Neutrophil Elastase and Proteinase 3 Cleavage Sites Are Adjacent to the Polybasic Sequence within the Proteolytic Sensitive Activation Loop of the SARS-CoV-2 Spike Protein

Abstract

Serine proteases neutrophil elastase (NE), protease 3 (PR3), cathepsin G (CatG), and neutrophil serine protease 4 (NSP4) are released by activated neutrophils swarming around the place of pathogen invasion to provoke an immune response. However, uncontrolled proteolytic activity of proteases results in various human diseases, including cardiovascular diseases, thrombosis, and autoimmunity. In addition, proteases can be hijacked by several viruses to prime virus-derived surface proteins and evade immune detection by entering into the host cell. Indeed, porcine elastase increases the suitability of host cells to be infected by SARS-CoV-1. We compared the cleavage sites of human NE, PR3, and CatG as well as porcine-derived trypsin within the amino acid sequence of the proteolytic sensitive activation loop at the interface of S1/S2 of the spike protein (S protein) of SARS-CoV-1 as well as SARS-CoV-2. As a result, NE and PR3, but not CatG, hydrolyze the scissile peptide bond adjacent to the polybasic amino acid sequence of the S1/S2 interface of SARS-CoV-2, which is distinctive from SARS-CoV-1. These findings suggest that neutrophil-derived NE and PR3 participate in priming of the S1/S2 interface during an immune response.

Increased elastase sensitivity and decreased intramolecular interactions in the more transmissible 501Y.V1 and 501Y.V2 SARS-CoV-2 variants’ spike protein–an in silico analysis

“…As there are several approved elastase inhibitors, including Sivelestat (which is approved for acute respiratory syndrome), Alvestat (α1-antitrypsin) and Roseltide, the use of these inhibitors, perhaps as intranasal spray or drops, should be considered. Neutrophil elastase appears to cleave near the RRAR (amino acids Arg682 to Arg685) of the furin cleavage site [ 42 ]. The variant amino acids that we have discussed here are likely too far to affect the affinity of neutrophil elastase for RRAR directly, but it is possible that those hydrophobic amino acids in the variants can increase the propensity for neutrophil elastase to cleave this region of spike protein [ 23 , 24 ].…”

Hindrance of the Proteolytic Activity of Neutrophil-Derived Serine Proteases by Serine Protease Inhibitors as a Management of Cardiovascular Diseases and Chronic Inflammation

“…The imbalance between NE and anti-elastase activity of alpha-1 antitrypsin (A1AT) in the serum of patients admitted to intensive care ( Zerimech et al, 2021 ) suggests a need to develop effective antiviral drugs to manage COVID-19 outcomes. NE and PR3 cleave the S protein of SARS-CoV-2 within the polybasic sequence of the proteolytically sensitive activation loop, suggesting a role in S protein priming by NSPs ( Mustafa et al, 2021 ). An in silico analysis determined that the SARS-CoV-2 D614G substitution harbors a potential new NE cleavage site within the S protein ( Bhattacharyya et al, 2021 ) and the SARS-CoV-2 Alpha variant harbors novel cleavage sites possibly between isoleucine 716 and asparagine 717 (716IN717) and between alanine 982 and arginine 983 (982AR983), and in the case of the SARS-CoV-2 Beta variant additional NE cleavages sites were proposed, increasing the propensity for NE to digest the S protein ( Pokhrel et al, 2021 ).…”

Capturing a crucial ‘disorder-to-order transition’ at the heart of the coronavirus molecular pathology – triggered by highly persistent, interchangeable salt-bridges

“…The collective interplay of these fleeting salt-bridges triggers a 'transient dynamics' in disordered protein regions that is indispensable in retaining their flexibility [49] and is also pivotal towards imparting a critical behavior in associated disorder transitions among multiple self-similar fractal states [50]. The presence of transient salt-bridges (pers<0.1) in significant fractions (70.5% in the dynamic ensemble of RR1 CoV-2 , see Table 1) signals for relatively ordered metastable Furinbound states of the FLCS Spike which together retain enough flexibility (see Video S1, Supplementary Materials) to favor the Spike-S1/S2 proteolytic cleavage [33,35].…”

“…This then is relatively slowly [1] followed by the host Furin cleavage of the said junction (Spike-S1/S2) eventually leading to a more efficient host cell entry of SARS-CoV-2 [33] (compared to SARS-CoV and other related respiratory viruses) and its variants [1,28,30]. Apart from Furin, SARS-CoV-2 entry is also primed by a cell surface protease TMPRSS2, lysosomal cathepsins [34], and, also by proteases (NE, PR3, CatG, NSP4) released by activated neutrophils [35] swarming around the invaded pathogen to elicit an immune response. They therefore can be hijacked by virus-derived surface proteins as a mean to escape the host immune surveillance.…”

“…Naturally, the later is much slower than the earlier, further making it (FLCS Spike -Furin) the rate-limiting step in the viral host cell entry [1]. Insertion of the polybasic activation loop ( 681 PRRAR 685 ) in the SARS-CoV-2 Spike as opposed to other related respiratory viruses has been pinned to increased virulence, transmissibility, and pathogenesis [35][36][37]. This makes it critical to understand in tandem the dynamics and transition of these loops into ordered conformers upon binding to Furin (or other proteases) to pinpoint the molecular interactions in play.…”