Heparitinase

Concentrations of urinary and plasma GAGs and KS were measured by tandem mass spectroscopy as described elsewhere (16 (link)). Briefly, the chromatographic system consists of an HP1100 system (Agilent Technologies) and a Hypercarb column (Thermo Electron). The API-4000 mass spectrometer (Applied Biosystems) was equipped with a turbo ionspray ion source. Disaccharides of keratan, heparan, dermatan sulfate were used as standards [Galß1,4GlcNAc(6S), ΔDiHS-0S, ΔDiHS-NS, ΔDi-4S, ΔDi-6S (Seikagaku). Chondrosine was used as internal standard (Glycosyn). Urine or plasma was centrifuged, and the supernatants were digested overnight with 1 mU of chondroitinase b, 1mU heparitinase, and 1mU keratanase II (Seikagaku). Recovered samples were analyzed by the LC-MS/MS system and normalized by creatinine concentration in urine. Total GAGs refer to total glycosaminoglycans or the additive sum of all the GAGs measured (HS, DS and KS). Disaccharide GAG concentrations were calculated by Analyst 1.5.1 software (AB SCIEX). Each sample was measured in triplicate with three injections for each sample.

Hip caps from 4-week-old BALB/C wild-type mice were decellularised by repeated freeze thawing. Hip caps were incubated overnight at 37°C either in phosphate buffered saline (PBS) or in PBS containing 5 mU/mL heparitinase (Seikagaku). Total proteins were precipitated from supernatants using trichloroacetic acid and assessed by western blotting.

Isolation and purification of GAG chains from the cell cultures were performed^{67 (link)}. Cells were homogenized with ice-cold acetone, extracted with ice-cold acetone three times, and air-dried thoroughly. The delipidated acetone powder was digested with actinase E (one-tenth the weight of acetone powder) in 0.1 M borate-sodium, pH 8.0, containing 10 mM CaCl₂ at 55 °C for 48 h. The samples were adjusted to 5% v/v in trichloroacetic acid and centrifuged. The GAG-containing materials were precipitated from the resultant supernatants by mixing with ethanol, dissolved in water, and subjected to gel filtration on a PD-10 desalting column (Cytiva) using water as an eluent. The flow-through fractions were collected and evaporated to dryness. The purified GAG fraction containing the CS and HS chains was digested with 5 mIU of chondroitinase ABC (Seikagaku), or a mixture of 0.5 mIU of heparinase (Seikagaku) and 0.5 mIU of heparitinase (Seikagaku), at 37 °C for 3 h. The digests were derivatized with the fluorophore 2-AB and then analyzed by anion-exchange HPLC using a YMC-Pack PA-G column (4.6 × 250 mm, YMC, Kyoto, Japan). A modular HPLC system (Shimadzu) was operated by LabSolutions LC/GC (Ver. 5.42, Shimadzu). The identification and quantification of the resulting disaccharides were achieved by comparison with authentic unsaturated CS and HS disaccharides (Seikagaku).