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• W3043458689 abstract "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. 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. Type II transmembrane serine proteases (TTSPs) are a group of enzymes with a similar domain structural arrangement, including a short N-terminal cytoplasmic tail, a single-span transmembrane domain, and an extended extracellular region with various modules and a C-terminal trypsin-like protease domain (1Bugge T.H. Antalis T.M. Wu Q. Type II transmembrane serine proteases.J. Biol. Chem. 2009; 284 (19487698): 23177-2318110.1074/jbc.R109.021006Abstract Full Text Full Text PDF PubMed Scopus (255) Google Scholar, 2Hooper J.D. Clements J.A. Quigley J.P. Antalis T.M. Type II transmembrane serine proteases: insights into an emerging class of cell surface proteolytic enzymes.J. Biol. Chem. 2001; 276 (11060317): 857-86010.1074/jbc.R000020200Abstract Full Text Full Text PDF PubMed Scopus (322) Google Scholar). TTSPs act in diverse tissues to participate in physiological and pathological processes, including iron metabolism (3Heeney M.M. Finberg K.E. Iron-refractory iron deficiency anemia (IRIDA).Hematol. Oncol. Clin. North Am. 2014; 28 (25064705): 637-65210.1016/j.hoc.2014.04.009Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar, 4Stirnberg M. Gütschow M. Matriptase-2, a regulatory protease of iron homeostasis: possible substrates, cleavage sites and inhibitors.Curr. Pharm. Des. 2013; 19 (23016685): 1052-106110.2174/1381612811319060007Crossref PubMed Google Scholar), liver metabolism (5Li S. Peng J. Wang H. Zhang W. Brown J.M. Zhou Y. Wu Q. Hepsin enhances liver metabolism and inhibits adipocyte browning in mice.Proc. Natl. Acad. Sci. U.S.A. 2020; 117 (32404422): 12359-1236710.1073/pnas.1918445117Crossref PubMed Scopus (14) Google Scholar), blood pressure regulation (6Chen S. Cao P. Dong N. Peng J. Zhang C. Wang H. Zhou T. Yang J. Zhang Y. Martelli E.E. Naga Prasad S.V. Miller R.E. Malfait A.M. Zhou Y. Wu Q. PCSK6-mediated corin activation is essential for normal blood pressure.Nat. Med. 2015; 21 (26259032): 1048-105310.1038/nm.3920Crossref PubMed Scopus (90) Google Scholar, 7Zhou Y. Wu Q. Corin in natriuretic peptide processing and hypertension.Curr. Hypertens. Rep. 2014; 16 (24407448): 41510.1007/s11906-013-0415-7Crossref PubMed Scopus (46) Google Scholar), food digestion (8Zheng X.L. Kitamoto Y. Sadler J.E. Enteropeptidase, a type II transmembrane serine protease.Front. Biosci. (Elite Ed.). 2009; 1 (19482641): 242-249PubMed Google Scholar), auditory function (9Guipponi M. Antonarakis S.E. Scott H.S. TMPRSS3, a type II transmembrane serine protease mutated in non-syndromic autosomal recessive deafness.Front. Biosci. 2008; 13 (17981648): 1557-156710.2741/2780Crossref PubMed Scopus (35) Google Scholar, 10Guipponi M. Tan J. Cannon P.Z. Donley L. Crewther P. Clarke M. Wu Q. Shepherd R.K. Scott H.S. Mice deficient for the type II transmembrane serine protease, TMPRSS1/hepsin, exhibit profound hearing loss.Am. J. Pathol. 2007; 171 (17620368): 608-61610.2353/ajpath.2007.070068Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar), vascular remodeling (11Cui Y. Wang W. Dong N. Lou J. Srinivasan D.K. Cheng W. Huang X. Liu M. Fang C. Peng J. Chen S. Wu S. Liu Z. Dong L. Zhou Y. et al.Role of corin in trophoblast invasion and uterine spiral artery remodelling in pregnancy.Nature. 