| Chapter One:Role of Cytoplasmic Amino Acid Sequences in Corin Cell Surface TargetingObjective:Corin is a type II transmembrane serine protease that is highly expressed in the heart.Corin regulates blood pressure by converting pro-atrial natriuretic peptide(pro-ANP)to mature atrial natriuretic peptide(ANP).ANP circulates in the blood.In distal organs,ANP promotes renal sodium and water excretion and relaxes peripheral resistance vessels,thereby lowering blood volume and pressure.In cardiomyocytes,corin is synthesized as an inactive single-chain proenzyme,i.e.zymogen,which undergoes posttranslational modifications in the endoplasmic reticulum(ER)and the Golgi apparatus before reaching the cell surface,where the zymogen corin is converted to an active enzyme by proteolytic cleavage.Corin anchors on the cell surface via its N-terminal transmembrane domain.The cell membrane anchoring is critical for corin activity and biological function.The goal of this study is to understand the molecular basis that regulates corin intracellular trafficking and cell surface targeting.In particular,we examined the role of specific amino acids in the cytoplasmic tail and near the transmembrane domain in regulating corin cell surface targeting.Methods:Plasmids expressing corin mutants were constructed by PCR-based site-directed mutagenesis using wild-type(WT)mouse corin plasmid as a template.HEK293 cells were transfected with these plasmids.Western blotting was performed to analyze corin expression level and zymogen activation in the transfected cells.Flow cytometric analysis and immunofluorescent staining were used to examine corin expression on the cell surface.Results:1.We transfected HEK293 cells with mouse WT corin(mWT)plasmid and,after 24 h,treated the cells with trypsin.In Western blotting,the level of a-40-kDa band(corin-p,representing the activated protease domain fragment)decreased after trypsin digestion.We also examined mWT corin expression on the cell surface by flow cytometry.Our data showed that mWT corin-positive cells were reduced after trypsin digestion(14.91± 0.74 vs.48.84± 2.16%,p<0.0001,n=5).These data indicate that corin is expressed and activated on the surface of the transfected cells.2.Mouse corin contains 112 amino acids in the N-terminal cytoplasmic tail.To identify the minimal length in the cytoplasmic tail that is required for mouse corin expression on the cell surface,we constructed a truncation mutant,mD106,deleting 106 amino acids from the N-terminus.Western analysis showed similar corin expression and activation levels for mD106 and mWT in transfected cells,suggesting that the remaining six amino acids in the cytoplasmic tail were sufficient for corin cell surface targeting.3.We next constructed corin truncation mutants mD111 and mD112,deleting 111 and 112 amino acids from the N-terminus,respectively.Flow cytometric analysis of corin cell surface expression showed that the level of the mD111 mutant was similar to that in mWT(56.69 ± 4.27 vs.53.37 ± 1.61%,p=0.24,n=6).However,we detected little mD112 mutant expression on the cell surface(1.69 ± 0,13 vs.53.37 ± 1.61%in mWT,p<0.0001,n=6).Western analysis showed that mD111 and mWT corin expression and activation levels were similar(26.89 ± 2.35 vs.23.64 ? 3.28%in corin-p levels,p=0.44,n=5)in these cells.In contrast,the corin-p band in mutant mD112 was undetectable.These results indicate that a single arginine residue at the position 112 in the cytoplasmic tail is sufficient for corin cell surface expression and zymogen activation.4.We further examined the possibility that an arginine residue N-terminal to a hydrophobic sequence may serve as a membrane anchor signal.We made pro-ANP mutants that contained an extra arginine residue or the corin cytoplasmic tail before the pro-ANP signal peptide.In transfected HEK293 cells,the pro-ANP mutants did not express on the cell surface,indicating that an arginine N-terminal to a hydrophobic sequence is not a membrane anchor signal and unable to convert a secreted protein into a transmembrane protein.Conclusions:The cytoplasmic tail is critical for corin expression and activation on the cell surface.Within the mouse corin cytoplasmic tail,an initiating methionine and a single arginine at corresponding 112 position were sufficient for corin cell surface expression and zymogen activation.Chapter Two:Role of Positively-Charged Cytoplasmic Residues in Corin Transmembrane OrientationObjective:In Chapter One,we found that arginine at 112 position in the mouse corin cytoplasmic tail was critical for corin cell surface expression and activation.We also excluded the possibility that an arginine residue at this position alone could function as a membrane anchor signal.These results raised the question regarding the role of the cytoplasmic arginine residue in corin intracellular trafficking and