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.

Figure 1

Antigen processing of the proteolytic sensitive activation loop. (a) SARS-CoV-1 or (b) SARS-CoV-2 peptides, covering the activation loop of the S protein, were incubated with CatG, NE, PR3, or trypsin, and the resulting digesting pattern was analyzed by HPLC and mass spectrometry. Red arrows indicate cleavage sites, and bars represent peptides detected by mass spectrometry. Asterisk, less dominant peptide; n = 3 independent experiments.

Figure 2

Summary of the proteolytic cleavage sites within the activation loop. The cleavage sites of NE, PR3, CatG, and trypsin are shown for the proteolytic sensitive activation loops of SARS-CoV-1 and SARS-CoV-2. NE, purple; PR3, blue; CatG, red; and trypsin, green. The three-dimensional (3D) structure of S protein (homotrimer) is based on SARS-CoV-1 (PDB ID: 5X5B) as well as SARS-CoV-2 (PDB ID: 7DDN), and the 3D structure was repainted by using AutoCAD software (AutoCAD 2014 for PC, Autodesk, Inc., San Rafael, CA, USA).