemical and cell-based functional studies,variants R530S and T924M,but not Y13C,D95Y,L334W,R349H,R525H and P866S,exhibited reduced pro-ANP processing activity.4.Compared with that in WT corin,levels of the-40-kDa band(activation band,corin-p)were reduced in variants R530S and T924M(21.5±1.1 and 18.5 ± 0.9%,respectively;n=9,both p values<0.001 vs.39.9±1.7%in WT).These results indicate that variants R530S and T924M had impaired zymogen activation.5.In flow cytometric analysis,the surface corin-positive cells were less in R530S-expressing cells(20.4 ± 1.9%,p<0.001 vs.41.8 ± 2.3%in WT),whereas the number of surface corin-positive cells in T924M-expressing cells was similar to that in WT(37.8 ±2.9%,p=0.53 vs.WT).6.In ELISA analysis,corin levels were lower in the conditioned media from the cells expressing variants R530S and T924M.Three fragments of 180-,160-,and 100-kDa,respectively,were detected in WT corin-expressing cells.In the cells expressing R530S variant,levels of all these three fragments were reduced.In the cells expressing T924M variant,levels of the 160-and 100-kDa,but not 180-kDa,fragments were reduced.7.By immunostaining and confocal microscopy,we found that in the transfected cells,R530S variant protein was mostly in the ER.In contrast,T924M variant protein was mostly on the cell membrane.8.Molecular modeling indicated that R530S substitution likely destabilizes Fz2 domain structure,leading to protein misfolding,whereas T924M substitution alters the structure of a surface loop adjacent to the R801 activation site,which may hinder corin zymogen activation.9.PCSK6 overexpression enhanced the activation cleavage in WT corin and the T924M variant,but not the variant R530S.10.In the cells co-expressing WT and the R530S variant,the level of the corin-p fragment was lower than that in WT alone(32.4 ± 1.9 vs.42.1 ±1.3%in WT;n=5,P<0.01),indicating a dominant-negative effect of the R530S variant on WT corin activation cleavage in these cells.11.Levels of plasma corin in 11 individuals with the R530S allele were lower than that in the normal group(0.59 ± 0.05 vs.0.86 ± 0.03 ng/mL,p=0.002).Plasma NT-pro-ANP levels in 10 randomly selected normal individuals with the WT allele was 0.85 ± 0.16 nmol/L,whereas the level in 10 hypertensive individuals with the R530S allele was higher at 2.06 ± 0.27 nmol/L(p=0.001).In contrast,plasma NT-pro-BNP levels in 6 normal individuals with the WT allele and 10 hypertensive individuals with the R530S allele were similar(27.67 ± 9.59 and 34.1 ±9.11 pg/mL,p=0.652).Conclusions:In this study,we sequenced CORIN exons and identified nine non-synonymous variants,of which eight were not characterized previously.Among them,variants Y13C,D95Y,L334W and R525H occurred similarly in both hypertensive and normal individuals.Variants R349H,P866S and T924M were found once each in hypertensive individuals.Variant R530S occurred preferentially in hypertensive.In biochemical and cell-based functional studies,variants R530S and T924M,but not Y13C,D95Y,L334W,R349H,R525H and P866S,exhibited reduced pro-ANP processing activity,which was caused by endoplasmic reticulum retention and poor zymogen activation,respectively.Together,our results indicate that genetic variants that impair corin function are not uncommon in general populations and that such variants may be an important contributing factor in hypertension.Studies of the naturally occurring variants have also provided important insights into the key steps in the regulation of corin biosynthesis and activation.Chapter Two:Intracellular Retention Signals in Corin Cytoplasmic Tail SequencesObjective:The transmembrane protease corin is synthesized as an inactive zymogen,which travels through the endoplasmic reticulum,the Golgi apparatus and finally reaches the cell membrane where it is activated by another protease,PCSK6.The intracellular trafficking and cell surface targeting are critical for corin zymogen activation and hence biological function.Previously,we identified a human CORIN variant,insA,that was associated with hypertensive patients.This variant shortened corin cytoplasmic tail and caused corin protein retention in the Golgi,thereby impairing corin function.The results are consistent with findings from other previous studies,indicating that corin cytoplasmic tail sequences are important in promoting corin intracellular trafficking and cell membrane expression.The purpose of this study is to analyze and identify specific amino acids in the corin cytoplasmic tail sequence that may regulate corin intracellular trafficking.Methods:1.In our previous studies,we found that human corin variant insA and mouse corin mutant mD99 had different cell membrane targeting efficiencies and that such a difference was probably due to different cytoplasmic tail sequences.We aligned cytoplasmic amino acid