cell surface targeting.In type ? transmembrane proteins,positively-charged residues are mostly found in the cytoplasmic side of the membrane.Based on the "positive-inside rule",which was proposed in the 1980s,the total number of positively-charged residues on the each side of the transmembrane domain determine the protein' s membrane orientation and the side with most positively-charged residues should be cytoplasmic.Corin is a type II transmembrane protein with an N-terminal membrane-proximal region containing positively-charged residues,which is consistent with the long-established "positive-inside rule".The goal of this part of study is to examine if the corin transmembrane orientation is determined by positively-charged residues in the cytoplasmic membrane-proximal region.Methods:1.We made plasmids expressing D-R112A,D-R112D,D-R112K,R112A,R112D and R112K mutants to test whether arginine-112 or other positively-charged amino acids at this position in the membrane-proximal region are critical for corin cell membrane targeting.2.HEK293 cells were transfected with the plasmids.Western blotting was performed to analyze corin expression and zymogen activation in the transfected cells.Immunoprecipitation and Western analysis were also performed to analyze corin protein fragments in the conditioned medium.3.The mutant corin proteins were tested for their activities in pro-ANP processing by cell transfection,immunoprecipitation,SDS-PAGE and Western blotting.4.Flow cytometric analysis and biotin-labeling of cell surface proteins were used to examine mutant corin expression on the cell surface.5.PNGase F digestion experiment was used to test if mutant corin proteins were N-glycosylated in the transfected cells.Results:1.In the transfected cells,the mutant D-R112K was expressed on the cell surface and activated.In contrast,mutants D-R112A and D-R112D were not express on the cell surface and not activated.Similar results were found when these mutations were made in the context of the full-length corin.These results show that a positively-charged amino acid,either an arginine or a lysine,at the 112 position in the cytoplasmic membrane-proximal region is critical for corin cell surface expression and zymogen activation.2.In the pro-ANP processing assay,mWT,mD111 and D-R112K mutants were active,whereas the mutants mD112,D-R112A and D-R112D were inactive in processing pro-ANP.3.The extracellular region of mouse corin contains 17 N-glycosylation sites.N-glycosylation occurs in the ER lumen.We used PNGase F digestion to examine corin transmembrane domain orientation.If corin remains in the type ? protein orientation(N-terminus inside and C-terminus outside),corin is expected to be N-glycosylated and thus its protein mass should be reduced after PNGase digestion.If the membrane orientation is reversed(C-terminus inside and N-terminus outside),corin should not be N-glycosylated and the protein mass should remain similar before and after PNGase treatment.Western analysis showed that protein bands in WT corin and mutants D-R112A,D-R112D,R112A and R112D migrated faster after PNGase F digestion.indicating that all these corin mutants were N-glycosylated.These results indicate that substitution of the positively-charged arginine to a natural or negatively-charged reside at 112 position did not alter corin transmembrane orientation,which is inconsistent with the long-established "positive-inside rule".4.We next examined mWT and mutants corin proteins in the conditioned medium.We found that mD112 corin(lacking the cytoplasmic tail and the 112 arginine)was secreted into the conditioned medium.Our results indicate that deleting the cytoplasmic tailing converts corin from a transmembrane protein into a secreted protein.Conclusions:A positively-charged residue,either an arginine or a lysine,at the 112 position in the cytoplasmic tail is critical for corin cell surface expression.Substitutions of arginine-112 to a natural or negatively-charged residue did not prevent N-glycosylation and thus did not alter corin transmembrane orientation in corin.These results indicate that the long-established "positive-inside rule" in determining the membrane orientation in type II transmembrane proteins may need to be re-considered.The detection of mD112,D-112A and D-112D corin proteins in the conditioned suggested a potential role of the positively-charged residues in the cytoplasmic membrane-proximal region of corin in regulating protein secretion.Chapter Three:Effect of Arginine Residue in Membrane-Proximal Region on Signal Peptidase CleavageObjective:In Chapter Two,we found that the corin transmembrane orientation was not altered when the positively-changed arginine at the 112 position in the cytoplasmic membrane-proximal region was changed to a natural or negatively-charged residue.Instead?we detected mD112,D-R112A