Degradation of Spike Glycoprotein of SARS CoV-2 Virus by Hydrolyzed Collagen trypsin

Our proprietary ingredient, Hydrolyzed Collagen Trypsin (HCT), is a protein and proteolytic enzyme derived from marine collagen. It is extracted from Crustacean sources through a proprietary extraction method to yield hydrolyzed collagen that contains trypsin. HCT has various benefits that could be used in biomedical applications, supplements, industrial use, and cosmetic applications. The only product that is formulated with HCT is ProFX Mouth Spray, which is intended to be used to protect the oral mucosa and aid in wound healing while moisturizing and hydrating oral mucosa.

In the light of SARS CoV-2 Virus and the pandemic, there is a need in understanding the infection of the virus and ways to prevent and/or treat them. The attachment of spike glycoprotein (S-protein) of SARS-CoV-2 to angiotensin-converting enzyme 2 (ACE2) receptor of the host cell is a critical step in the infection route of this virus. Blocking this attachment is therefore considered a promising strategy to prevent the virus replication inside the host cells. In this respect, the degradation of S-protein by proteases should prevent the attachment, which puts the proteases as a promising compound for combating COVID-19. Reports by Sagar et al. (2020) convincingly showed that the degradation of S-protein by bromelain protease prevented the infection of SARS-CoV-2 towards the VeroE6 cells line. This also was supported by Akhter et al. (2020), who reported that bromelain protease disintegrates the spike and envelope protein from SARS-CoV-2 and may render it non-infective.

Hence, with recent research on proteolytic activity on SARS CoV-2 spike glycoprotein, this research was conducted to identify the degradation of spike protein by hydrolyzed collagen trypsin, the proprietary active ingredient. The result of this research prompted future product research and development.

Methodology:

In this research, 5 μl (concentration = 9.86 mg/mL) of HCT solution was added into 10 μl of S1 and S2 subunit (final concentration was 0.5 mg/ml each) at 37 ̊C. Initial assay of 0 hour of incubation was run on SDS PAGE and then it was incubated for 2 hours. The reaction was stopped by adding 2X SDS-Sample buffer and heated to 100 ̊C for 10 minutes and the mixture is loaded onto SDS-PAGE. The gel was stained with Coomassie Blue solution for 30 minutes followed by acetic acid staining for another 30 minutes. The protein fragments can be seen on the band visualized on Bio-rad Gel Doc.

1. Degradation activity of S1 and S2 subunits by hydrolyzed collagen trypsin.

Figure 1 SDS page of S1 and S2 subunits in presence of hydrolyzed collagen trypsin

Figure 1 showed that the thickness of the band corresponding to S1 and S2 subunits was almost completely degraded in 0 hour and 2 hour by HCT at 37 ̊C. It can be assumed that degradation of spike protein may have initiated on contact time, resulting in instant degradation of spike proteins. It is important to note that the concentration of hydrolyzed collagen trypsin here is approximately 1,500-fold higher than standard PRO-FX. 

2. The cleaving site of S1 and S2 subunits of SARS CoV-2 Virus by trypsin enzyme

Figure 2 Cleavage position of trypsin in S1 and S2 subunit.

Computation analysis, as shown in Figure 2, indicated that trypsin indeed cleaved S1 and S2 subunits. The cleaving position at S1 and S2 subunits were 16 and 11, respectively. This confirmed the SDS-Page result which showed that both subunits were degraded by trypsin and PRO-FX. This result also justified the degradation rate of S1 subunits are higher than S2 subunits. Result of degradation of S1 and S2 subunits by ProFX mouth spray can be found here.

Limitations:

  • The exact duration taken for S1 and S2 subunits to be degraded were not taken due to the nature of the methodology used. Hence, It can only be assumed that 0 hour is equivalent to contact time and has almost completely degradation of spike proteins.
  • Due to limited lab equipment in the biotech lab, spectrophotometers were not used in this test for spectrophotometric enzyme assay. Instead, SDS-PAGE was used to identify the protein degradation fragments. This would result in limited data and result due to the methodology used.