Fucosylated chondroitin sulfate (FCS) from Holothuria mexicana (FCSHm) was selected for investigation because of its intriguing branch features. Selective β-eliminative depolymerization and the bottom-up assembly were performed to unravel that FCSHm consisted of a {D-GlcA-β1,3-D-GalNAc4S6S} backbone and branches of alternating FucS (55 %) and D-GalNAcS-α1,2-L-FucS (45 %), the highest proportion of disaccharide branch reported to date. In branches, sulfation could occur at every free -OH site except O-3 of GalNAc, being the most complex and various structure features of natural FCS. Detailed structure-activity relationship analyses showed that FCSHm and its depolymerized products (>8 kDa) effectively competed with SLeX and PSGL-1 to bind with P-sel at nano-molar level and the inhibition potency increased with Mw increasing. For the structural trisaccharide unit, di-O-sulfation of the FucS (Fuc2S4S and Fuc3S4S) was almost 10-fold more potent than mono-O-sulfation (Fuc4S). Unexpectedly, higher sulfation of the disaccharide-branched tetrasaccharide unit reduced inhibition. The reversal may attribute to fewer interactions with P-sel by molecular docking study. These results suggested that the specific configuration underpinned the potent inhibition, whereas the size and sulfate number of branches were not the key factors for the specific binding. dHmF4 (8.0 kDa) potently blocked the platelet-leukocyte aggregates formation, further verifying the potential value in use.
Keywords: Chemical depolymerization; Fucosylated chondroitin sulfate; Holothuria mexicana; Oligosaccharide; P-selectin inhibition.
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