2012; 484 (22437503): 246-25010.1038/nature10897Crossref PubMed Scopus (228) Google Scholar), epithelial development (12Miller G.S. List K. The matriptase-prostasin proteolytic cascade in epithelial development and pathology.Cell Tissue Res. 2013; 351 (22350849): 245-25310.1007/s00441-012-1348-1Crossref PubMed Scopus (34) Google Scholar, 13Szabo R. Bugge T.H. Membrane-anchored serine proteases as regulators of epithelial function.Biochem. Soc. Trans. 2020; 48 (32196551): 517-52810.1042/BST20190675Crossref PubMed Scopus (13) Google Scholar), intestinal barrier function (14Buzza M.S. Johnson T.A. Conway G.D. Martin E.W. Mukhopadhyay S. Shea-Donohue T. Antalis T.M. Inflammatory cytokines down-regulate the barrier-protective prostasin-matriptase proteolytic cascade early in experimental colitis.J. Biol. Chem. 2017; 292 (28490634): 10801-1081210.1074/jbc.M116.771469Abstract Full Text Full Text PDF PubMed Scopus (15) Google Scholar, 15Lahey K.A. Ronaghan N.J. Shang J. Dion S.P. Désilets A. Leduc R. MacNaughton W.K. Signaling pathways induced by serine proteases to increase intestinal epithelial barrier function.PLoS ONE. 2017; 12 (28671992): e018025910.1371/journal.pone.0180259Crossref PubMed Scopus (9) Google Scholar), and cancer development (16Martin C.E. List K. Cell surface-anchored serine proteases in cancer progression and metastasis.Cancer Metastasis Rev. 2019; 38 (31529338): 357-38710.1007/s10555-019-09811-7Crossref PubMed Scopus (59) Google Scholar, 17Tanabe L.M. List K. The role of type II transmembrane serine protease-mediated signaling in cancer.FEBS J. 2017; 284 (27870503): 1421-143610.1111/febs.13971Crossref PubMed Scopus (85) Google Scholar). In recent years, TTSPs have been implicated in coronavirus (CoV) infection. In particular, TTSPs expressed in the human respiratory tract, including human airway trypsin-like protease (HAT) (18Bertram S. Glowacka I. Müller M.A. Lavender H. Gnirss K. Nehlmeier I. Niemeyer D. He Y. Simmons G. Drosten C. Soilleux E.J. Jahn O. Steffen I. Pöhlmann S. Cleavage and activation of the severe acute respiratory syndrome coronavirus spike protein by human airway trypsin-like protease.J. Virol. 2011; 85 (21994442): 13363-1337210.1128/JVI.05300-11Crossref PubMed Scopus (217) Google Scholar), the TTSP mosaic serine protease large form (19Zmora P. Blazejewska P. Moldenhauer A.S. Welsch K. Nehlmeier I. Wu Q. Schneider H. Pöhlmann S. Bertram S. DESC1 and MSPL activate influenza A viruses and emerging coronaviruses for host cell entry.J. Virol. 2014; 88 (25122802): 12087-1209710.1128/JVI.01427-14Crossref PubMed Scopus (54) Google Scholar), differentially expressed in squamous cell carcinoma 1 (DESC1) (19Zmora P. Blazejewska P. Moldenhauer A.S. Welsch K. Nehlmeier I. Wu Q. Schneider H. Pöhlmann S. Bertram S. DESC1 and MSPL activate influenza A viruses and emerging coronaviruses for host cell entry.J. Virol. 2014; 88 (25122802): 12087-1209710.1128/JVI.01427-14Crossref PubMed Scopus (54) Google Scholar), transmembrane protease serine 2 (TMPRSS2) (18Bertram S. Glowacka I. Müller M.A. Lavender H. Gnirss K. Nehlmeier I. Niemeyer D. He Y. Simmons G. Drosten C. Soilleux E.J. Jahn O. Steffen I. Pöhlmann S. Cleavage and activation of the severe acute respiratory syndrome coronavirus spike protein by human airway trypsin-like protease.J. Virol. 