Structural characterization of chondroitin sulfate from Italian cheese Parmigiano Reggiano

Chondroitin sulfate (CS) was purified for the first time from the Italian cheese Parmigiano-Reggiano and analyzed to evaluate its structure and properties. Two main polysaccharides were identified as CS, ~72%, and fast moving-heparin/heparan sulfate, ~28%. Quantitative analyses yielded ~1.5-3.0 µg of total GAGs per gram of Parmigiano-Reggiano (0.15-0.30%). By means of specific chondroitinases and HPLC separation of generated unsaturated disaccharides, CS was found to be composed of ~9% of nonsulfated disaccharide, ~26% of disaccharide monosulfated in 6 of the GalNAc, ~65% of disaccharide monosulfated in 4 of the GalNAc, with a charge density of ~0.91 and a 4/6-sulfated ratio of 2.45. The ratio of 4/6 sulfated residues was confirmed by 13C-NMR experiments. Susceptibility to cleavage catalyzed by chondroitinase B confirmed that the purified Parmigiano-Reggiano CS contains mainly GlcA (~94%) as uronic acid. PAGE analysis showed a CS having a molecular mass with an average value of 15400.