Heparinase

Heparitinase I (Flavobacterium heparinum, EC 4.2.2.8), Heparitinase II (F. heparinum, no number assigned), heparinase (F. heparinum, EC 4.2.2.7), chondroitinase ABC, chondroitinase ACII, unsaturated disaccharides kit for HS (heparan sulfate) or CS were purchased from Seikagaku Corp (Tokyo, Japan). Pronase and collagenase D were from Roche Applied Science (Germany). Deoxyribonuclease I was from Sigma-Aldrich Japan (Tokyo, Japan). Anti-type I collagen antibody was from Cosmo Bio Corp (Tokyo, Japan). Anti-decorin monoclonal antibody (Anti dermatan sulfate proteoglycan mAb, clone 6-B-6) and anti-versican monoclonal antibody (Anti large proteoglycan mAb clone 2-B-1) were from Seikagaku Corp (Tokyo, Japan). AlexaFluor546 conjugated anti-rabbit IgG antibody was from Thermo Fisher Scientific (Yokohama, Japan). Histofine (mouse staining kit) for mouse monoclonal antibody to mouse tissue was from Nichirei (Tokyo, Japan). Envision kit/HRP (DAB) was from DAKO Japan (Tokyo, Japan). Recombinant human TGF-β1 and recombinant murine TNF-α were purchased from PeproTeck (NJ, USA)

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).