Biological Studies Of Type Ⅱ Transmembrane Serine Proteases Corin And TMPRSS11A

Chapter 1:Molecular mechanism of corin apical specific expression in polarized renal epithelial cellsObjective:Type Ⅱ transmembrane serine proteases are a group of membrane-anchored proteases essential for many physiological functions.Atrial natriuretic peptide(ANP)converting enzyme corin is a type Ⅱ transmembrane serine protease that regulates blood pressure and salt-water balance.In addition to the heart,corin is expressed on the apical membrane of renal proximal tubular epithelial cells that are the main sites of renal reabsorption.Studies in animal models and patients indicate that renal corin expression is important in kidney function.To date,the molecular mechanism underlying apical corin expression in polarized renal epithelial cells remains unclear.In this chapter,we will conduct molecular and cellular studies to determine corin subcellular localization in renal proximal tubular epithelium and to identify key elements responsible for apical corin expression in polarized renal epithelial cells.Methods:1.Immune electron microscopy(EM)was used to examine the subcellular localization of corin protein in human renal proximal tubular epithelial cells.2.Transfection,immunofluorescent staining,and confocal microscopy were used to study corin expression in Madin-Darby Canine Kidney(MDCK)cells3.Plasmids expressing corin mutants and TMPRSS3/4 were constructed by site-directed mutagenesis using ClonExpress One Step Cloning kit4.Immunofluorescent staining was used to examine the cell membrane expression pattern of corin,TMPRSS3/4,enterokinase(EK),and hepsin in MDCK cells.5.SWISS-MODEL and PyMoL software were used to build homology models of LDL receptor(LDLR)modules from corin and other cell receptorsResults:1.In immune EM studies with human renal sections,corin was localized in the apical membrane region underneath the brush border of the proximal tubular epithelium,but not on basolateral membranes.2.In transfected MDCK cells,specific corin expression on the apical membrane was confirmed by immunofluorescent staining and confocal microscopy.3.Studies with series of domain deletion mutants indicated that corin LDLR8 domain is a key element for the specific apical membrane expression in MDCK cells.4.Experiments with TMPRSS 3/4 and EK chimeric proteins supported the importance corin LDLR8 domain in the apical membrane expression.5.Further site-directed mutagenesis studies identified a conserved DSSDE motif in corin LDLR8 domain as an import motif in the apical membrane expression.6.In computer simulation of protein module structure,the DSSDE motif of corin LDLR8 domain was located in a corner position with the first Ser(S)residue in the DSSDE motif being surface exposed,indicating the potential for protein-protein interaction.Conclusions:Our results show that corin is specifically expressed on the apical membrane in polarized renal epithelial cells.Our results also identify the DSSDE motif in LDLR8 domain is a key element in the specific apical membrane expression of corin.Chapter 2:Role of the DSSDE motif in CD320 apical specific expressionObjective:The results of the first chapter show that the DSSDE motif in corin LDLR8 domain is the key element for the specific expression of corin.Many cell surface receptors and proteolytic enzymes contain LDLR domains with a conserved DxSDE motif,where x can be any amino acid.CD320 is a transcobalamin receptor that is expressed in the kidney.CD320 contains two LDLR domains and the second LDLR domain(LDLR2)also has a DSSDE motif.This chapter,we will study the membrane expression of CD320 in renal epithelial cells and determine if the DSSDE motif in LDLR2 domain of CD320 plays a similar role in supporting apical specific expression.Methods:1.Immunofluorescent staining was performed to study the subcellular expression of CD320 and corin in proximal tubules in human kidney sections.2.Immunofluorescent staining was used to examine the cell membrane targeting of CD320 and corin in MDCK cells.3.SWISS-MODEL was used to build homology models to analyze CD320 LDLR2 modular structure.4.Site-directed mutagenesis and immunofluorescent staining were used to study the membrane expression pattern of human CD320 and its mutants in MDCK cells.Results:1.Immunofluorescent staining identified CD320 in the apical membrane region underneath the brush border in proximal tubules in human renal sections,a pattern overlapping that that of corin.2.In transfected MDCK cells,CD320 was found on the apical,but not basolateral,membrane.3.In computer modeling,CD320 LDLR2 exhibited a topology similar to that of corin LDLR8 domain,with the DSSDE motif in a comparable corner position.4.Site-directed mutagenesis and immunostaining studies indicated that the DSSDE motif in LDLR2 is critical for the specific apical membrane expression of human CD320 in MDCK cells.Conclusions:Human CD320 exhibits a specific apical expression pattern in renal epithelial cells,which is similar to that of corin.LDLR2 domain of CD320 contains a conserved DSSDE motif that is important for the specific apical expression of CD320 in MDCK cells.These results indicate that the DSSDE motif may play a similar role in other LDLR-containing proteins that are expressed on the apical membrane in polarized renal epithelial cells.Chapter 3:Role of Rablla in the apical specific expression of