Transmembrane serine protease TMPRSS2 implicated in SARS-CoV-2 infection is autoactivated intracellularly and requires N-glycosylation for regulation

Characterization of spike processing and entry mechanisms of seasonal human coronaviruses NL63, 229E and HKU1

“…TMPRSS2 is a type II transmembrane glycoprotein that belongs to the type 2 transmembrane serine protease (TTSP) family and is composed of an N-terminal intracellular domain, a single-pass transmembrane (TM) domain and an ectodomain comprising three subdomains: a low-density lipoprotein receptor type A (LDLRA) domain, a Class A Scavenger Receptor Cysteine-Rich (SRCR) domain and a C-terminal trypsin-like Serine protease (SP) domain with a canonical H296-D345-S441 catalytic triad (Figure 3A) (17, 18). TMPRSS2 is synthesized as a single chain zymogen (∼65kD) and is intracellularly autoactivated at R 292 -I 293 junction to generate cleaved protease fragment (∼31kD) and two additional lighter bands of (∼45 to 48 kDa), possibly generated upon cleavages between the LDLRA and the SRCR domains (19).…”

“…To investigate if domains other than SP domain might support HCoV-HKU1 spike-mediated entry, we engineered stop codons in TMPRSS2-Flag-N plasmid within nucleotide sequences between LDLRA and SRCR (Y198 aa position), or SRCR and SP domains (R 292 I 293 aa position) to allow expression of N-terminally Flag-tagged LDLRA or LDLRA-SRCR domains respectively (Figure 3A). To investigate if HCoV-HKU1 spike-mediated entry is independent of TMPRSS2 SP activity, we introduced single amino acid substitutions H296A and S441A in the catalytic triad of SP domain to abolish catalytic activity (Figure 3A) (19, 20). Structural basis of TMPRSS2 catalysis involves formation of an oxyanion hole by the backbone amine groups of G439 and S441 in the GDSGG motif to help activate and stabilize the carbonyl of the scissile bond (21).…”

“…Plasmids encoding LDLRA and LDLRA-SRCR domains, and TMPRSS2 proteins defective for SP catalytic activity (H296A, S441A and GS-AA) were transfected into 293T cells and confirmed for protein expression by Western Blotting (Figure 3B). In transfected 293T cells, full-length TMPRSS2 is expressed as ∼54kDa band which is autoactivated possibly at the R 292 -I 293 junction to generate an N-terminal cleavage product of ∼24kDa and a cleaved protease fragment (∼31kDa) (17, 19). Since we probed for TMPRSS2 using an antibody directed against Flag tag at the N-terminus, full-length TMPRSS2 and several cleavage products were detected.…”

SARS-CoV2 infection triggers reactive astrocyte states and inflammatory conditions in long-term Human Cortical Organoids

“…Next, we analyzed the level of expression of known SARS-CoV2 receptors and co-receptors in the hCOs. RNA sequencing of late stage (6M) hCOs confirmed low expression of the major SARS-CoV2 receptor, ACE2 2,16,17 , and higher expression of some of the SARS-CoV2 coreceptors such as NRP1, CD147/BSG; GRP78/HSPA5; DPP4 and AXL 7,16,[18][19][20][21][22][23][24][25][26] as well as some of the enzymes involved in the cleavage of the SARS-CoV2 virus spike protein to mediate viral entry to the cell, such as transmembrane serine protease 2 (TMPRSS2), cathepsin B (CTSB), cathepsin L (CTSL) and furin 14,23 (Fig. 1M).…”

“…SARS-CoV2 viral entry into target cells has been proposed to be mediated by the biding of the spike S protein to the receptor angiotensin converting enzyme 2 (ACE2). This event is followed by the activation of the spike S protein by a cleavage enacted by the host proprotein convertase furin and by membrane-associated serine proteinases (MASPs) 14,15 , which leads to the fusion of the virus membrane with the host membrane 2,16 . Whereas high levels of ACE2 expression have been detected in major target tissues of SARS-CoV2 infection such as lung, kidney and heart, ACE2 levels are reported to be low or undetectable in the brain 17 .…”