Coronavirus disease 2019 (COVID-19) is caused by severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2). The initial interaction between Transmembrane Serine Protease 2 (TMPRSS2) primed SARS-CoV-2 spike (S) protein and host cell receptor angiotensin-converting enzyme 2 (ACE-2) is a pre-requisite step for this novel coronavirus pathogenesis. Here, we expressed a GFP-tagged SARS-CoV-2 S-Ectodomain in Tni insect cells. That contained sialic acid-enriched N- and O-glycans. Surface resonance plasmon (SPR) and Luminex assay showed that the purified S-Ectodomain binding to human ACE-2 and immunoreactivity with COVID-19 positive samples. We demonstrate that bromelain (isolated from pineapple stem and used as a dietary supplement) treatment diminishes the expression of ACE-2 and TMPRSS2 in VeroE6 cells and dramatically lowers the expression of S-Ectodomain. Importantly, bromelain treatment reduced the interaction between S-Ectodomain and VeroE6 cells. Most importantly, bromelain treatment significantly diminished the SARS-CoV-2 infection in VeroE6 cells. Altogether, our results suggest that bromelain or bromelain rich pineapple stem may be used as an antiviral against COVID-19.
Bromelain cleaves SARS-CoV-2 Spike protein
Studies have shown that the SARS-CoV-2 spike protein has a 10 to 20-fold higher binding affinity with ACE-2 as compared to SARS-CoV-1 spike protein for viral pathogenesis (28). Hence, we cloned and expressed SARS-CoV-2 spike protein ectodomain that contains insect cell secretion signal (SP), NTD, RBD, FP, HR1, CH, and CD domains (1 −1220 amino acids) along with C-terminal GFP and 12X His tag and a mutated furin cleavage site (Figure 2A and Figure S1). The calculated molecular weight of the purified S-Ectodomain-GFP protein is ~165 kDa; however, we observed a higher molecular weight of S-Ectodomain (~215 kDa), which may be due to heavy N- and O-linked glycosylation (Figure 2B intent). Next, we determined the interaction between the purified S-Ectodomain and human recombinant ACE-2 by using surface plasmon resonance (SPR) technology. The SPR analysis revealed that S-Ectodomain binds with the receptor ACE-2. Further, the binding increased in a concentration-dependent manner and has a comparable binding affinity as control RBD binding to ACE-2 (Figure 2B). We also validated whether the expressed S-Ectodomain can be recognized by antibodies from COVID-19 patient samples; for this, we performed a Luminex serological assay with de-identified COVID-19 positive (n=6) and COVID-19 negative (n=6) UNMC patient samples. Luminex assay results showed a significantly increased median fluorescent intensity (MFI) of S-Ectodomain with COVID-19 positive patients’ samples (P<0.0001) (Figure 2C). These results indicate that the purified S-Ectodomain is correctly folded and active. Since S-Ectodomain has 22 N-glycosylation and 2 O-glycosylation sites (12–15), we explored the type of glycosylation on the purified S-Ectodomain. Figure 2D shows the schematic representation of the N- and O-linked glycosylation sites in S-protein (12, 13). Treatment of S-Ectodomain with N-glycanase (PNGase F) showed increased mobility shift and destabilization of the protein, which may be due to the loss of heavy N-glycans (Figure 2E). However, the treatment of S-Ectodomain with O-glycanase (specific for core 1 O-glycan) showed a slightly decreased protein mobility shift, which supports the previous findings that S-protein has only two O-linked core 1 derived glycans in its RBD backbone (Figure 2E). Conversely, treatment of S-Ectodomain with Sialidase A (which removes sialic acid group) either alone or in combination with either N-glycanase or O-glycanase or together induced a noticeable change in the migration of S-Ectodomain. This decreased mobility shift of S-Ectodomain may be due to the loss of negatively charged sialic acid groups in the N- and O-linked glycans (Figure 2E). These results indicate that the SARS-CoV-2 Spike protein has both highly sialylated N- and O-linked glycans.