2011; 85 (21994442): 13363-1337210.1128/JVI.05300-11Crossref PubMed Scopus (217) Google Scholar, 20Gierer S. Bertram S. Kaup F. Wrensch F. Heurich A. Krämer-Kühl A. Welsch K. Winkler M. Meyer B. Drosten C. Dittmer U. von Hahn T. Simmons G. Hofmann H. Pöhlmann S. The spike protein of the emerging betacoronavirus EMC uses a novel coronavirus receptor for entry, can be activated by TMPRSS2, and is targeted by neutralizing antibodies.J. Virol. 2013; 87 (23468491): 5502-551110.1128/JVI.00128-13Crossref PubMed Scopus (251) Google Scholar, 21Glowacka I. Bertram S. Müller M.A. Allen P. Soilleux E. Pfefferle S. Steffen I. Tsegaye T.S. He Y. Gnirss K. Niemeyer D. Schneider H. Drosten C. Pöhlmann S. Evidence that TMPRSS2 activates the severe acute respiratory syndrome coronavirus spike protein for membrane fusion and reduces viral control by the humoral immune response.J. Virol. 2011; 85 (21325420): 4122-413410.1128/JVI.02232-10Crossref PubMed Scopus (731) Google Scholar, 22Hoffmann M. Kleine-Weber H. Schroeder S. Krüger N. Herrler T. Erichsen S. Schiergens T.S. Herrler G. Wu N.H. Nitsche A. Müller M.A. Drosten C. Pöhlmann S. SARS-CoV-2 cell entry depends on ACE2 and TMPRSS2 and is blocked by a clinically proven protease inhibitor.Cell. 2020; 181 (32142651): 271-280.e27810.1016/j.cell.2020.02.052Abstract Full Text Full Text PDF PubMed Scopus (11601) Google Scholar, 23Matsuyama S. Nao N. Shirato K. Kawase M. Saito S. Takayama I. Nagata N. Sekizuka T. Katoh H. Kato F. Sakata M. Tahara M. Kutsuna S. Ohmagari N. Kuroda M. et al.Enhanced isolation of SARS-CoV-2 by TMPRSS2-expressing cells.Proc. Natl. Acad. Sci. U.S.A. 2020; 117 (32165541): 7001-700310.1073/pnas.2002589117Crossref PubMed Scopus (797) Google Scholar, 24Shang J. Wan Y. Luo C. Ye G. Geng Q. Auerbach A. Li F. Cell entry mechanisms of SARS-CoV-2.Proc. Natl. Acad. Sci. U.S.A. 2020; 117 (32376634): 11727-1173410.1073/pnas.2003138117Crossref PubMed Scopus (1847) Google Scholar, 25Shirato K. Kawase M. Matsuyama S. Middle East respiratory syndrome coronavirus infection mediated by the transmembrane serine protease TMPRSS2.J. Virol. 2013; 87 (24027332): 12552-1256110.1128/JVI.01890-13Crossref PubMed Scopus (304) Google Scholar, 26Shulla A. Heald-Sargent T. Subramanya G. Zhao J. Perlman S. Gallagher T. A transmembrane serine protease is linked to the severe acute respiratory syndrome coronavirus receptor and activates virus entry.J. Virol. 2011; 85 (21068237): 873-88210.1128/JVI.02062-10Crossref PubMed Scopus (460) Google Scholar), and TMPRSS11A (27Kam Y.W. Okumura Y. Kido H. Ng L.F. Bruzzone R. Altmeyer R. Cleavage of the SARS coronavirus spike glycoprotein by airway proteases enhances virus entry into human bronchial epithelial cells in vitro.PLoS ONE. 2009; 4 (19924243): e787010.1371/journal.pone.0007870Crossref PubMed Scopus (118) Google Scholar, 28Zmora P. Hoffmann M. Kollmus H. Moldenhauer A.S. Danov O. Braun A. Winkler M. Schughart K. Pöhlmann S. TMPRSS11A activates the influenza A virus hemagglutinin and the MERS coronavirus spike protein and is insensitive against blockade by HAI-1.J. Biol. Chem. 2018; 293 (29976755): 13863-1387310.1074/jbc.RA118.001273Abstract Full Text Full Text PDF PubMed Scopus (28) Google Scholar), were shown to cleave the severe acute respiratory syndrome (SARS) CoV, the Middle East respiratory syndrome (MERS) CoV or SARS-CoV-2 spike (S) proteins in cell-based studies. As the S proteins are major determinants for receptor binding and membrane fusion in host cells (29Li F. Structure, function, and evolution of coronavirus spike proteins.Annu. Rev. Virol. 