Fig 1.
Expression of mucins in respiratory epithelial cells.
(A) scRNA-seq analysis of ACE2 and MUC1 expression in different cell types in the respiratory mucosa. Dataset include samples from nasal cavity (N), upper, intermediate and lower respiratory tract [16]. (B) Expression of TM mucins MUC1, MUC4 and MUC16 and gel-forming mucins MUC5AC and MUC5B in ACE2 positive cells. MUC1 is the most highly expressed mucin in ACE2-positive cells. (C) Immunofluorescence confocal microscopy images showing expression of TM mucins MUC1 (214D4, green), MUC4 (8G7, green) and gel-forming mucin MUC5AC (MUC5AC, green) in permeabilized Calu-3 cells. Maximum projections and side views of Z-stacks are shown. (D) Immunofluorescence confocal microscopy without permeabilization showing expression of MUC1 on the surface of Calu-3 cells. MUC4 and MUC5AC could barely be detected suggesting intracellular localization. (E) Immunofluorescence confocal microscopy imaging for α-2,6 sialic acids (SNA, red), α-2,3 sialic acids (MALII, red) and fucose (UEAI, red) in combination with MUC1 (214D4 antibody, green) demonstrates high levels of sialic acid and fucose in Calu-3 cells. Nuclei were stained with DAPI (blue). White scale bars represent 20 μm.
Fig 2.
StcE specifically cleaves the glycosylated MUC1 ED and does not affect ACE2 expression.
(A) Immunofluorescence confocal microscopy images showing Calu-3 cells treated with StcE or E447D stained for the glycosylated part of the MUC1 extracellular domain (214D4, green) and α-2,6-linked sialic acids (SNA, red). Complete loss of 214D4 signal was observed after treatment with StcE. (B,C) Immunofluorescence confocal microscopy images of Calu-3 cells as above stained for the MUC1 SEA domain (α-MUC1-SEA antibody 232A1, green) or cytoplasmic tail of MUC1 (α-MUC1-CT antibody CT2, green) in combination with α-2,6-linked sialic acids (SNA, red). The SEA domain and CT were not affected by StcE treatment. Nuclei were stained with DAPI (blue). White scale bars represent 20 μm. Western blot and dotblot analysis of 7-day grown Calu-3 cells incubated with indicated enzymes for 3 h at 37°C stained with α-MUC1-ED antibody 214D4 (D), β-actin loading control (E), the MUC1 cytoplasmic tail with α-MUC1 CT antibody CT2 (F), and ACE2 (G). StcE treatment removes the MUC1 ED but does not affect the MUC1 CT or ACE2 receptor. (H) Immunofluorescence confocal microscopy images showing Calu-3 cells treated with StcE stained for the glycosylated part of the MUC1 extracellular domain (214D4, green) and fluorescently labelled mucin binding domain derived from StcE (X409-GFP) (X409, red). More continuous surface staining for MUC1 ED and limited punctate staining with X409 on the non-permeabilized cells Calu-3 cells. The MUC1 signal was completely removed after StcE treatment, while some staining remained for X409. (I) Immunofluorescence confocal microscopy images showing, a comparable result with a higher level of remaining X409 signal with permeabilized cells. White scale bars represent 20 μm.
Fig 3.
Removal of the glycosylated MUC1 extracellular domain enhances SARS-CoV-2 entry.
(A) Microscopy images of Calu-3 cells treated with StcE, E447D, neuraminidase or fucosidase infected with SARS2-S pseudotyped VSV-GFP without or with neutralizing monoclonal antibody (mAb) against SARS2-Spike. White scale bars represent 200 μm. (B) Quantification of SARS2-S pseudotyped VSV-GFP signal in Calu-3 cells using EVOS software. StcE treatment resulted in a 5.4-fold increase in infection. (C) Quantification of luciferase signal (RLU) in Calu-3 cells after treatment with indicated enzymes and infection with SARS2-S pseudotyped VSV-Luc in the absence or presence of mAb against spike. A 4-fold increase in RLU value was observed when cells were treated with StcE. (D) Quantification of Calu-3 cell infection with VSV-G pseudotyped VSV-Luc lacking the spike protein. Infection was not blocked by the anti- spike mAb. (E) Infection of Calu-3 cells with authentic SARS-CoV-2 after treatment with indicated enzymes. StcE treatment resulted in a 2-fold increase in infected cell count. Neuraminidase and fucosidase treatment did not significantly impact viral entry. Represented values are the mean ± SEM of three biological replicates performed in triplicate. Statistical analysis was performed by repeated measures one way-ANOVA with Dunnett’s post-hoc test. p > 0.05 [ns, not significant], p<0.05 [*], p<0.01 [**], p<0.001 [***], p<0.0001 [****].
Fig 4.
MUC1 and MUC16 are expressed on the surface of human airway organoid-derived air-liquid interface cultures and decreases upon StcE treatment.
