Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is extensively N-glycosylated, and unlike the receptor-binding domain of the S1 subunit which undergoes frequent mutations, the glycosylation sites remain conserved across most variants of concern. In this study, we cloned and purified EndoE, an endo-β-N-acetylglucosaminidase enzyme from an Enterococcus faecalis strain isolated in our laboratory (E8 strain). The purified EndoE effectively removed glycans from the S1 protein of SARS-CoV-2 spike. We constructed a catalytically inactive mutant form of EndoE, termed EndoE (Mut). Both wild-type EndoE and the EndoE (Mut) demonstrated neutralizing activity against SARS-CoV-2 S pseudotyped virus infection, with IC50 values of 81.26 ± 8.42 nM and 63.15 ± 5.06 nM, respectively. Enzyme-linked immunosorbent assay revealed that both forms of EndoE bound to the S1 protein. Moreover, commercial EndoH enzyme, which also cleaves N-glycosylation, did not exhibit neutralizing activity against SARS-CoV-2 S pseudotyped virus at any tested concentration. In contrast, the plant lectin Concanavalin A demonstrated the most potent neutralization ability, with an IC50 of 40.89 ± 24.04 nM. Importantly, neither form of EndoE displayed toxicity even at the highest tested concentration (6,250 nM), whereas Concanavalin A exhibited toxicity to cells at a concentration as low as 157 nM. These findings shed light on the role of glycosidases in SARS-CoV-2 infection and offer a novel avenue for the development of antiviral strategies.

This work is part of the Grant PID2020-115403RB (C21 and C22) funded by the Spanish Ministry of Science and Innovation (MICIN)/Spanish State Research Agency (AEI)/10.13039/501100011033. The study was also supported by Valencian Government grant AICO/2021/033. RG acknowledged funding from grants SGL2021-03-009 and SGL2021-03-052 from European Union NextGenerationEU/PRTR through the CSIC Global Health Platform established by EU Council Regulation 2020/2094. EMM-G was supported by the Grant PRE2018-085768 funded by MICIN/AEI/10.13039/501100011033 and by “ESF Investing in your future”. SL-N was supported by the FPU22/00443 grant from the Spanish Ministry of Science and Innovation (MICIN). IATA-CSIC is a Centre of Excellence Severo Ochoa (CEX2021-001189-S MCIN/AEI/10.13039/501100011033).

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