2016; 3 (27578435): 237-26110.1146/annurev-virology-110615-042301Crossref PubMed Scopus (1595) Google Scholar), it appears that human airway TTSPs have been exploited by the CoVs to enhance their infectivity. TTSPs are synthesized in a precursor or zymogen form with little catalytic activity. Proteolytic cleavage at a conserved activation site converts the zymogen to an active enzyme. To date, how CoV-activating TTSPs are activated in cells is not well understood. In this study, we analyzed the activation cleavage of TMPRSS11A, which is expressed in airway epithelial cells (28Zmora P. Hoffmann M. Kollmus H. Moldenhauer A.S. Danov O. Braun A. Winkler M. Schughart K. Pöhlmann S. TMPRSS11A activates the influenza A virus hemagglutinin and the MERS coronavirus spike protein and is insensitive against blockade by HAI-1.J. Biol. Chem. 2018; 293 (29976755): 13863-1387310.1074/jbc.RA118.001273Abstract Full Text Full Text PDF PubMed Scopus (28) Google Scholar, 30Plasschaert L.W. Žilionis R. Choo-Wing R. Savova V. Knehr J. Roma G. Klein A.M. Jaffe A.B. A single-cell atlas of the airway epithelium reveals the CFTR-rich pulmonary ionocyte.Nature. 2018; 560 (30069046): 377-38110.1038/s41586-018-0394-6Crossref PubMed Scopus (485) Google Scholar) and activates SARS and MERS CoV S proteins (27Kam Y.W. Okumura Y. Kido H. Ng L.F. Bruzzone R. Altmeyer R. Cleavage of the SARS coronavirus spike glycoprotein by airway proteases enhances virus entry into human bronchial epithelial cells in vitro.PLoS ONE. 2009; 4 (19924243): e787010.1371/journal.pone.0007870Crossref PubMed Scopus (118) Google Scholar, 28Zmora P. Hoffmann M. Kollmus H. Moldenhauer A.S. Danov O. Braun A. Winkler M. Schughart K. Pöhlmann S. TMPRSS11A activates the influenza A virus hemagglutinin and the MERS coronavirus spike protein and is insensitive against blockade by HAI-1.J. Biol. Chem. 2018; 293 (29976755): 13863-1387310.1074/jbc.RA118.001273Abstract Full Text Full Text PDF PubMed Scopus (28) Google Scholar). By immunostaining, flow cytometry, Western blotting, protease digestion, and site-directed mutagenesis, we show that TMPRSS11A is autoactivated inside the cell before reaching the cell surface. This mechanism of intracellular activation cleavage differs from the extracellular activation cleavage reported in other TTSPs. Moreover, we found that TMPRSS11A exhibited the activity in cleaving the SARS-CoV-2 S protein. Human TMPRSS11A consists of 418 amino acids. Fig. 1A shows the domain structure of TMPRSS11A, including an N-terminal cytoplasmic tail, a transmembrane domain (TM), and an extracellular region containing a SEA (sea urchin sperm protein/enteropeptidase/agrin) domain and a C-terminal serine protease domain. The conserved activation cleavage site is at Arg186–Ile187 (Fig. 1A and Fig. S1). There is a disulfide bond (Cys175–Cys292) linking the protease domain to the propepide region after the cleavage at the Arg186–Ile187 (Fig. 1A). To study TMPRSS11A, we expressed human TMPRSS11A with a C-terminal V5 tag in transfected human embryonic kidney 293 (HEK293) cells. In flow cytometry, we found TMPRSS11A on the cell surface (Fig. 1B). In immunostaining, we detected TMPRSS11A on the surface of nonpermeabilized cells (Fig. 1C). The immunofluorescent signal was stronger when the cells were permeabilized, which allows staining both the cell surface and intracellular TMPRSS11A protein (Fig. 1C). These results are consistent with the predicted membrane topology of TMPRSS11A