The Effect of Glycosylation Modulators on the Trafficking and Interaction of Spike Protein S1 Subunit and Angiotensin-Converting Enzyme 2.

The betacoronavirus severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the cause of the ongoing worldwide pandemic. The spike (S) glycoprotein of SARS-CoV-2 is a heavily glycosylated trimer that binds to host cell receptor angiotensin-converting enzyme 2 (ACE2), initiating viral entry. Evidence also shows that the S glycoprotein interacts with other receptors including dipeptidyl peptidase IV (DPPIV). Glycosylation is a post-translational modification important for proper folding and trafficking of membrane and secretory proteins. It plays a critical role not only for host cell proteins, but also in viral virulence, immunity evasion and receptor binding capabilities. This study focused on the modulation of glycosylation and its effects on the trafficking of the S1 subunit, ACE2 and DPPIV, in addition to its effect on their interactions. For this purpose, the S1 subunit was expressed in COS-1 cells in the presence or absence of N-butyldeoxynojirimycin (NB-DNJ), an inhibitor of the ER-located a-glucosidases I and II, 1-deoxymannojirimycin (dMM), an inhibitor of Golgi-mannosidase I, or benzyl-N-acetyl-α-galactosaminide (benzyl-GalNac), an inhibitor of O-glycosylation. The data demonstrate that NB-DNJ affects substantially the trafficking and secretion of S1 protein, while its overall synthetic levels remained unchanged. A similar effect was observed upon treatment of the cells with the benzyl-GalNac. On the contrary, the presence of dMM in the culture medium did not affect the secretion of S1. Together, the data suggest that folding events implicating calnexin in the ER, the site of action of NB-DNJ, as well as O-glycans that are modulated by benzyl-GalNac are crucial for the secretion of the S1 protein. We further investigated the trafficking of ACE2 and DPPIV in lung Calu-3 and intestinal Caco-2 cells in the presence or absence of the glycosylation modulators and their interaction with S1. The data demonstrate a reduced binding capacity of S1 to ACE2 in the presence of NB-DNJ and dMM, while this interaction was enhanced in Calu-3 cells upon inhibition of O-glycosylation by benzyl-GalNac. We conclude that glycosylation modulators differentially act on the secretory pathway of S1 and the receptors. By virtue of its negative impact on both the secretion of S1 as well as interaction with its receptor ACE2, NB-DNJ may be considered as a potential therapeutic drug that may act at the level of viral entry and exit.