2 fluro l fucose

P. clara 1C4 was cultured overnight in the presence of Tunicamycin (Sigma-Aldrich; final concentration, 10 µg ml⁻¹), 2-fluro-l-fucose (Cayman Chemical; final concentration, 250 µM) or DMSO control. Cultured bacteria were then pelleted, washed once with PBS and lysed with 1% SDS solution (in 50 mM Tris-HCl buffer supplemented with 5 mM EDTA). SDS–PAGE was conducted using the Novex NuPAGE SDS–PAGE Gel system (Thermo Fisher Scientific). Glycan-containing proteins were stained with the Pro-Q Emerald 300 Glycoprotein Gel and Blot Stain Kit (Thermo Fisher Scientific) according to the manufacturer’s protocol. The protein contents of the whole-cell lysates were stained using the Colloidal Blue Staining kit (Thermo Fisher Scientific). Supernatant proteins were first condensed using Amicon Ultra Centrifugal Filters (10 kDa NMWL) and then stained using the Colloidal Blue Staining kit (Thermo Fisher Scientific).

Overnight bacterial cultures were incubated with recombinant mouse trypsin (final concentration 1 µg ml⁻¹) for 1 h or human trypsin (final concentration 20 µg ml⁻¹) for 4 h. The recombinant trypsin isoforms used in this study were as follows: mouse recombinant PRSS2 (50383-M08H, Sino Biological), human recombinant PRSS1 (LS-G135640), human recombinant PRSS2 (LS-G20167) and human recombinant PRSS3 (His-tag) (NBP2-52220). In some experiments, recombinant mouse PRSS2 was first treated with one of the following trypsin inhibitors for 30 min before incubation with P. clara: AEBSF (Sigma-Aldrich; final concentration, 2 mM), Leupeptin (Sigma-Aldrich; final concentration, 100 µM) and TLCK (Abcam; final concentration, 100 µM). In some of the experiments P. clara was grown overnight in the presence of tunicamycin (Sigma-Aldrich; final concentration, 10 µg ml⁻¹), 2-fluro-l-fucose (Cayman Chemical; final concentration, 250 µM) or DMSO control before incubation with recombinant mouse PRSS2. For the experiments assessing the effect of Ca²⁺, P. clara was grown in a low-Ca²⁺ mGAM medium with or without supplementation with 1 mM Ca²⁺ before incubation with mouse recombinant PRSS2. For experiments using P. clara supernatant, the P. clara overnight culture was filtered with a sterile filter unit with a PVDF membrane (0.22 µm pore size).