being a TTSP. In Western blotting of lysates from the transfected HEK293 cells, we detected a single ∼57-kDa band under nonreducing conditions (Fig. 1D, left panel). When Western blotting was done under reducing conditions, three bands of ∼57, ∼37, and ∼28 kDa, respectively, were detected (Fig. 1D, right panel). In Western blotting with cell surface-labeled proteins, a single band (∼57 kDa) and two bands (∼57 and ∼37 kDa) were detected under nonreducing and reducing conditions, respectively (Fig. 1E). Based on the calculated molecular mass, the ∼57-kDa band represents the one-chain TMPRSS11A zymogen, whereas the ∼37-kDa band represents the protease domain fragment cleaved at the conserved activation site (Fig. 1A). Because the V5 tag was at the C terminus, the cleaved N-terminal fragment was not detected by the anti-V5 antibody in Western blotting. These results indicate that TMPRSS11A is activated and present on the surface of the transfected HEK293 cells. The identity of the ∼28-kDa band detected in Western blotting (Fig. 1D, right panel) was unclear. This band was observed under reducing conditions in cell lysates, but not among cell surface-labeled proteins (Fig. 1E, right panel), indicating that this fragment remained inside the cells. Human TMPRSS11A contains two N-glycosylation sites: one at Asn153 in the SEA domain and the other at Asn303 in the protease domain (Fig. 2A). To exclude the possibility that the ∼28-kDa band was an unglycosylated fragment, we treated HEK293 cell lysates with PNGase F to remove N-glycans on proteins. In Western blotting under reducing conditions, the ∼57-, ∼37-, and ∼28-kDa TMPRSS11A bands all migrated faster compared with those in untreated samples (Fig. 2B), indicating that the ∼28-kDa band may be a proteolytically cleaved fragment but not an unglycosylated fragment. Based on the calculated molecular mass, the ∼28-kDa band could be generated from a cleavage at Arg265 in the protease domain (Fig. 2C). To test this hypothesis, we made a plasmid expressing the mutant R265A, in which Arg265 in TMPRSS11A was replaced by Ala. In Western blotting with lysates from transfected HEK293 cells, the ∼28-kDa band was detected in TMPRSS11A WT but not the mutant R265A (Fig. 2D), indicating that the ∼28-kDa band is created by proteolytic cleavage at Arg265 in the protease domain. In a recent study in transfected 293T cells, more than seven major TMPRRSS11A fragments were detected by Western blotting (28Zmora P. Hoffmann M. Kollmus H. Moldenhauer A.S. Danov O. Braun A. Winkler M. Schughart K. Pöhlmann S. TMPRSS11A activates the influenza A virus hemagglutinin and the MERS coronavirus spike protein and is insensitive against blockade by HAI-1.J. Biol. Chem. 2018; 293 (29976755): 13863-1387310.1074/jbc.RA118.001273Abstract Full Text Full Text PDF PubMed Scopus (28) Google Scholar). It is difficult to know if those fragments were derived by TMPRSS11A autocatalysis or unknown protease(s) or both in 293T cells. To circumvent this problem, we made plasmids expressing mutants R186A and S368A, in which the activation cleavage site at Arg186 and the catalytic Ser368 were mutated to Ala, respectively (Fig. 3A). In Western blotting of lysates from HEK293 cells expressing the R186A mutant, only the ∼57-kDa zymogen band was detected (Fig. 3B), indicating that the ∼37-kDa band was derived from cleavage at the conserved activation site Arg186 and that the single-chain TMPRSS11A was