Neuraminidase from clostridium perfringens

The role of O-GalNAc-glycoproteins for rmE-selectin binding was investigated by adding 20 mM GalNAc-α-O-benzyl (Sigma) for 72 h to the standard tumor cell culture prior to the E-selectin binding assay. Likewise, tumor cell cultures were treated for 1 h with 10 mU/mL neuraminidase from Clostridium perfringens (Roche, Mannheim, Germany) or with 1 mg/mL pronase from Streptomyces griseus (Roche) under serum-free conditions at 37 °C for 1 h or 45 min before the E-selectin binding assay to analyze the role of sialic acid residues or glycoproteins, respectively. Detrimental effects of all treatments were excluded by propidium iodide staining immediately before flow cytometry. To test which linkage of sialic acid was mainly affected by neuraminidase, lectin binding flow cytometry was performed using biotinylated Maackia amurensis lectin II (MAA-II, detecting α-2,3-linked sialic acid) and Sambucus nigra agglutinin I (SNA-I, detecting α-2,6-linked sialic acid) (both from Vector Labs, Burlingame, USA). Both lectins were marked with streptavidin-APC (Sigma) before incubation with the tumor cells. To test for carbohydrate-specific binding, one tumor cell sample was pre-treated with periodic acid, which oxidizes free sialic acid residues and by this cracks the ring structure of the carbohydrate, which is required for the binding of MAA-II and SNA-I.

The optimal conditions for cleavage of terminal sialic acid residues were
determined in a series of pre-experiments. To this end, freshly drawn
human whole blood was centrifuged (10 min, 500 g) at room temperature
to isolate platelet rich plasma, which was re-centrifuged (10 min,
800 g) to obtain platelets. Platelets were resuspended in PBS to a
final concentration of 3 × 10⁸/ml. Aliquots of 500 μl of
the platelet suspension were incubated for 60 min at 37°C with 1, 2.5,
5, 10, and 20 mU neuraminidase from Clostridium
perfringens (Roche, Mannheim, Germany) per
10⁸ platelets, or were left untreated. Platelets
treated with 5 mU of neuraminidase were additionally analyzed at
different time points (30, 60, 90, and 120 min). The presence of
terminal sialic acid or galactose residues was analyzed by flow
cytometry as described below.