Abstract MP10: Furin-Mediated Modification Is Required For Epithelial Sodium Channel-Activating Activity Of Soluble (Pro)Renin Receptor In Cultured Collecting Duct Cells

(Pro)renin receptor (PRR) contains overlapping cleavage site for site-1 protease (S1P) and furin for generation of soluble PRR (sPRR). Although S1P-mediated cleavage mediates the release of sPRR, the functional implication of furin-mediated cleavage is unclear. Here we tested whether furin-mediated cleavage was required for the activity of sPRR in activating ENaC in cultured M1 cells. M1 cells were transfected with pcDNA3.4 containing full-length PRR with (Furin Mut) or without (WT) mutagenesis of furin cleavage site; empty vector (EM) served a control. As compared with EM, overexpression of WT and Furin Mut vectors induced a more than 16-fold comparable increase in the release of sPRR. Amiloride-sensitive short circuit current as assessed by Ussing chamber technique was elevated by overexpression of WT PRR which was reduced by 37% by Furin Mut (ENaC activity: 1.00 + 0.06 μA/cm 2 in EM, 1.67 + 0.05 μA/cm 2 in WT, and 1.04 + 0.07 μA/cm 2 in Furin Mut, p < 0.05). In parallel, the expression of α-ENaC but not β or γ subunit as assessed by immunoblotting and qRT-PCR analysis was elevated by WT PRR and this increase was blunted by Furin Mut. In a separate experiment, M1 cells were transfected with pcDNA3.4 containing cDNA for sPRR with S1P cleavage (AA 1-282) (sPRR-S1P) or with furin cleavage (AA 1-279) (sPRR-furin); empty vector was used as a control. Overexpression of cDNA for the two types of sPRR induced a significant and comparable increase in the release of sPRR. By Ussing chamber technique, ENaC activity was 1.00 + 0.09 μA/cm 2 in EM, 1.03 + 0.10 μA/cm 2 in sPRR-S1P, and 1.39 + 0.14 μA/cm 2 in sPRR-furin, p < 0.05. Lastly, 293 cells were pretreated for 1 h with furin inhibitor α1-antitrypsin Portland followed by transfection with empty vector, WT PRR, or Furin Mut vectors. sPRR in the condition medium was enriched by using protein centrifugal filter devices and applied to M1 cells for 10 min followed by measurement of ENaC activity. Pretreatment with furin inhibition attenuated ENaC-acting activity induced by overexpression of WT PRR. Overall, the three independent approaches consistently demonstrated that furin-mediated modification is required for the activity of sPRR to increase ENaC-mediated Na + transport in the CD cells.

Download Full-text

Furin cleavage of the SARS-CoV-2 spike is modulated by O-glycosylation

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.2109905118 ◽

2021 ◽

Vol 118 (47) ◽

pp. e2109905118

Author(s):

Liping Zhang ◽

Matthew Mann ◽

Zulfeqhar A. Syed ◽

Hayley M. Reynolds ◽

E. Tian ◽

...

Keyword(s):

Cleavage Site ◽

Protein S ◽

Viral Infectivity ◽

The Novel ◽

Furin Cleavage ◽

Global Pandemic ◽

Furin Cleavage Site ◽

Respiratory Cells ◽

Syncytia Formation

The SARS-CoV-2 coronavirus responsible for the global pandemic contains a novel furin cleavage site in the spike protein (S) that increases viral infectivity and syncytia formation in cells. Here, we show that O-glycosylation near the furin cleavage site is mediated by members of the GALNT enzyme family, resulting in decreased furin cleavage and decreased syncytia formation. Moreover, we show that O-glycosylation is dependent on the novel proline at position 681 (P681). Mutations of P681 seen in the highly transmissible alpha and delta variants abrogate O-glycosylation, increase furin cleavage, and increase syncytia formation. Finally, we show that GALNT family members capable of glycosylating S are expressed in human respiratory cells that are targets for SARS-CoV-2 infection. Our results suggest that host O-glycosylation may influence viral infectivity/tropism by modulating furin cleavage of S and provide mechanistic insight into the role of the P681 mutations found in the highly transmissible alpha and delta variants.

Download Full-text

SARS-CoV-2 Origin: An Affair of Codons?