incapable of cleaving at Arg265. Similarly, Western blotting of lysates from HEK293 cells expressing the S368A mutant showed the ∼57-kDa band only (Fig. 3B), indicating that cleavages at Arg186 (generating the ∼37-kDa band) and Arg265 (generating the ∼28-kDa band) depended on the catalytic activity of TMPRSS11A. TMPRSS11A, also called ECRG1 (esophageal cancer-related gene 1), was first identified in human esophageal cancers (31Li Y. Zhang X. Huang G. Miao X. Guo L. Lin D. Lu S.H. Identification of a novel polymorphism Arg290Gln of esophageal cancer related gene 1 (ECRG1) and its related risk to esophageal squamous cell carcinoma.Carcinogenesis. 2006; 27: 798-80210.1093/carcin/bgi258Crossref PubMed Scopus (21) Google Scholar, 32Zhao N. Huang G. Guo L. Lu S.H. ECRG1, a novel candidate of tumor suppressor gene in the esophageal carcinoma, triggers a senescent program in NIH3T3 cells.Exp. Biol. Med. (Maywood). 2006; 231: 84-9010.1177/153537020623100110Crossref PubMed Scopus (11) Google Scholar). To verify our results, we expressed TMPRSS11A WT and mutants R186A and S368A in EC9706 cells, a human esophageal cancer cell line (33Wang H.T. Kong J.P. Ding F. Wang X.Q. Wang M.R. Liu L.X. Wu M. Liu Z.H. Analysis of gene expression profile induced by EMP-1 in esophageal cancer cells using cDNA microarray.World J. Gastroenterol. 2003; 9 (12632483): 392-39810.3748/wjg.v9.i3.392Crossref PubMed Scopus (47) Google Scholar). In Western blotting of lysates from transfected EC9706 cells, we detected three bands of ∼57, ∼37, and ∼28 kDa, respectively, in TMPRSS11A WT, but a single ∼57-kDa band in mutants R186A and S386A (Fig. 3C). Similar results were observed in additional experiments with human bronchial (16HBE) and alveolar basal (A549) epithelial cells (Fig. S2). These results are consistent, indicating that TMPRSS11A undergoes autoactivation at Arg186 and subsequent autocleavage at Arg265 in the protease domain. To understand if the detected TMPRSS11A autoactivation cleavage occurred intracellularly or on the cell surface, we expressed TMPRSS11A WT in HEK293 cells and treated the cells with trypsin to remove surface proteins. In flow cytometry, TMPRSS11A was detected on the surface of the transfected HEK293 cells (Fig. 4A). The expression was reduced to the background level in the cells treated with trypsin (Fig. 4A). When the cells were lysed and lysates were analyzed by Western blotting, we observed the ∼57-, ∼37-, and ∼28-kDa bands in the cells without or with trypsin treatment (Fig. 4B). These results indicate that TMPRSS11A activation cleavage occurred intracellularly. We next treated HEK293 cells expressing TMPRSS11A with brefeldin A (BFA) and monensin, which inhibit protein trafficking in the endoplasmic reticulum (ER) and the Golgi (34Dinter A. Berger E.G. Golgi-disturbing agents.Histochem. Cell Biol. 1998; 109 (9681636): 571-59010.1007/s004180050256Crossref PubMed Scopus (320) Google Scholar). In Western blotting, we found the ∼57-, ∼37-, and ∼28-kDa bands in TMPRSS11A-expressing cells without or with BFA or monensin treatment (Fig. 4C). In these studies, we did parallel control experiments with corin (Fig. S3), a TTSP known to be activated on the cell surface but not intracellularly (6Chen S. Cao P. Dong N. Peng J. Zhang C. Wang H. Zhou T. Yang J. Zhang Y. Martelli E.E. Naga Prasad S.V. Miller R.E. Malfait A.M. Zhou Y. Wu Q. PCSK6-mediated corin activation is essential for normal blood pressure.Nat. Med. 