Identification of keratan sulfate disaccharide at C-3 position of glucuronate of chondroitin sulfate from Mactra chinensis

Abstract

Glycosaminoglycans (GAGs), including chondroitin sulfate (CS), dermatan sulfate, heparin, heparan sulfate and keratan sulfate (KS) are linear sulfated repeating disaccharide sequences containing hexosamine and uronic acid [or galactose (Gal) in the case of KS]. Among the GAGs, CS shows structural variations, such as sulfation patterns and fucosylation, which are responsible for their physiological functions through CS interaction with CS-binding proteins. Here, we solved the structure of KS-branched CS-E derived from a clam, Mactra chinensis KS disaccharide [d-GlcNAc6S-(1→3)-β-d-Gal-(1→] was attached to the C-3 position of GlcA, and consecutive KS-branched disaccharide sequences were found in a CS chain. KS-branched polysaccharides clearly exhibited resistance to degradation by chondroitinase ABC or ACII (at low concentrations) compared with typical CS structures. Furthermore, KS-branched polysaccharides stimulated neurite outgrowth of hippocampal neurons. These results strongly suggest that M. chinensis is a rich source of KS-branched CS, and it has important biological activities.

Keywords: Glycobiology; chondroitin sulfate; chondroitinase; keratan sulfate; proteoglycan.

Figure 1.. Disaccharide composition of CS of M. chinensis after chondroitinase ABC, ACI and ACII treatment.

Chromatograms of unsaturated disaccharides of Fr. 3 (A) and Fr. 4 (B) of CS obtained by weak anion-exchange chromatography (see Supplementary Figure S1). Unsaturated disaccharide analysis was performed as follows. CS (5 µg) were incubated in the reaction mixture (35 µl), which contained 28.6 mM Tris/acetate (pH 8.0) and 25 mU of Chase ABC, ACI or ACII. After incubation, depolymerized samples were submitted to gradient HPLC with fluorescence detection as described previously [17]. Experiments were repeated in triplicate with reproducible results. Peaks: 1, ΔDi-0S; 2, ΔDi-4S; 3, ΔDi-6S; 4, ΔDi-diS_E; a–d, unknown peaks.

Figure 2.. Different sensitivities of existing and unknown polysaccharides to chondroitinase ACII.

(A) Chromatogram of unsaturated disaccharides of clam CS (Fr. 4) obtained by Chase ACII. Clam CS (2.5 μg) in reaction mixture (17.5 μl) was treated with Chase ACII at the specified concentrations, and resulting unsaturated disaccharides were analyzed by HPLC. (B) Clam CS has consecutive repeating unknown structures. After incubation of RT mix (17.5 μl) containing 2.5 μg of clam CS and 1.6 mU of Chase ACII, remaining polysaccharides and unsaturated disaccharides were separated using HiTrap™ Desalting column. The isocratic elution condition was as follows: eluent, 10 mM ammonium bicarbonate; flow rate, 1.0 ml/min. To obtain the remaining polysaccharide, 3 mg of CS (Fr. 4) was treated with 3 units of Chase ACII. (C) Chromatogram of unknown structure treated with Chase ACII. Remaining polysaccharides (2.5 μg) were treated with 12.5 mU of Chase ACII in RT mix (17.5 μl). To collect the unknown peaks (c) and (d), 200 μg of remaining polysaccharides was degraded and fractionated (see Supplementary Figure S2). Peaks: 1, ΔDi-0S; 2, ΔDi-4S; 3, ΔDi-6S; 4, ΔDi-diS_E; a–d, unknown peaks.

Figure 3.. LC–MS/MS analysis of AMAC-labeled unknown peak (d).

(A) Extracted ion chromatograms (EICs) of unknown peak (d) obtained by partial degradation of Chase ACII (see Figure 2C). AMAC labeling and LC–MS analysis were performed according to the method of Yang et al. [23]. A single peak (m/z 588) at 23.4 min was also observed in TIC. (B) Mass spectra of unknown peak (d). MS² or MS³ was performed using m/z 588.17²⁻ or 548.29²⁻ as a precursor ion.

Figure 4.. Methylation analysis of unknown (d) peak.

PMAA from unknown (d) peak were performed as described in the Materials and methods section. To obtain PMAAs, 100 μg of unknown (d) peak was used. (A) EIC (m/z 233) of unknown (d) peak. (B) Mass spectrum of PMAA at 28.68 min. (C) Theoretical mass fragment pattern of 1,3,5-tri-O-acetyl-2,4,6-tri-O-methyl-galactitol [26].

Figure 5.. 2D COSY spectra of remaining polysaccharides obtained after the digestion of clam CS by chondroitinase ACII.

COSY spectra were recorded in D₂O at 30°C. Proton signals of GlcNAc were assigned according to the result of Hounsell et al. [28]. The cross-peaks are assigned as: (A1) ΔUA-H1/H2; (A2) ΔUA-H2/H3; (A3) ΔUA-H3/H4; (B1) GalNAc-H1/H2; (B2) GalNAc-H2/H3; (B3) GalNAc-H3/H4; (C1) GlcNAc-H1/H2; (C2) GlcNAc-H2/H3; (C3) GlcNAc-H3/H4; (C4) GlcNAc-H4/H5; (C5) GlcNAc-H5/H6.

Figure 6.

Proposed structure of KS disaccharide-branched CS (A) and of consecutively branched disaccharides in CS of M. chinensis (B).

Figure 7.. Effect of clam CS (Fr. 4) or KS-branched polysaccharides on neurite outgrowth of hippocampal neurons.

(A) Representative morphological features of E16 hippocampal neurons cultured with clam CS (Fr.4) or KS-branched polysaccharides. E16 hippocampal neuronal cells (16 000 cells/cm²) were cultured for 18 h on various substrates coated on poly-d,l-ornithine, fixed and immunostained as described in the Materials and methods section. (B) The length of the longest neurite of the randomly selected 50–100 individual neurons was measured. The values obtained from the three separate experiments are expressed as the means ± SEM. Mann–Whitney's U-test was used to evaluate the significance of differences between means (**P < 0.01).