Enzymatic protein deglycosylation kit

Wildtype recombinant human prostasin zymogen was prepared and activated by matriptase as described above. 100 ng of zymogen or of activated, double-chain prostasin was then subjected to removal of N- and O-linked carbohydrates using Enzymatic Protein Deglycosylation Kit (EDEGLY, Sigma-Aldrich, St. Louis, MO) according to manufacturer’s instructions. Resulting products were then analyzed by reducing SDS-PAGE and anti-prostasin Western blot analysis as indicated above.

In order to remove all glycans of glycoconjugates present in the different protein extracts of T. cruzi, the Enzymatic Protein Deglycosylation kit (Sigma) was used. Briefly, 100 μg of proteins was diluted in 30 μL of miliQ water. 10 μL of 5x reaction buffer was added, as well as 2.5 μL of denaturation solution, then the mixure was gently mixed. Extracts were heated at 100°C for 5 min and cooled to room temperature, after which 2.5 μL of the Triton X-100 solution was added. In order to remove sialic acid residues and O-glycans, 1 μL of α(2 → 3,6, 8,9)-neuraminidase and 1 μL of O-glycosidase were added. To remove galactose and N-acetylglucosamine residues, 1 μL of both β(1 → 4)-galactosidase and β-N-acetylglucosaminidase was added. Each extract was incubated for 3 h at 37°C and then analyzed by electrophoresis or electrotransferred into a nitrocellulose membrane for Western blot assays.

To determine the level of PD‐L1 protein glycosylation, the deglycosylation of PD‐L1 protein was performed using the enzymatic protein deglycosylation kit from Sigma (St. Louis, MO, USA), and assay of GLT1D1 deglycosylation was performed using a PNGase F digestion kit (NEB, New England Biolabs, Beverly, MA, USA) according to the manufacturer instructions. The protein extraction reagents did not contain SDS. The protein after deglycosylation was assayed by SDS/PAGE.

For deglycosylation assays, protein extracts were treated with different combinations of enzymes from an enzymatic protein deglycosylation kit (Sigma-Aldrich, St Louis, MO) according to the manufacturer's instructions. O-Deglycosylation assays required the use of sialidase A for cleavage of terminal sialic acid residues, O-glycosidase to remove the core Galβ(1 → 3)-GalNAc, β(1 → 4)-galactosidase and β-N-acetylglucosaminidase to remove sugars associated with specific O-linked glycan structures. To remove all N-linked glycans, we used the PNGase F.

Membrane glycoproteins were detected by lectin blot. Proteins were extracted with a RIPA buffer and were treated with PNGase F (Enzymatic Protein Deglycosylation Kit; Sigma‐Aldrich). An electrophoresis analysis was performed as explained below except that only 25 µg of proteins was loaded. After transfer on nitrocellulose blotting membrane (Premium 0.2 µm NC; GE Healthcare Life Sciences) at 130 mA for 1.5 h, a treatment was applied with PBS supplemented with 10% (v/v) blocking reagent (Roche) for 30 min at room temperature. After three washings with TBS and lectin buffer 1 for MAA lectin or with TBS (for SNA binding), membranes were incubated with lectins MAA (125 µg·mL⁻¹, DIG glycan differentiation kit; Roche) and SNA (5 µg·mL⁻¹; Vector Laboratories). Then, we performed three washes of 10 min in TBS for MAA and PBS‐0.05% Tween‐20 for SNA. We incubated the MAA membrane in α‐digoxigenin‐alkaline phosphatase or SNA membrane in streptavidin‐HRP during 1 h. We finished by three 10‐min washes in TBS 1X for MAA and PBS‐0.05% Tween‐20 solution for SNA.
Two control glycoproteins were also used: carboxypeptidase Y (an asialoprotein) as a negative control and fetuin (a sialylated protein) as a positive control.

Lyophilized glycoproteins (100μg) were dissolved in 30μl deionized water, heated to 100ºC for 5 min, cooled to room temperature and deglycosylated using the Enzymatic Protein Deglycosylation Kit (Sigma-Aldrich, St. Louis, MO, USA), according to the manufacturer’s instruction.