2015; 21 (26259032): 1048-105310.1038/nm.3920Crossref PubMed Scopus (90) Google Scholar). In Western blotting, the ∼40-kDa corin protease domain fragment from activation cleavage was detected in the cells without, but not with, trypsin, BFA, or monensin treatment (Fig. 4D). These results indicate that, unlike corin, TMPRSS11A is activated intracellularly before reaching the cell surface. To examine if the transmembrane domain in TMPRSS11A is required for activation cleavage, we made a plasmid expressing a soluble form of TMPRSS11A, in which the cytoplasmic tail and the transmembrane domain were replaced with the signal peptide of IgK (Fig. 5A) (35Knappe S. Wu F. Masikat M.R. Morser J. Wu Q. Functional analysis of the transmembrane domain and activation cleavage of human corin: design and characterization of a soluble corin.J. Biol. Chem. 2003; 278 (14559895): 52363-5237010.1074/jbc.M309991200Abstract Full Text Full Text PDF PubMed Scopus (99) Google Scholar). In addition, we made another plasmid expressing an inactive soluble TMPRSS11A mutant (soluble S368A), in which the catalytic Ser368 was replaced by Ala (Fig. 5A). As expected, TMPRSS11A WT, but not soluble TMPRSS11A (s11A), was found on the surface of transfected HEK293 cells in flow cytometry (Fig. 5B). In Western blotting, we found all three bands of ∼57/53, ∼37, and ∼28 kDa, respectively, in lysates from HEK293 cells expressing TMPRSS11A WT and s11A (Fig. 5C), indicating that soluble TMPRSS11A undergoes similar intracellular activation cleavage and that the transmembrane domain is not required for TMPRSS11A autoactivation. We next incubated the conditioned medium containing s11A with a recombinant SARS–CoV-2 S protein fragment corresponding to the nearly entire extracellular region (residues 16-1213) produced from insect cells. We detected a ∼60-kDa band (Fig. 5D), which is close to the calculated molecular mass of the cleaved S2 fragment (59 kDa). The ∼60-kDa band was not detected in samples from vector-transfected cells or cells expressing the inactive soluble TMPRSS11A S368A (Fig. 5D). In a positive control, a similar ∼60-kDa band was observed when we used the conditioned medium from cells expressing a soluble form of TMPRSS2, which cleaves SARS–CoV-2 S protein (22Hoffmann M. Kleine-Weber H. Schroeder S. Krüger N. Herrler T. Erichsen S. Schiergens T.S. Herrler G. Wu N.H. Nitsche A. Müller M.A. Drosten C. Pöhlmann S. SARS-CoV-2 cell entry depends on ACE2 and TMPRSS2 and is blocked by a clinically proven protease inhibitor.Cell. 2020; 181 (32142651): 271-280.e27810.1016/j.cell.2020.02.052Abstract Full Text Full Text PDF PubMed Scopus (11601) Google Scholar) (Fig. 5D). We did not detect any SARS–CoV-2 S protein fragments of ∼45 kDa, which is the calculated molecular mass of the S2' fragment. These results indicate that TMPRSS11A is capable of cleaving SARS–CoV-2 S protein at least in vitro. The results described above support TMPRSS11A autoactivation. It was unclear if the autoactivation cleavage of TMPRSS11A occurs in cis (intramolecular) or in trans (intermolecular). To address this question, we further analyzed the soluble S368A mutant (sS368A) (Fig. 5A). In transfected HEK293 cells, sS368A was detected in the conditioned medium, as expected (Fig. 6A). On Western blots, the sS368A fragment in the conditioned medium had a higher molecular mass than that in lysates (∼57 versus ∼53 kDa) (Fig. S4). When the samples were