Chondroitinase b

IdoA in radiolabeled CS/DS was quantified after degradation by chondroitinase B that only cleaves the N-acetyl galactosamine~iduronic acid linkages. Incubations were in 50 μL of 20 mM Hepes, pH 7.2, 50 mM NaCl, 4 mM CaCl₂, 0.1 mg/mL BSA and 2 mIU chondroitinase B (R&D System). After 2 h at 37 °C the products were separated on a Superdex Peptide column, radioactivity was measured by scintillation counting, and the percentage of iduronic acid over the total iduronic acid + glucuronic acid was calculated.

Small, full-thickness samples of bovine cornea were cut from the central 0–3 mm zone, the 3–6 mm zone, the 6–9 mm zone, and the 9–12 mm zone and embedded in OCT. Cryosections, 8 μm thick, were blocked with an Image IT Signal Enhancer (Invitrogen, UK)/5% normal goat serum for 20 min then incubated with the anti-keratan sulphate primary antibodies 5D4 (1:100 final dilution) and 1B4 (1:50 final dilution). Additional sections were also incubated for 1–2 h at 37 °C with antibody, 2B6 (1:20 dilution)—specific for CS/DS GAGs, with or without enzyme pretreatment to expose the CS and DS epitopes: chondroitinase ABC (0.4 U/ml) for CS and DS GAGs, or chondroitinase ACII (0.1 U/ml) (Seikagaku Corp., Tokyo, Japan) for CS GAGs, or chondroitinase B (0.5 U/ml) (R & D Systems, Inc., MN) for DS GAGs. Sections were then labelled with Alexa Fluor 488-conjugated goat anti-mouse IgG secondary antibody, mounted with Prolong Gold containing the nuclear stain DAPI (Invitrogen, UK) and examined on an Olympus BX61 microscope and F-View digital camera.

The analysis was conducted as in Stachtea et al. (2015) (link) with modifications. The cells were plated in DMEM medium and the day after, at a confluency of 90%, changed to sulfate-free DMEM, 10% FBS, 10 units/mL penicillin and 10 μg/mL streptomycin, with the addition of 100 μCi/mL ³⁵S-sulfate and ebselen. After 24 h, the medium was recovered for purification of proteoglycans (PGs) by DEAE-column chromatography in the presence of 6 M urea. The eluted PGs were desalted and subjected to pronase digestion followed by deaminative cleavage of HS at pH 1.5. CS/DS was re-isolated on Superose 6 and the degraded HS disaccharides were removed. The purity of CS/DS was verified by analytical digestion with chondroitinase ABC (Sigma C3667). CS/DS was degraded by chondroitinase B, which only cleaves the iduronic acid~N-acetyl galactosamine linkages, (R&D System; 2 mIU/incubation) in 20 mM Hepes, pH 7.2, 50 mM NaCl, 4 mM CaCl₂ and 0.1 mg/mL BSA for 2 h at 37°C. The split products were separated on a Superdex Peptide column and the percentage of iduronic acid over the total iduronic acid + glucuronic acid was calculated.