sequences of variants insA and mD99 corin:insA:M-G-N-G-C-S-Q-K-L-A-T-A-N-L-L-R-mD99:M-G-C-P-Q-K-L-V-T-A-N-L-L-R-There is an extra GN motif(in red)in the amino terminal end(N-terminal)of insA corin compared with that in mD99 corin.The amino acids S-6 and A-10(in blue)in the cytoplasmic tail of insA also differ from the corresponding residues P-4 and V-8 in mD99.2.Plasmids expressing variants insAAGN(in which the GN motif in the insA cytoplasmic tail was deleted),mD99GN(in which GN motif was added to the mD99 cytoplasmic tail N-terminus),mD99-SA(in which the residues P-4 and V-8 in the mD99 cytoplasmic tail were replaced with S-4 and A-8,respectively)were constructed using plasmids expressing insA and mD99 as templates.3.The plasmids expressing WT corin and corin variants were transfected into HEK293 cells.Corin expression and activation in the transfected cells were analyzed by Western blotting.4.Flow cytometry was used to examine corin expression on the cell surface.5.To examine protein half-lives,HEK293 cells expressing corin WT and variants were treated with cycloheximide.After 4,6,8,10,12 hours(h),the cells were collected and lysed.Corin proteins in the cells were analyzed by Western blotting.6.To examine the intracellular distribution of corin proteins,immunofluorescent staining was performed in the transfected HEK293 cells expressing corin WT and variants.The stained cells were analyzed using a confocal microscope.7.Endoglycosidase H(Endo H)digestion experiment was used to distinguish corin proteins in the ER and the early Golgi(Endo H sensitive)or the late Golgi(Endo H resistant).WT and variant corin proteins from the transfected cells were treated with Endo H followed by Western blotting analysis.The migration pattern of the corin proteins on the Western blots was analyzed to understand the subcellular localization of the corin proteins.Results:1.In Western blotting analysis,levels of the activated corin protease domain band(corin-p)in variant insA was lower than that of human WT corin(hWT)(27.7 ±1.7 vs.47.7 ± 1.7%,p<0.01,n=6).The variant insA?GN lacking the GN motif had a higher level of corin-p(38.6 ±3.3%)than that in insA but similar to that in hWT.In the variant mD99(no GN motif),the corin-p level was higher than that of mouse WT corin(mWT)(59.8±2.9 vs.42.8 ± 2.5%,p<0.01,n=6).In the variant mD99GN(adding the GN motif to mD99),the corin-p level was lower(44.0 ± 4.1%)than that in mD99,These results indicate that the GN motif in the corin cytoplasmic tail may act as an intracellular retention signal,preventing corin targeting to the cell membrane where corin zymogen activation occurs.2.Amino acids P-4 and V-8 in the cytoplasmic tail of mD99 were replaced by S-4 and A-8(mD99-SA),respectively.In the transfected cells,the corin-p level of the mD99-SA variant was similar to that in the variant mD99,indicating that these two residues are not critical for corin intracellular trafficking,cell surface targeting and zymogen activation.3.In flow cytometric analysis,HEK293 cells expressing hWT and variants insA,insA?GN,had 67.4 ± 2.9,43.3 ± 4.9 and 63.7±4.3%of cells,respectively,that were corin-positive on the surface.In similar studies,HEK293 cells expressing mWT and variants mD99,mD99GN had 44.6±9.9,67.1±11.0 and 48.6 ± 12.4%of cells,respectively,that were corin-positive on the surface.These results support the idea that the GN motif reduces corin intracellular trafficking,thereby decreasing corin expression on the cell surface.4.In cycloheximide-based experiments,human and mouse corin variants with the GN motif stayed longer in the transfected HEK293 cells,as indicated by longer half-lives.These results are also consistent with idea that the GN motif in the corin cytoplasmic tail acts as an intracellular retention signal.5.By immunofluorescent staining,we found that levels of human and mouse corin variants with the GN motif were increased in the Golgi,compared with that of corin variants without the GN motif.The results indicate that the GN motif acts as a Golgi retention signal.6.In Endo H digestion and Western blotting experiments,intracellular corin bands in the variants with the GN motif were Endo H-sensitive,indicating that the GN motif increases corin protein retention in the early Golgi.Conclusions:In this study,we identified a novel GN motif in the corin cytoplasmic tail that acts as an early Golgi retention signal.The GN motif slows corin trafficking in the early Golgi,thereby reducing corin expression on the cell surface and subsequently corin zymogen activation.Our results provide new insights into the mechanisms that regulate corin biosynthesis,post-translational modifications,and biological function.Our findings also help to understand how naturally occurring variants may impair corin function and contribute to hypertension and heart disease.