Heparinase 1 3

At first, the native cells were measured. To block the specific interactions, a 100 ng/mL anti-ACE2 (BioVision) blocking antibody solution was added to the native cell and incubated for 20 min in AFM liquid cell. After this time, the buffer was replaced, AFM probe coated with S-protein was mounted and the force-distance (FD) curves were immediately measured. To block S-proteins, the AFM probe covered with S-protein and cells were exposed to 50 U low molecular weight heparin (LMWM, Sigma) for 30 min. After this time, FD curves were measured. For reduction of glycocalyx layer, 100 U Heparinase I/III (Sigma) was added to the native cells immersed in HBSS and incubated for 45 min at 36.2 °C in AFM liquid cell. After this time, the buffer was replaced and the AFM measurement started. In the end, the anti-ACE2 was added to block the ACE2 receptors. All experiments were repeated two or three times, as described in Supplementary Table 1.

293T and L929 cells were treated in complete growth medium with 2 Units/ml Heparinase I/III (Sigma-Aldrich) for 2 h at 37°C. Cleavage and reduction of cell surface heparan sulfate (HS) was verified by flow cytometry analysis.
293T and L929 cells were passaged for 2 weeks in complete growth medium supplemented with 30 or 50 mM sodium chlorate (Roth).
Post-treatment, supernatant was removed and cells were infected with VSV WT or VSV-GP variants for 1 h at 37°C in complete growth medium at an MOI of 1. Infection was assessed 15 h p.i. by flow cytometry (eGFP) and normalized to untreated controls.

Sdc2 detection in mouse ECs and HUVEC: heparinase pre-treatment is required to unveil epitopes on core protein and remove nonspecific sugar epitopes (Supplementary Figure 7 for uncut WB images). Briefly, confluent ECs on 6 cm tissue culture dishes were rinsed twice with DPBS that was then replaced with 2 ml of serum-free media (Opti-MEM, ThermoFisher) containing 0.5 U/ml of Heparinase I-III (Sigma-Aldrich, #H2519 and #H8891) and 0.2 U/ml of Heparinase II (Sigma-Aldrich, #H6512). After 2 h digestion at 37 °C, cells were washed three times with ice-cold PBS, lysed in RIPA buffer and samples prepared for western blot analysis. Specific band at ~42 kDa is the naked core protein in a dimeric form. Additional specific smears (50–250 kDa) are Sdc2 glycosylation isoforms. Sdc4 detection in mouse ECs and HUVEC: heparinase pretreatment is not necessary. A specific band below 20 kDa is detected corresponding to the naked core in a monomeric form. HA-Tag syndecans: heparinase pretreatment is not required. HA detection reveals naked core proteins in a dimeric form and additional higher molecular weight smear bands due to glycosylation (Fig. 4e for comparison of all syndecans).

293TT cells were seeded at 4.10⁵ cells/well in a 12-well Falcon plate for binding experiments and at 10⁴ cells/well in 96-well Falcon plate for infectivity experiments. 293TT cells were treated for 1h at 37°C, with 0.5U/mL of Neuraminidase V from Clostridium perfringens in DMEM +20 mM HEPES +0.1% BSA or without Neuraminidase V for control conditions. The VLP binding protocol was the same as previously described. For infectivity, cells were inoculated with different PSVs for 3 h and then washed 3 times with complete medium before incubation for 48–72 h at 37°C.
For glycosaminoglycan (GAG) removal, heparinase I/III (Sigma-Aldrich) or chondroitinase ABC (Sigma-Aldrich) were applied to 293TT cells for 2h in digestion medium (20 mM HEPES, pH 7.5, 150 mM NaCl, 4 mM CaCl2 and 0.1% BSA) at 37°C. Diluted PSVs were then added to the wells, and incubated for 3h at 37°C. Enzymes and PSV were then removed, cells were washed 3x in complete medium, then replaced in the incubator in 150μL per well fresh complete medium.