Mechanism Of TMPRSS2 Activation And Cleavage Of SARS-CoV-2 Spike Protein

Chapter 1:Study on the activation mechanism of TMPRSS2Research Objectives:Transmembrane serine proteinase 2(TMPRSS2)is a type II Transmembrane serine protease,which is widely distributed in respiratory tract,gastrointestinal tract and urogenital tract.TMPRSS2 is involved in the occurrence and development of prostate cancer and mediates the invasion of human cells by a variety of viruses,which is closely related to the occurrence of COVID-19,which is currently spreading worldwide.Like other type Ⅱ transmembrane serine proteases,TMPRSS2 is synthesized as a proenzyme and requires activation to function.So far,the molecular mechanism of TMPRSS2 activation is not completely understood.In this chapter,we will focus on the molecular mechanism of TMPRSS2 activation in cells,including exploring its activation mode and location in cells.Research methods:1.Human Wild-type(WT)TMPRSS2 expression vector was constructed,and a V5 label was attached to the C-terminal.2.Using WT TMPRSS2 expression vector as template,mutant R292A and S478A were constructed by site-directed mutagenesis.3.The constructed plasmid was transfected into HEK293 cells,A549 cells and 16HBE cells.Western blotting,flow cytometry,immunofluorescence and biotin-labeling of cell surface proteins were used to detect the expression and activation of TMPRSS2 and its mutants in cell lysates.Small molecule fluorescence substrate incubation experiments was used to test the enzyme activity of TMPRSS2.4.To detect the activation position of TMPRSS2 in cells,HEK293 cells expressing TMPRSS2 were treated with trypsin,brefeldin A(BFA)and monensin.After lysis,Western blotting was used to detect the expression and activation of TMPRSS2 in cell lysates.Results:1.Western blotting was used to analyze HEK293 cells transfected with WT TMPRSS2.A～65kDa band could be detected under non-reduction conditions.Under the reduction condition,a～65 kDa proenzyme band and a～31 kDa activated protease band were detected.These results indicated that TMPRSS2 could be activated in HEK293 cells.2.Western blotting results showed that only the～65 kDa band was detected after transferring TMPRSS2 mutants R292A and S478A into HEK293,16HBE and A549 cells under reduction conditions,and no～31 kDa activated band was detected.Small molecule fluorescence substrate incubation experiments showed that WT TMPRSS2 had enzyme activity,while mutants R292A and S478A had no enzyme activity.The results suggest that the active site of TMPRSS2 is located in R292,and the activation process depends on its own enzyme activity.3.Immunofluorescence,flow cytometry and biotin-labeling of cell surface proteins were used to detect WT TMPRSS2 protein on HEK293 membrane.Anti-TMPRSS2 antibody detection showed that WT and its mutants were expressed on the membranes of HEK293,A549 and 16HBE,which was different from inactive mutants R292A and S478A by V5 antibody.,suggesting that activation of WT TMPRSS2 would remove the V5 label from its carboxyl terminal,while mutants R292A and S478A did not have such self-cutting phenomenon.4.The results of Trypsin digestion and Western blotting experiments showed that～31 kDa protease bands could be detected in HEK293 cells before and after trypsin treatment,indicating that the activation of TMPRSS2 occurred in cells.5.After monensin and BFA treated cells expressing TMPRSS2,Western blotting detection showed that the activated bands of TMPRSS2 could still be detected after monensin treatment,while the activation of TMPRSS2 was inhibited after BFA treatment.The results suggest that the activation of TMPRSS2 is not in the endoplasmic reticulum,but in the process of transport to the membrane of cells behind the endoplasmic reticulum,probably in the Golgi apparatus.Conclusion:Our results suggest that TMPRSS2 is activated by self-cleavage and that activation of TMPRSS2 occurs during transport behind the endoplasmic reticulum,most likely in the Golgi apparatus.Chapter 2:The regulation of hepatocyte growth factor activator inhibitors and N-glycosylation on TMPRSS2 activityResearch Objectives:TMPRSS2,as a serine protease,plays an important role in the cell infection process of influenza virus,SARS-COV,MERS-CoV and SARS-CoV-2.Therefore,the study on the regulation mechanism of TMPRSS2 enzyme activity is of great significance for the prevention and treatment of virus infection.Hepatocyte growth factor activator inhibitors 1 and 2(HAI-1 and HAI-2)are widely expressed protease inhibitors in human body,which can inhibit the activity of various proteases.N-glycosylation is a common post-translational modification of proteins and plays a role in the regulation of protein structure and function.In this chapter,we will explore the effects of HAI-1 and HAI-2,as well as N-glycosylation on the activation and transport of TMPRSS2.Research methods:1.HEK293 cells were co-transfected with hepatocyte growth factor activator inhibitors HAI-1/HAI-2 and TMPRSS2 plasmids.Western blotting was used to detect whether HAI-1 and HAI-2 could affect the activation of TMPRSS2.2.Cell lysates expressing TMPRSS2 were treated with glycosidase PNGase F,and the N-glycosylation of TMPRSS2 protein was detected by Western blotting.3.Using WT TMPRSS2 expression vector as template,N250Q,N286Q and