treated with PNGase F, the sS368A fragment from the conditioned medium and lysates migrated faster at ∼53 and ∼51 kDa, respectively (Fig. S4). The results suggest that other conformational changes or post-translational modifications may account for the higher molecular mass observed in the sS368A fragment from the conditioned medium. We next transfected HEK293 cells with the plasmid expressing the sS368A mutant together with plasmids expressing TMPRSS11A WT and mutants R186A and S368A or a control vector. In Western blotting, the ∼37-kDa band derived from activation cleavage was observed in the conditioned medium from HEK293 cells co-expressing TMPRSS11A WT, but not mutants R186A and S368A (Fig. 6B), indicating that intermolecular cleavage of TMPRSS11A did occur under our experimental conditions. We then did another experiment, in which plasmid expressing the sS368A mutant was co-transfected with plasmids expressing TMPRSS11A, hepsin, TMPRSS2, and corin in HEK293 cells. In Western blotting of the conditioned medium, the ∼37-kDa band from the sS368A mutant was detected in samples from TMPRSS11A-, hepsin- and TMPRSS2-expressing, but not corin-expressing, cells. The levels of this band, however, were much lower in samples from hepsin- and TMPRSS2-expressing cells than that in TMPRSS11A-expressing cells (Fig. S5). These results support the idea of TMPRSS11A autoactivation, although the possibility that other TTSP-mediated transactivation cleavage may occur cannot be excluded. Hepatocyte growth factor activator inhibitors 1 and 2 (HAI-1 and HAI-2) are structurally related type I transmembrane serine protease inhibitors (36Kataoka H. Kawaguchi M. Fukushima T. Shimomura T. Hepatocyte growth factor activator inhibitors (HAI-1 and HAI-2): Emerging key players in epithelial integrity and cancer.Pathol. Int. 2018; 68 (29431273): 145-15810.1111/pin.12647Crossref PubMed Scopus (38) Google Scholar). Recently, HAI-1 was shown to inhibit HAT and DESC1, but not TMPRSS11A, activity in cell-based studies (28Zmora P. Hoffmann M. Kollmus H. Moldenhauer A.S. Danov O. Braun A. Winkler M. Schughart K. Pöhlmann S. TMPRSS11A activates the influenza A virus hemagglutinin and the MERS coronavirus spike protein and is insensitive against blockade by HAI-1.J. Biol. Chem. 2018; 293 (29976755): 13863-1387310.1074/jbc.RA118.001273Abstract Full Text Full Text PDF PubMed Scopus (28) Google Scholar). To examine the effect of HAI-1 and HAI-2 on TMPRSS11A activation cleavage, we co-transfected HEK293 cells with plasmids expressing TMPRSS11A WT and human HAI-1 or HAI-2. In Western blotting of lysates from the transfected cells, we detected the ∼37-kDa TMPRSS11A band in cells co-expressing HAI-1 (Fig. 7, top panel). However, the level of the ∼37-kDa band was lower than that in control vector co-transfected cells. In cells co-expressing TMPRSS11A and HAI-2, the ∼37-kDa band was barely visible (Fig. 7, top panel). The ∼28-kDa TMPRSS11A band w" @default.
• W3043458689 created "2020-07-23" @default.
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• W3043458689 date "2020-09-01" @default.
• W3043458689 modified "2023-10-09" @default.
• W3043458689 title "Intracellular autoactivation of TMPRSS11A, an airway epithelial transmembrane serine protease" @default.
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• W3043458689 doi "https://doi.org/10.1074/jbc.ra120.014525" @default.
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