Chitin hydrolysate

The glycosylation profile of UDA15a and UDA15b cell lines was studied by lectin blotting using ConA and TL. Both, sVSG (2 μg) and cell pellets (1 x 10⁶ cell equivalents/sample) coming from hypotonic lysis were denatured in SDS-sample buffer containing 8 M urea and 50 mM DTT, subjected to electrophoresis on a NuPAGE Bis-Tris 4–12% gradient gel (Invitrogen) in MOPS buffer and transferred to a nitrocellulose membrane. Proteins were stained with Ponceau S (Sigma) as loading control and blocked with 3% BSA in PBS. Membranes were probed with biotinylated TL (0.33 μg/mL, Vector Laboratories, Inc.) in a solution containing 50 mM Tris-HCl pH 7.4, 0.5 M NaCl, 0.05% IGEPAL and 0.25% BSA, or biotinylated ConA (0.05 μg/mL, Sigma) in PBS containing 1 mM MgCl₂, 1 mM CaCl₂, 1 mM MnCl₂, 0.05% IGEPAL and 0.25% BSA. Specific inhibitors of lectin binding such as chitin hydrolysate (1:10 dilution, Vector Laboratories, Inc.) for TL and methyl α-D- mannopyranoside (0.5 M, Sigma) for ConA were also used as carbohydrate-specific binding controls. Finally, glycoproteins were detected with Extravidin-peroxidase conjugated (Sigma) by chemiluminescent detection ECL Western Blotting Detection Reagents (GE Healthcare).

The canonical E-selectin ligands were analyzed at the tumor cell surface by flow cytometry using mAbs against sLeX (CSLEX1) or sLeA (121SLE) as described.⁵¹ (link) β-1,6-GlcNAc branches and poly-LacNAc were determined using biotinylated PHA-L and DSL, respectively (Vector Labs). As “isotype” controls, lectins were applied after sugar inhibition with bovine thyroglobulin (Sigma) and chitin hydrolysate (Vector Labs), respectively. Lectins were labeled with streptavidin-APC (Sigma) for flow cytometry.
To test their functional importance for endothelial adhesion and E-selectin binding, sialic-acid-containing sugar residues were enzymatically cleaved using 10 mU/mL ND (from Clostridium perfringens, Roche) added to the tumor cell culture at 37°C for 1h under serum-free conditions. Likewise, glycoproteins were cleaved by 1 mg/mL pronase (from Streptomyces griseus) added to the tumor cell culture at 37°C for 45 min under serum-free conditions. O-GalNAc-glycosylation was chemically inhibited by adding 2 mM GOB (Sigma) for 72 h to the culture medium (solvent control: cell culture medium). N-glycosylation was inhibited using 2 μM synthetic Sw (Sigma) for 72 h (solvent control: methanol). Detrimental effects of all treatments on tumor cell viability were excluded by propidium iodide uptake analyses (flow cytometry [FC]).