corin and CD320Objective:In chapter 1 and 2,we found that the DSSDE motif in LDLR was involved in corin and CD320 apical trafficking.However,it is not determined by a single factor that the polarized expression of proteins in polarized renal epithelial cells,but intracellular multi-molecular interactions.Rab small molecule proteins are distributed in the intracellular recycling endosomes and play an important role in intracellular vesicle trafficking and cell membrane targeting.Among them,Rabll(include Rab11a and Rab11b)has been shown to be involved in the regulation of apical transport of various proteins in polarized renal epithelial cells.In this chapter,we will study the role of Rab11 in the apical specific expression of corin and CD320 in polarized renal epithelial cells.Methods:1.Plasmids expressing Rablla,Rabllb and their dysfunctional mutants DNRab11a and DNRabllb were constructed by cloning and site-directed mutagenesis.2.Transfection and immunofluorescent staining were used to analyze the effects of Rab11a,Rabllb and mutants DNRab11a and DNRabllb on the apical specific expression of corin in MDCK cells.3.Endogenous Rab11a expression was inhibited by shRNA-based gene targeting in stably transfected MDCK cells.Quantitative RT-PCR was used to measured Rab11a mRNA expression levels.4.Immunofluorescent staining was used to examine corin and CD320 expression in Rablla-knockdown MDCK cellsResults:1.Transfection of the dominant-negative Rablla mutant(DNRablla)resulted corin expression on both apical and basolateral membranes in MDCK cells2.In MDCK cells transfected with the dominant-negative Rabllb mutant(DNRabllb),corin expression remained on the apical,but not basolateral,membrane.3.In the Rabl la-knocked down MDCK cells,the expression of corin and CD320 was found on both apical and basolateral membranes,as indicated by immunofluorescent staining.Conclusions:Rablla is important for the apical specific expression of corin and CD320 in polarized renal epithelial cells.Based on the results in this chapter and those in Chapters 1 and 2,we propose a model,in which the DSSDE motif in corin LDLR8 and CD320 LDLR2 domains is recognized by a Rabl la-dependent mechanism in protein intracellular trafficking to allow apical,but not basolateral,membrane targeting in polarized renal epithelial cells.Chapter 4:Biochemical and cellular studies of TMPRSS11A activationObjective:In the former three chapters,we explored the molecular mechanism of corin apical specific expression in polarized renal epithelial cells the type Ⅱ transmembrane serine proteinase(Type Ⅱ transmembrane serine proteases,TTSPs).corin is not the only one in TTSPs,also includes 16 other family members,which participate in a variety of normal physiological and pathological processes.Recent studies have shown that epithelial TTSPs are involved in coronavirus(CoV)infection by cleaving the viral spike(S)protein.TMPRSSllA is a TTSP expressed in human airway epithelial cells.Previous experiments showed that TMPRSS11A activated SARS(severe acute respiratory syndrome)and MERS(Middle East respiratory syndrome)CoV S proteins.To date,how TMPRSS11A is activated remains unknown.In this chapter,we will study the mechanism of TMPRSS11A activation in HEK293 and EC9706 cells.Methods:1.Plasmids expressing human TMPRSS11A WT,inactive mutants(R186A and S368A),and soluble TMPRSS11A(sTMPRSS11A)were constructed by cDNA cloning and site-directed mutagenesis.2.Flow cytometry,immunofluorescent staining,Western blotting,and biotin labeling were used to analyze TMPRSS11A expression and cleavage in HEK293 and EC9706 cells.3.N-glycosylation in TMPRSS11A was examined by PNGase F treatment and Western blotting.4.Trypsin digestion,brefeldin A(BFA)and monensin treatment,and Western blotting were used to study subcellular location of TMPRSS11A activation.5.Effects of hepatocyte growth factor activator inhibitor(HAI)1 and 2 on TMPRSS11A activation were studied in co-transfected HEK293 cells.Results:1.Flow cytometry and immunofluorescent staining detected TMPRSS11A on the surface of HEK293 cells,confirming the membrane topology of TMPRSS11A2.Western blotting detected the cleavage protease domain fragment in TMPRSSllA-expressing HEK293 cells,an indication of TMPRSS11A activation in the cells.3.Biotin-labeling and Western blotting experiments showed that the activated TMPRSS11A molecules were both in cell lysates and on the cell surface.4.Studies with the inactive mutants R186A and S368A indicated that TMPRSS11A was activated by autocatalysis.7.Experiments with trypsin digestion and BFA and monensin treatment showed that TMPRSS11A activation occurred before the protein reached the cell surface.8.Studies with sTMPSSllA lacking the transmembrane domain confirmed the intracellular activation and intermolecular cleavage of TMPRSS11A.9.In co-transfection experiments,HAI-2 was found to be more potent than HAI-1 in inhibiting TMPRSS11A autoactivation in HEK293 cells.Conclusions:Our results show that TMPRSS11A undergoes autoactivation that occurs inside the cells.This intracellular autoactivation mechanism is unique among TTSPs whose activation occurs mostly in the extracellular space.Our results also show that the protease inhibitor HAI-2 may play an an important role in regulating TMPRSS11A activation.These findings provide new insights into a potential role of TMPRSS11A in epithelial cell function and CoV infection.