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.
Highlights
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 [1, 2]
TMPRSS11A is an airway epithelial TTSP implicated in severe acute respiratory syndrome (SARS) and Middle East respiratory syndrome (MERS) CoV infection [27, 28]
We examined the activation cleavage of human TMPRSS11A in human embryonic kidney 293 (HEK293) cells, esophageal cancer EC9706 cells, and lung epithelial 16HBE and A549 cells
Summary
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 [1, 2]. 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.
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