(A) Microscopy of permeabilized human airway organoid-derived air-liquid interface cultures showing combined extracellular and intracellular staining of MUC1, MUC4, MUC5AC and MUC16. (B) Microscopy of live stained air-liquid culture for MUC1, MUC4, MUC5AC and MUC16 without permeabilization. MUC1 and MUC16 are detectable demonstrating expression on the cell surface, whereas MUC4 staining is negative and MUC5AC only stains positive in occasional mucus strands on top of the cells. (C) Immunoblot analysis of MUC1 levels in human airway organoid-derived ALI cultures from donor 1 and donor 2 treated with StcE, E447D or no treatment. The high molecular weight MUC1 is removed upon StcE treatment. (D) Microscopy of MUC1 and MUC16 in permeabilized ALI cultures along with O-glycan probe X409-GFP. Arrows indicate co-localization of X409 with MUC16, but not MUC1. (E) Quantification of colocalization of MUC1 and MUC16 with X409 staining in ALI cultures of two donors as performed in D. Mander’s overlap coefficient plots are included in S3 Fig. (F) Microscopy of surface binding of X409 on untreated, 10ug/ml StcE and 10ug/ml E447D treated ALI cultures. All white scale bars indicate 50 μm. (G) Quantification of X409 signal intensity per imaged field from experiment performed in E. Statistical analysis was performed by repeated measures one way-ANOVA with Tukey’s post-hoc test. p<0.05 [*].
Fig 5.
StcE treatment of human airway organoid-derived air-liquid interface cultures increases SARS-CoV-2 replication.
(A-D) Replication kinetics of SARS-CoV-2 in ALI cultures in terms of RNA copies (A, C) and infectious virus (B, D) in two donors. Represented values are the mean ± SD of three replicates. Statistical analysis was performed for donor 2 by repeated measures two way-ANOVA with Tukey’s post-hoc test. p<0.05 [*], p<0.01 [**], p<0.001 [***], p<0.0001 [****]. p<0.01 was found between NC and StcE and E447D and StcE treated cells at 1 day post infection and at 2 days post infection between E447D and StcE treated cells (C). p<0.05 was found between NC and StcE and E447D and StcE treated cells at 2 days post infection (D). (E-F) Microscopy images of untreated, 10ug/ml E447D or 10ug/ml StcE treated cells from donor 1 (E) or donor 2 (F), infected with SARS-CoV-2 at two days post-infection. White scale bars represent 100 μm. NP = nucleoprotein.
Fig 6.
Removal of the MUC1 extracellular domain increases spike and virus attachment.
(A) Immunofluorescence confocal microscopy of Calu-3 cells incubated with 2.5 ug/ml SARS-CoV-2 spike (Fc-tagged SARS2-S1B-Fc, red) at 4°C for 1 h. Spike was incubated with the cell without permeabilization. Increased spike binding and higher spike signal intensity was observed after treatment with StcE in comparison to E447D treatment and control. White scale bars represent 20 μm (B) Quantification of spike fluorescence signal as depicted in A. Fluorescence intensity along the edge of cell island was determined in control, StcE- and E447D-treated cells using ImageJ. Mean ± SEM raw integrated density/length from three random fields from three independent experiments are plotted. The area of spike binding was significantly higher in StcE-treated cells. White scale bars represent 50 μm.
Fig 7.
Protective functions of MUC1 and MUC16 at the respiratory surface.
(A) Immunofluorescence confocal microscopy analysis of expression and localization of ACE2 (green) and TM mucin MUC1 (214D4, red) in Calu-3 cells. (B) Proximity ligation assay (PLA) for MUC1-ED (214D4; light blue) and ACE2 (green) (left) and MUC1-SEA (232A1; light blue) and ACE2 (green) (right) in Calu-3 cells. A positive PLA signal (red) was detectable for both combinations in double positive cells, demonstrating that MUC1 and ACE2 are in close proximity. (C) Immunofluorescence confocal microscopy to determine expression of MUC1 (214D4; red) in ciliated cells (AcTub; green). MUC1 is highly expressed in non-ciliated cells and is expressed in cells with short cilia. (D) Immunofluorescence confocal microscopy for MUC16 (red) and cilia (AcTub; green). MUC16 is expressed in some non-ciliated cells and ciliated cells. (E) Immunofluorescence confocal microscopy for MUC16 (red) and goblet cells (MUC5AC; green). Several goblet cells are positive for MUC16. White scale bars represent 20 μm for A and B and 50 μm for C-E. (F) Schematic model describing the expression and localization of transmembrane mucins MUC1 and MUC16 in different cell types within the respiratory epithelium. MUC1 is highly expressed in non-ciliated cells and MUC16 is expressed in some ciliated cells and enriched in goblet cells. (G) Schematic model describing the protective functions of the extracellular domains of transmembrane mucins MUC1 and MUC16 during SARS-CoV-2 infection. The extended glycosylated extracellular domains prevent access of the virus to the ACE2 receptor (left). Enzymatic removal of the glycosylated part of the extracellular domains with the StcE mucinase allows the viral spike protein to connect with the ACE2 receptor resulting in viral entry into lung epithelial cells (right).