Chondroitin sulfate b

Heparin (from porcine intestinal mucosa, product ID H3149), hyaluronic acid (HA; from Streptococcus equi, product ID 94137), chondroitin sulfate A (CS-A; from bovine trachea, product ID C9819), chondroitin sulfate B (CS-B; from porcine intestinal mucosa, product ID C3788), chondroitin sulfate C (CS-C; from shark cartilage, product ID C4384), sodium chlorate (product ID 244147), Heparinase III (hepIII; from Flavobacterium Heparinum, product ID H8891), neuraminidase (neu; from Vibrio cholerae, product ID N7885), and chondroitinase ABC (ChABC; from Proteus vulgaris, product ID C3667) were purchased from Sigma (St. Louis, USA). Keratan sulfate (from bovine cornea) was a gift from Prof. Robert J Linhardt. Heparin oligosaccharide 14-mer (H014) and CSC-14 mer (CS-C 14-mer; CS014) were purchased from Iduron (Macclesfield, UK). Heparin hexamer (GAG 012) and dimer (GAG 063), and GD1a-glycan were purchased from Elicityl (Crolles, France).

Glycosidase activity assays were performed essentially as previously described (44 (link)). HA sodium salt from rooster comb, chondroitin sulfate sodium salt from shark cartilage, heparan sulfate sodium salt from bovine kidney, and chondroitin sulfate B (also known as dermatan sulfate) sodium salt were purchased from Sigma. The substrates were dissolved in 50 mm ammonium acetate buffer (pH 6.5), 10 mm calcium chloride (45 (link)). Recombinant HylA (500 or 5,000 pm) and 0.05–0.30 or 1.0 mg/ml substrates were incubated in the ammonium acetate buffer at 37 °C. The rate of substrate degradation was measured by monitoring the increase of A₂₃₂ over time. The kinetic parameters of M4 HylA with concentration ranges of 0.05–0.30 mg/ml of HA at 37 °C were calculated using the following formula and Lineweaver-Burk double-reciprocal plots,

V₀ is the initial reaction rates (A₂₃₂/min), K_m is the Michaelis-Menten constant, V_max is the maximum reaction velocity, and [S] is the substrate concentration.

Five micrograms of HA of different sizes (high, medium and low molecular weight, which correspond to > 950 kDa, 75–350 kDa and 15–40 kDa, respectively, RD Systems, Minneapolis, MN) were incubated for 1 h at 37°C with SGE (equivalent of 1 SG pair) or LuloHya (10 nM). Other commercial hyaluronidases were included as controls: bovine hyaluronidase (Sigma) and hyaluronidase from Streptomyces hyalurolyticus (Sigma), both at 1 Unit per reaction. Samples were run on a 1.2% agarose gel for 30 min at 20 V followed by 2 h 30 min at 40 V. Agarose gels were stained with 0.05% Stains-all (Sigma).
For hyaluronidase activity specificity, other glycosaminoglycan components of the extracellular matrix were also tested, including chondroitin sulfate B, dextran sulfate and heparin (5 μg/sample, Sigma).

Recombinant human cytokines used in this study [CCL1, CCL2, CCL3, CCL3L1, CCL4, CCL4L1, CCL5, CCL7, CCL8, CCL11, CCL13, CCL14, CCL15, CCL16, CCL17, CCL18, CCL19, CCL20, CCL21, CCL22, CCL23, CCL24, CCL25, CCL26, CCL27, CCL28, CXCL1, CXCL2, CXCL3, CXCL4, CXCL5, CXCL6, CXCL7, CXCL8, CXCL9, CXCL10, CXCL11, CXCL12α, CXCL12β, CXCL13, CXCL14, CXCL16, XCL1, CX3CL1, interleukin-1α (IL-1α), IL-1β, IL-6, IL-6Rα, IL-10, IL-12p70, IL-13, IL-17a, IL-18BP-Fc, IL-23, IL-27, IL-35, tumor necrosis factor–α (TNF-α), TNF-β, interferon-β (IFN-β), IFN-γ, IFN-λ1, and IFN-ω] from PeproTech and IFN-α2 and IL-18 from Sino Biological were reconstituted in DPBS 0.1% BSA at 10 μM, aliquoted, and stored at −80°C. Heparin (no. 2106), heparan sulfate from bovine kidney (no. H7640), chondroitin sulfate A (no. C9819), and chondroitin sulfate B (no. C3788) were obtained from MilliporeSigma. Heparan sulfate from porcine mucosa (no. AMS.GAG-HS01) and keratan sulfate (no. AMS.CSR-NAKPS2-SHC-1) were purchased from AMSBIO. We assumed an average molecular weight of 30 kDa for heparan sulfate from porcine mucosa and 15 kDa for heparin (37 ).