Clostridium perfringens sialidase

A mixture of mono- and di-sialogangliosides containing 5–10 μg sialic acid were incubated with Clostridium perfringens sialidase (2U/ml) (Sigma Chemical Co., St. Louis, MO) in 0.5 ml of 50mM sodium citrate-phosphate buffer, pH 5.5, at 37oC, for 2h, as previously described (25 (link)). A duplicate sample of equal quantity of gangliosides was incubated in buffer alone. Reactions were terminated by addition of 0.1 M NaOH, neutralized with 0.1 M HCl, desalted on SepPak (Waters Assoc., Milford, MA) columns and evaluated on TLC for hydrolytic products, with resorcinol. The presence of resorcinol-negative spots on sialidase-treated samples was confirmed on TLC’s, by reversible staining with iodine vapor, prior to resorcinol spraying (25 (link)). Efficacy of enzymatic activity was determined with gangliosides with sialidase-susceptible external sialic acid residues (GM3, GD3, GD1a, GD1b) and gangliosides with sialidase-resistant internal sialic acid residues (GM1a, GM2), respectively (Matreya LLC, Pleasant Gap, PA).

Disaggregated mTECs were loaded into cuvettes (1 × 10⁵ cells/slide) then centrifuged (600 rpm, 4 min, RT). After centrifugation, samples were fixed for 1 min in methanol at RT. Ligand expression was studied by incubation with Siglec-F-Fc or irrelevant humanized monoclonal IgG1 (each at 1 μg/mL, 1 hr, 37°C), then incubating with secondary biotinylated polyclonal anti-human IgG antibody. Samples were then washed and incubated with streptavidin/alkaline phosphatase linker, followed by visualization with Vector red chromogen (Vector Laboratories, Burlingame, CA). In some experiments, samples were pretreated with or without Clostridium perfringens sialidase (10 mU/mL, 24 hr, 37°C; Sigma-Aldrich). To permeabilize cells, samples were boiled in 10 mM sodium citrate buffer, pH 6.0 followed by 10 minutes at 95°C before staining. Biotinylated Maackia amurensis (MAA) lectin (specific for α2,3-linked sialic acid) and Sambucus nigra (SNA) lectin (specific for α2,6-linked sialic acid), each at 10 mg/mL (EY Laboratories, Inc., San Mateo, CA) were also used in some experiments as previously described.^{14 (link)}Tissue distribution and localization of potential Siglec-F ligand was studied by histochemistry using Siglec-F-Fc (1 μg/mL, 1 hr, 37°C, R&D Systems) detected with alkaline phosphatase imaging as previously described.