N250Q/N286Q mutants were constructed by site-directed mutation technique.4.The constructed plasmid was transfected into HEK293 cells,and Western blotting was used to detect the expression and activation of WT and N-glycosylation site mutants in HEK293 cells to explore the N-glycosylation of TMPRSS2 and the effect of N-glycosylation on the activation of TMPRSS2.5.HEK293 cells expressing WT TMPRSS2 and N-glycosylated mutants were treated with cycloheximide(CHX).Cells were collected at different time to prepare lysate.Western blotting was used to detect protein expression and calculate proteins’half-lives.6.Co-localization of WT TMPRSS2,N250Q,N286Q and N250Q/N286Q with ER marker KDEL and Golgi marker GM130 in HEK293 cells was detected by immunofluorescence.Results:1.Western blotting analysis of HEK293 cell samples co-transfected with serine protease inhibitors and TMPRSS2 plasmid showed that the activated bands of TMPRSS2 in cell samples co-transfected with HAI-1/HAI-2 and TMPRSS2 were significantly reduced compared with the control group.These results suggest that both HAI-1 and HAI-2 can inhibit the self-activation of TMPRSS2.2.Western blotting results showed that the migration of WT TMPRSS2 was faster after treatment with glycosidase PNGase F,indicating that TMPRSS2 was modified by N-glycosylation.3.Western blotting results showed that the migration of N-glycosylated mutants was accelerated compared with WT,indicating that TMPRSS2 had glycosylations at N250 and N286 respectively.In addition,N250Q and N250Q/N286Q did not detect the active band,while N286Q,similar to WT,could detect the active band.The results showed that the deletion of N-glycosylation in N250 position prevented the activation of TMPRSS2.4.By immunofluorescence analysis of the localization of WT and its N-glycosylation site mutants in cells,it was found that N250Q and N250Q/N286Q had obvious co-localization with the ER,while WT and N286Q did not detect co-localization with the ER.These results indicated that the deletion of N-glycosylation at N250 position would cause TMPRSS2 to remain in the endoplasmic reticulum,thus preventing its activation,while the deletion of N-glycosylation at N286 position would not affect the activation and transport of TMPRSS2 in cells.Conclusion:Our results showed that protease inhibitors HAI-1 and HAI-2 can inhibit the activation of TMPRSS2.N-glycosylation at the N250 of TMPRSS2 is critical for protein transport and activation,and its deletion results in retention of TMPRSS2 in the endoplasmic reticulum and inactivation.Chapter 3:Study on the mechanism of SARS-CoV-2 Spike protein cleaved by TMPRSS2Research Objectives:TMPRSS2 is a transmembrane serine protease distributed in the epithelial cells of nasal,bronchial and lung respiratory organs.Spike(S)proteins on the surface of coronaviruses can be cleaved to participate in the process of coronaviruses infecting host cells.TMPRSS2 has two shear isoforms.TMPRSS2 isoform 1 has a longer cytoplasmic segment than isoform 2.Currently,studies on TMPRSS2 mainly focus on isoform 2,and reports on TMPRSS2 isoform 1 are few.The molecular mechanism of TMPRSS2 cleaving Spike protein,such as the specific cleaving site,is not completely clear.In this chapter,we used the expression vector of TMPRSS2 isoform 1(WT)to study the cleaving effect of TMPRSS2 on SARS-CoV-2 Spike protein and the specific molecular mechanism.Research methods:1.The Spike protein expression plasmid of SARS-CoV-2 was transfected into HEK293 cells and its expression was detected by western blotting.2.TMPRSS2 and Spike protein expression plasmid were co-transfected into cells,and the Spike protein cleavage by TMPRSS2 was detected by western blotting.3.Construct S1/S2 mut(mutant),S2’ mut,S2’ mut and S1/S2-S2’ mut,and then co-transfer these plasmids with TMPRSS2 into HEK293 cells to explore the mechanism of Spike protein cleavage by TMPRSS2.Results:1.Western blotting analysis of HEK293 cells transfected with SARS-CoV-2 Spike expression plasmid showed that the full length Spike protein band(～207 kDa),S2 fragment(～92 kDa)and Spike protein polymer band of large molecular weight were detected.These results indicated that the Spike expression plasmid of SARS-CoV-2 could be expressed in HEK293 cells.2.When TMPRSS2 and Spike were transferred into HEK293 cells,S2’ band(68 kDa)could be detected.It suggested that TMPRSS2 could cleave Spike protein at S2’position and then produce the S2’ fragment.3.Co-transfer TMPRSS2 with WT Spike and its mutants,Western blotting results showed that:The full length Spike protein could be detected,but S2 fragment could not be detected in S1/S2 mut group,S2’mut group and S1/S2-S2’mut group could not detect the S2 and S2’ fragment regardless of whether TMPRSS2 existed or not.The results suggest that S2’ is crucial for Spike protein cleavage,and the mutation at this site not only affects Spike protein cleavage by TMPRSS2,but also affects Spike protein cleavage at S1/S2 sites.Conclusion:We demonstrate that TMPRSS2 isoform 1 can cleave the Spike protein of SARS-COV-2 at S2’,and the cleavage site of S1/S2 can only be cleaved if Spike protein can be cleaved at S2’.Given the importance of Spike protein S1/S2 and S2’cleavage sites in infection,these findings provide new insights into the role of TMPRSS2 in SARS-CoV-2 infection.