10.20944/preprints202106.0121.v1 ◽

2021 ◽

Author(s):

Antonio Ramón Romeu ◽

Enric Ollé

Keyword(s):

Human Genome ◽

Genetic Markers ◽

Cleavage Site ◽

Cleavage Sites ◽

Furin Cleavage ◽

Large Sample ◽

The Core ◽

Furin Cleavage Site

The furin cleavage site, with an arginine doublet (RR), is one of the clues of the SARS-CoV-2 origin. This furin-RR is encoded by the CGG-CGG sequence. Because arginine can be encoded by six codons, in a previous work we found that in SARS-CoV-2, CGG was the minority arginine codon (3%). Also, analyzing the RR doublet from a large sample of furin cleavage sites of several kinds of viruses, we found that none of them were encoded by CGG-CGG. Here, we come back to the core of the matter, but from the perspective that in the human genome, in contrast, CGG is the majoroty arginine codon (21%). Here, we highlighted that the 6 arginine codons provide genetic markers to a traceability on the RR origin in the furin site, as well as, to weigh the probability of the theories about the origin of the virus.

Download Full-text

Furin-Mediated Cleavage of the Feline Foamy Virus Env Leader Protein

Journal of Virology ◽

10.1128/jvi.78.24.13573-13581.2004 ◽

2004 ◽

Vol 78 (24) ◽

pp. 13573-13581 ◽

Cited By ~ 25

Author(s):

Verena Geiselhart ◽

Patrizia Bastone ◽

Tore Kempf ◽

Martina Schnölzer ◽

Martin Löchelt

Keyword(s):

Cleavage Site ◽

Transmembrane Protein ◽

Foamy Virus ◽

Proteolytic Processing ◽

Signal Peptidase ◽

Amino Acid Residues ◽

Furin Cleavage ◽

Furin Cleavage Site ◽

Leader Protein ◽

Distinguishing Features

ABSTRACT The molecular biology of spuma or foamy retroviruses is different from that of the other members of the Retroviridae. Among the distinguishing features, the N-terminal domain of the foamy virus Env glycoprotein, the 16-kDa Env leader protein Elp, is a component of released, infectious virions and is required for particle budding. The transmembrane protein Elp specifically interacts with N-terminal Gag sequences during morphogenesis. In this study, we investigate the mechanism of Elp release from the Env precursor protein. By a combination of genetic, biochemical, and biophysical methods, we show that the feline foamy virus (FFV) Elp is released by a cellular furin-like protease, most likely furin itself, generating an Elp protein consisting of 127 amino acid residues. The cleavage site fully conforms to the rules for an optimal furin site. Proteolytic processing at the furin cleavage site is required for full infectivity of FFV. However, utilization of other furin proteases and/or cleavage at a suboptimal signal peptidase cleavage site can partially rescue virus viability. In addition, we show that FFV Elp carries an N-linked oligosaccharide that is not conserved among the known foamy viruses.

Download Full-text

Abstract 095: Mutation in the Furin Cleavage Site of the Prorenin Receptor Attenuates Angiotensin-II Induced Hypertension and Albuminuria

Hypertension ◽

10.1161/hyp.74.suppl_1.095 ◽

2019 ◽

Vol 74 (Suppl_1) ◽

Author(s):

Nirupama Ramkumar ◽

Deborah Stuart ◽

Will Wheatley ◽

Donald E Kohan

Keyword(s):

Angiotensin Ii ◽

Cleavage Site ◽

Furin Cleavage ◽

Furin Cleavage Site ◽

Prorenin Receptor ◽

Induced Hypertension

Download Full-text

Site-1 Protease-Derived Soluble (Pro)Renin Receptor Contributes to Angiotensin II–Induced Hypertension in Mice

Hypertension ◽

10.1161/hypertensionaha.120.15100 ◽

2020 ◽

Author(s):

Ye Feng ◽

Kexin Peng ◽

Renfei Luo ◽

Fei Wang ◽

Tianxin Yang

Keyword(s):

