Fucosylated chondroitin sulfate (FCS) extracted from Phyllophorella kohkutiensis (PkFCS) is composed of d-GalNAc, d-GlcA, l-Fuc and -SO42-. According to the defined structures revealed by NMR spectra of the branches released by mild acid hydrolysis and oligosaccharides generated by β-eliminative depolymerization, the backbone of PkFCS is CS-E, and the branch types attached to C-3 of d-GlcA include l-Fuc2S4S, l-Fuc3S4S, l-Fuc4S, and the disaccharide α-d-GalNAc-1,2-α-l-Fuc3S4S with the ratio of 43:13:22:22. Notably, novel heptasaccharide and hendecasaccharide were identified that are branched with continuous distribution of the disaccharide. The structural sequences of the oligosaccharides indicate that three unique structural motifs are present in the entire PkFCS polymer, including a motif branched with randomly distributed different sulfated l-Fuc units, a motif containing regular l-Fuc2S4S branches and a motif enriched in α-d-GalNAc-1,2-α-l-Fuc3S4S. This is the first report about the distribution pattern of diverse branches in natural FCS. Natural PkFCS exhibited potent anticoagulant activity on APTT prolonging and anti-iXase activity. Regarding the structurally defined oligosaccharides with sulfated fucosyl side chains, octasaccharide (Pk4b) is the minimum fragment responsible for its anticoagulant activity correlated with anti-iXase. However, further glycosyl modification with a non-sulfated d-GalNAc at the C-2 position of l-Fuc3S4S could significantly decrease the anticoagulant and anti-iXase activity.
Keywords: Anticoagulant; Disaccharide branch; Distribution pattern; Fucosylated chondroitin sulfate; Oligosaccharides; iXase.
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