and D-R112D proteins in the conditioned medium,suggesting that these corin mutants may become secreted proteins.Secreted proteins are synthesized in the ribosome with a signal peptide sequence at the N-terminus.The signal peptide guides the newly-synthesized polypeptide into the ER,and then is cleaved by signal peptidase in the ER lumen.The remaining polypeptide undergoes protein folding and posttranslational modifications in the ER and the Golgi apparatus before secreted from the cell.The goal of this part of study is to determine if signal peptidase is involved in the secretion of the corin mutants and to understand the role of the positively-charged residues in the cytoplasmic membrane-proximal region in this process.Methods:We constructed CRISPR-Cas9-based targeting vectors and established HEK293 cells lines,in which the expression of signal peptidase complex(SPC)subunits 18 and 21 was abolished separately.The SPC 18 and SPC21 knockout(KO)cells were verified by PCR-based genotyping,DNA sequencing,and Western blotting.Parental HEK293 cells and SPC 18 and SPC21 KO cells were transfected with plasmids expressing mWT and corin mutants.Western blotting was performed to analyze corin expression and zymogen activation in the transfected cells.Conditioned medium was collected and analyzed for corin secretion.Flow cytometric analysis was done to examine corin protein expression on the surface of parental HEK293 and SPC 18 and SPC21 KO cells.Immunofluorescent staining was used to examine the intracellular distribution of WT and mutant corin proteins in the transfected cells.Results:1.Western analysis confirmed the lack of SPC18 and SPC21 protein expression in the KO cells,indicating that SPC18 and SPC21 genes were disrupted in those cells.2.mWT,mD111 and mD112 corin proteins were expressed in parental HEK293 cells and the SPC18 and SPC21 KO cells.Western analysis showed that mD112 protein was secreted in parental HEK293 and SPC21 KO cells,but not in SPC18 KO cells,indicating that SPC18 subunit in the signal peptidase complex is required for mD112 corin secretion.In flow cytometry,no mD112 corin protein expression was detected on the cell surface.By immunofluorescent staining and confocal microscopy.we found that mD112 corin was retained in the ER in SPC18 KO cells.Similar results were found in corin mutants D-R112A and D-R112D,but not D-R112K.These results indicate a positively-charged residue at the 112 position in the cytoplasmic membrane-proximal region prevents the cleavage by SPC18 subunit in the signal peptidase complex.In SPC18 KO cells.lack of a signal peptidase catalytic subunit prevented the cleavage of mD1 12,D-R1 1A and D-R112D proteins,causing the ER retention of these mutant corin proteins.Conclusions:Our results indicate that positively-charged residues in the cytoplasmic membrane-proximal region of corin serve as a protective mechanism,preventing corin from being cleaved by signal peptidase in the ER.Substitutions of the positively-charged residues to natural or negatively-charged residues disrupt such a protective mechanism,resulting in corin secretion after signal peptidase cleavage.In the signal peptidase complex,the catalytic subunit SPC18,but not SPC21,is required for signal peptidase-mediated corin cleavage.The ER retention of the mutant corin proteins in SPC KO cells indicate that additional proteins in the ER may be involved in the interactions between mutant corin proteins and the signal peptidase complex.In summary,corin is a transmembrane serine protease that acts on the cell surface to convert pro-ANP to active ANP.We show that a single arginine at the 112 position in the cytoplasmic tail was sufficient for corin expression on the cell surface and zymogen activation.Such a function of arginine-112 can be substituted by another positively-charged residue,such as lysine,but not a natural or negatively-charged residue at this position.We also show that the presence of the positively-charged residues in the cytoplasmic membrane-proximal region is not critical for corin transmembrane domain orientation,suggesting that the long-standing "positive-inside rule" in deciding the membrane topology of single-spanning transmembrane proteins may need to be reconsidered.Our results also indicate an unsuspected role of the positively-charged residues in the cytoplasmic membrane-proximal region in preventing corin being cleaved on the ER lumen by SPC18 catalytic subunit in the signal peptidase complex.Such a protective mechanism is important for corin trafficking in the ER before reaching the cell surface.As positively-charged residues are present in most transmembrane proteins,the novel"protective function" of the positively-charged residues in corin revealed in our studies may apply to many transmembrane proteins. |
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