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J Biol Chem
2014 Oct 10;28941:28284-98. doi: 10.1074/jbc.M114.572297.
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Fucosylated chondroitin sulfates from the body wall of the sea cucumber Holothuria forskali: conformation, selectin binding, and biological activity.
Panagos CG, Thomson DS, Moss C, Hughes AD, Kelly MS, Liu Y, Chai W, Venkatasamy R, Spina D, Page CP, Hogwood J, Woods RJ, Mulloy B, Bavington CD, Uhrín D.
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Fucosylated chondroitin sulfate (fCS) extracted from the sea cucumber Holothuria forskali is composed of the following repeating trisaccharide unit: → 3)GalNAcβ4,6S(1 → 4) [FucαX(1 → 3)]GlcAβ(1 →, where X stands for different sulfation patterns of fucose (X = 3,4S (46%), 2,4S (39%), and 4S (15%)). As revealed by NMR and molecular dynamics simulations, the fCS repeating unit adopts a conformation similar to that of the Le(x) blood group determinant, bringing several sulfate groups into close proximity and creating large negative patches distributed along the helical skeleton of the CS backbone. This may explain the high affinity of fCS oligosaccharides for L- and P-selectins as determined by microarray binding of fCS oligosaccharides prepared by Cu(2+)-catalyzed Fenton-type and photochemical depolymerization. No binding to E-selectin was observed. fCS poly- and oligosaccharides display low cytotoxicity in vitro, inhibit human neutrophil elastase activity, and inhibit the migration of neutrophils through an endothelial cell layer in vitro. Although the polysaccharide showed some anti-coagulant activity, small oligosaccharide fCS fragments had much reduced anticoagulant properties, with activity mainly via heparin cofactor II. The fCS polysaccharides showed prekallikrein activation comparable with dextran sulfate, whereas the fCS oligosaccharides caused almost no effect. The H. forskali fCS oligosaccharides were also tested in a mouse peritoneal inflammation model, where they caused a reduction in neutrophil infiltration. Overall, the data presented support the action of fCS as an inhibitor of selectin interactions, which play vital roles in inflammation and metastasis progression. Future studies of fCS-selectin interaction using fCS fragments or their mimetics may open new avenues for therapeutic intervention.
FIGURE 1. a, 800 MHz 1H NMR spectrum of H. forskali fCS. b, conformation of fCS I, highlighting the GalNAc/fucose protons showing inter-residue NOEs (H-2/H-5 (purple), H-6/H-3 (yellow), H-6/H-4 (black), and H-2/H-6 (red)), as well as the distance (green) between fucose H-5 and the oxygen involved in the GalNAcβ4,6S(1→4)GlcAβ glycosidic bond in both fCS I and fCS II. c and d, expansions of an overlay of the two-dimensional 1H-13C HSQC (red) and the two-dimensional 1H-13C HSQC-NOESY (blue) spectra. The inter-residue NOE cross-peaks are circled. The methyl resonances of proteins are marked by an asterisk.
FIGURE 2. Closest to average structures generated by MD simulations of 2,4 Fuc- (a) and 3,4 Fuc-sulfated fCS (b). The insets show an expansion of the respective trisaccharide repeating units.
FIGURE 3. Negative ion electrospray mass spectra of the tri- and tetrasaccharide fractions.
FIGURE 4. Microarray analyses of the binding of human L-, P-, and E-selectins with NGLs derived from H. forskali fCS oligosaccharides. The results shown are the means of the fluorescent intensities of duplicate spots at 2 and 5 fmol/spot with error bars. The insets in the L- and P-selectin panels are an expansion of the fluorescence signals of the control probes 1–4 in relation to the fCS hexasaccharide probes 9, 10, and 12.The asterisks indicate measured values that are off-scale. The details of fCS oligosaccharides are shown in Table 1, and sequences of the control NGLs are given under “Experimental Procedures.†High performance TLC of NGL fractions of P-fCS-dp3, -dp4, and -dp6 (S, solvent front; O, origin) is shown on the bottom right.
FIGURE 5. Activation of PK by H. forskali fCS and fCS oligosaccharides. The elevated OD represents the increasing presence of kallikrein in blood plasma, which is directly correlated with PK activation. Dextran sulfate, a known PK activator, was included for comparison. The native H. forskali fCS polysaccharide caused activation similar to that seen with dextran sulfate, but the oligosaccharides F-fCS-dp9 and F-fCS-dp20 did not activate PK at these concentrations.
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