Blood Pressure ◽

Angiotensin Ii ◽

Collecting Duct ◽

Short Circuit ◽

Ussing Chamber ◽

Short Circuit Current ◽

Duct Cell ◽

Ang Ii ◽

Cortical Collecting Duct ◽

Induced Hypertension

Activation of PRR ([pro]renin receptor) contributes to enhancement of intrarenal RAS and renal medullary α-ENaC and thus elevated blood pressure during Ang II (angiotensin II) infusion. The goal of the present study was to test whether such action of PRR was mediated by sPRR (soluble PRR), generated by S1P (site-1 protease), a newly identified PRR cleavage protease. F1 B6129SF1/J mice were infused for 6 days with control or Ang II at 300 ng/kg per day alone or in combination with S1P inhibitor PF-429242 (PF), and blood pressure was monitored by radiotelemetry. S1P inhibition significantly attenuated Ang II–induced hypertension accompanied with suppressed urinary and renal medullary renin levels and expression of renal medullary but not renal cortical α-ENaC expression. The effects of S1P inhibition were all reversed by supplement with histidine-tagged sPRR termed as sPRR-His. Ussing chamber technique was performed to determine amiloride-sensitive short-circuit current, an index of ENaC activity in confluent mouse cortical collecting duct cell line cells exposed for 24 hours to Ang II, Ang II + PF, or Ang II + PF + sPRR-His. Ang II–induced ENaC activity was blocked by PF, which was reversed by sPRR-His. Together, these results support that S1P-derived sPRR mediates Ang II–induced hypertension through enhancement of intrarenal renin level and activation of ENaC.

Download Full-text

TMPRSS4-dependent activation of the epithelial sodium channel requires cleavage of the γ-subunit distal to the furin cleavage site

AJP Renal Physiology ◽

10.1152/ajprenal.00330.2011 ◽

2012 ◽

Vol 302 (1) ◽

pp. F1-F8 ◽

Cited By ~ 30

Author(s):

Christopher J. Passero ◽

Gunhild M. Mueller ◽

Michael M. Myerburg ◽

Marcelo D. Carattino ◽

Rebecca P. Hughey ◽

...

Keyword(s):

Sodium Channel ◽

Cleavage Site ◽

Epithelial Sodium Channel ◽

Cleavage Sites ◽

Furin Cleavage ◽

Channel Activation ◽

Α Subunit ◽

Γ Subunit ◽

Furin Cleavage Site ◽

Unique Mechanism

The epithelial sodium channel (ENaC) is activated by a unique mechanism, whereby inhibitory tracts are released by proteolytic cleavage within the extracellular loops of two of its three homologous subunits. While cleavage by furin within the biosynthetic pathway releases one inhibitory tract from the α-subunit and moderately activates the channel, full activation through release of a second inhibitory tract from the γ-subunit requires cleavage once by furin and then at a distal site by a second protease, such as prostasin, plasmin, or elastase. We now report that coexpression of mouse transmembrane protease serine 4 (TMPRSS4) with mouse ENaC in Xenopus oocytes was associated with a two- to threefold increase in channel activity and production of a unique ∼70-kDa carboxyl-terminal fragment of the γ-subunit, similar to the ∼70-kDa γ-subunit fragment that we previously observed with prostasin-dependent channel activation. TMPRSS4-dependent channel activation and production of the ∼70-kDa fragment were partially blocked by mutation of the prostasin-dependent cleavage site (γRKRK186QQQQ). Complete inhibition of TMPRSS4-dependent activation of ENaC and γ-subunit cleavage was observed when three basic residues between the furin and prostasin cleavage sites were mutated (γK173Q, γK175Q, and γR177Q), in addition to γRKRK186QQQQ. Mutation of the four basic residues associated with the furin cleavage site (γRKRR143QQQQ) also prevented TMPRSS4-dependent channel activation. We conclude that TMPRSS4 primarily activates ENaC by cleaving basic residues within the tract γK173-K186 distal to the furin cleavage site, thereby releasing a previously defined key inhibitory tract encompassing γR158-F168 from the γ-subunit.

Download Full-text

Regulation of CFTR Expression and Arginine Vasopressin Activity Are Dependent on Polycystin-1 in Kidney-Derived Cells

Cellular Physiology and Biochemistry ◽

10.1159/000438606 ◽

2016 ◽

Vol 38 (1) ◽

pp. 28-39 ◽

Cited By ~ 4

Author(s):

Carolina Monteiro de Lemos Barbosa ◽

Jackson Souza-Menezes ◽

Andressa Godoy Amaral ◽

Luiz Fernando Onuchic ◽

Liudmila Cebotaru ◽

...

