Glycosylated extracellular mucin domains protect against SARS-CoV-2 infection at the respiratory surface

Author summarySARS-CoV-2, the virus that has caused the devastating COVID-19 pandemic, causes a range of symptoms in infected individuals, from mild respiratory illness to acute respiratory distress syndrome. A fundamental understanding of host factors influencing viral entry is critical to elucidate SARS-CoV-2-host interactions and identify novel therapeutic targets. In this study, we investigated the role of host mucins and mucin glycans on SARS-CoV-2 entry into the airway epithelial cells. Mucins are a family of high molecular weight O-glycosylated proteins that play an essential role in protecting the respiratory tract against viral and bacterial infections. The gel-forming mucins MUC5AC and MUC5B clear pathogens by mucociliary clearance while transmembrane mucins MUC1, MUC4, and MUC16 can restrict or facilitate microbial invasion at the apical surface of the epithelium. The mucin-selective protease StcE specifically cleaves the glycosylated extracellular part of the mucins without perturbing the underlying domains. We show that removal of mucins from the surface of Calu-3 cells with StcE mucinase increases binding of the SARS-CoV-2 spike protein to the respiratory surface and greatly enhances infection. Enhanced viral replication was also significantly increased in primary airway epithelial cultures treated with StcE mucinase. This study demonstrates the important role of glycosylated extracellular mucin domains as a host defense mechanism during SARS-CoV-2 entry. Future efforts should be focused on characterizing the expression and role of specific soluble and transmembrane mucins in different cell types during the different stages of SARS-CoV-2 infection. Mucins play an essential role in protecting the respiratory tract against microbial infections while also acting as binding sites for bacterial and viral adhesins. The heavily O-glycosylated gel-forming mucins MUC5AC and MUC5B eliminate pathogens by mucociliary clearance. Transmembrane mucins MUC1, MUC4, and MUC16 can restrict microbial invasion at the apical surface of the epithelium. In this study, we determined the impact of host mucins and mucin glycans on epithelial entry of SARS-CoV-2. Human lung epithelial Calu-3 cells express the SARS-CoV-2 entry receptor ACE2 and high levels of glycosylated MUC1, but not MUC4 and MUC16, on their cell surface. The O-glycan-specific mucinase StcE specifically removed the glycosylated part of the MUC1 extracellular domain while leaving the underlying SEA domain and cytoplasmic tail intact. StcE treatment of Calu-3 cells significantly enhanced infection with SARS-CoV-2 pseudovirus and authentic virus, while removal of sialic acid and fucose from the epithelial surface did not impact viral entry. In Calu-3 cells, the transmembrane mucin MUC1 and ACE2 are located to the apical surface in close proximity and StcE treatment results in enhanced binding of purified spike protein. Both MUC1 and MUC16 are expressed on the surface of human organoid-derived air-liquid interface (ALI) differentiated airway cultures and StcE treatment led to mucin removal and increased levels of SARS-CoV-2 replication. In these cultures, MUC1 was highly expressed in non-ciliated cells while MUC16 was enriched in goblet cells. In conclusion, the glycosylated extracellular domains of different transmembrane mucins might have similar protective functions in different respiratory cell types by restricting SARS-CoV-2 binding and entry.