Keyword(s):

Arginine Vasopressin ◽

Chloride Channel ◽

Collecting Duct ◽

Short Circuit ◽

Ussing Chamber ◽

Renal Cysts ◽

Fluid Secretion ◽

Intracellular Camp ◽

Mrna Levels ◽

Cortical Collecting Duct

Background: Autosomal dominant polycystic kidney disease (ADPKD) is characterized by the development of multiple, progressive, fluid-filled renal cysts that distort the renal parenchyma, leading to end-stage renal failure, mainly after the fifth decade of life. ADPKD is caused by a mutation in the PKD1 or PKD2 genes that encode polycystin-1 (PC-1) and polycystin-2 (PC-2), respectively. PC-1 is an important regulator of several signaling pathways and PC-2 is a nonselective calcium channel. The CFTR chloride channel is responsible for driving net fluid secretion into the cysts, promoting cyst growth. Arginine vasopressin hormone (AVP), in turn, is capable of increasing cystic intracellular cAMP, contributing to cell proliferation, transepithelial fluid secretion, and therefore to disease progression. The aim of this study was to assess if AVP can modulate CFTR and whether PC-1 plays a role in this potential modulation. Methods: M1 cells, derived from mouse cortical collecting duct, were used in the current work. The cells were treated with 10-7 M AVP hormone and divided into two main groups: transfected cells superexpressing PC-1 (Transf) and cells not transfected (Ctrl). CFTR expression was assessed by immunodetection, CFTR mRNA levels were quantified by quantitative reverse transcription-polymerase chain reaction, and CFTR net ion transport was measured using the Ussing chamber technique. Results: AVP treatment increased the levels of CFTR protein and mRNA. CFTR short-circuit currents were also increased. However, when PC-1 was overexpressed in M1 cells, no increase in any of these parameters was detected. Conclusions: CFTR chloride channel expression is increased by AVP in M1 cells and PC-1 is capable of regulating this modulation.

Download Full-text

Spike glycoprotein and host cell determinants of SARS-CoV-2 entry and cytopathic effects

Journal of Virology ◽

10.1128/jvi.02304-20 ◽

2020 ◽

Cited By ~ 1

Author(s):

Hanh T. Nguyen ◽

Shijian Zhang ◽

Qian Wang ◽

Saumya Anang ◽

Jia Wang ◽

...

Keyword(s):

Host Cell ◽

Cell Fusion ◽

Cleavage Site ◽

Virus Entry ◽

Cytoplasmic Tail ◽

Syncytium Formation ◽

Spike Glycoprotein ◽

Furin Cleavage ◽

Cytopathic Effects ◽

Furin Cleavage Site

SARS-CoV-2, a betacoronavirus, is the cause of the COVID-19 pandemic. The SARS-CoV-2 spike (S) glycoprotein trimer mediates virus entry into host cells and cytopathic effects (syncytium formation). We studied the contribution of several S glycoprotein features to these functions, focusing on those that differ among related coronaviruses. Acquisition of the furin cleavage site by the SARS-CoV-2 S glycoprotein decreased virus stability and infectivity, but greatly enhanced syncytium-forming ability. Notably, the D614G change found in globally predominant SARS-CoV-2 strains increased infectivity, modestly enhanced responsiveness to the ACE2 receptor and susceptibility to neutralizing sera, and tightened association of the S1 subunit with the trimer. Apparently, these two features of the SARS-CoV-2 S glycoprotein, the furin cleavage site and D614G, have evolved to balance virus infectivity, stability, cytopathicity and antibody vulnerability. Although the endodomain (cytoplasmic tail) of the S2 subunit was not absolutely required for virus entry or syncytium formation, alteration of palmitoylated cysteine residues in the cytoplasmic tail decreased the efficiency of these processes. As proteolytic cleavage contributes to the activation of the SARS-CoV-2 S glycoprotein, we evaluated the ability of protease inhibitors to suppress S glycoprotein function. Matrix metalloprotease inhibitors suppressed S-mediated cell-cell fusion, but not virus entry. Synergy between inhibitors of matrix metalloproteases and TMPRSS2 suggests that both host proteases can activate the S glycoprotein during the process of syncytium formation. These results provide insights into SARS-CoV-2 S glycoprotein-host cell interactions that likely contribute to the transmission and pathogenicity of this pandemic agent. IMPORTANCE The development of an effective and durable SARS-CoV-2 vaccine is essential for combating the growing COVID-19 pandemic. The SARS-CoV-2 spike (S) glycoprotein is the main target of neutralizing antibodies elicited during virus infection or following vaccination. Knowledge of the spike glycoprotein evolution, function and interactions with host factors will help researchers to develop effective vaccine immunogens and treatments. Here we identify key features of the spike glycoprotein, including the furin cleavage site and the D614G natural mutation, that modulate viral cytopathic effects, infectivity and sensitivity to inhibition. We also identify two inhibitors of host metalloproteases that block S-mediated cell-cell fusion, a process that contributes to the destruction of the virus-infected cell.

Download Full-text