A 3D-structural model of unsulfated chondroitin from high-field NMR: 4-sulfation has little effect on backbone conformation

Abstract

The glycosaminoglycan chondroitin sulfate is essential in human health and disease but exactly how sulfation dictates its 3D-structure at the atomic level is unclear. To address this, we have purified homogenous oligosaccharides of unsulfated chondroitin (with and without (15)N-enrichment) and analysed them by high-field NMR to make a comparison published chondroitin sulfate and hyaluronan 3D-structures. The result is the first full assignment of the tetrasaccharide and an experimental 3D-model of the hexasaccharide (PDB code 2KQO). In common with hyaluronan, we confirm that the amide proton is not involved in strong, persistent inter-residue hydrogen bonds. However, in contrast to hyaluronan, a hydrogen bond is not inferred between the hexosamine OH-4 and the glucuronic acid O5 atoms across the beta(1-->3) glycosidic linkage. The unsulfated chondroitin bond geometry differs slightly from hyaluronan by rotation about the beta(1-->3) psi dihedral (as previously predicted by simulation), while the beta(1-->4) linkage is unaffected. Furthermore, comparison shows that this glycosidic linkage geometry is similar in chondroitin-4-sulfate. We therefore hypothesise that both hexosamine OH-4 and OH-6 atoms are solvent exposed in chondroitin, explaining why it is amenable to sulfation and hyaluronan is not, and also that 4-sulfation has little effect on backbone conformation. Our conclusions exemplify the value of the 3D-model presented here and progress our understanding of glycosaminoglycan molecular properties.

Graphical abstract

Figure 1

The repeating disaccharide units of unsulfated chondroitin and hyaluronan (grey). The glucuronic acid (GlcA; U) and N-acetyl-d-galactosamine (GalNAc; N) residues of chondroitin are joined by β(1→3) and β(1→4) glycosidic linkages. In this report the IUPAC ring numbering convention is used, that is, residues are numbered starting from the reducing end, which is capped by a hydroxyl. The adopted nomenclature for identifying carbon atoms is indicated. Hydrogen atoms assume the number of the closest bonded carbon atom.

Figure 2

[¹H, ¹⁵N]-HSQC spectra of CN oligosaccharides of different lengths. (A) Complete (broken-axis) spectrum of CN₆. Panels (B, C and D) illustrate internal amide resonances in magnified portions of CN₄, CN₆ and CN₈ spectra, respectively. The ³J_HN,H2 coupling constant of the N3 residue in CN₄ (also see Table 4) is annotated in panel (B). Standard errors: ¹H ±0.001 ppm, ¹⁵N ±0.003 ppm. Data were acquired at 600 MHz, pH 6.0, 25 °C in 5–10% (v/v) D₂O and referenced indirectly relative to δ_DSS (¹H).

Figure 3

(A) The [¹H]–1D NMR spectrum of the ¹⁵N-enriched unsulfated chondroitin tetrasaccharide. (B) The amide region magnified. Resonances attributed to residues GalNAc-1α, GalNAc-1β and GalNAc-3 are labelled. The large (≈90 Hz) ¹J_H,N and small (≈10 Hz) ³J_HN,H2 couplings are listed in Table 4. Data were acquired at 600 MHz, pH 6.0, 25 °C in 5–10% (v/v) D₂O and referenced relative to δ_DSS (¹H). Standard error: ¹H ±0.001 ppm.

Figure 4

Two conformationally dependent NOEs used in the structure calculations: (A) the β(1→4) glycosidic linkage NOE, and (B) the β(1→3) glycosidic linkage NOE. Data were obtained from a 900 MHz [¹H, ¹H]-NOESY spectrum of natural abundance isotope CN₆ with the axes transposed. The CN₆ chemical shift assignments are available as Supplementary data. The sample was at pH 6.0, 25 °C in 5–10% (v/v) D₂O and referenced relative to δ_DSS (¹H). Standard error: ¹H ±0.001 ppm.

Figure 5

Superposition of the 25 most energetically favourable CN₆ model conformers, PDB code 2KQO (following 750 ns restrained simulated annealing). The highlighted distances (Å) were measured from the lowest energy conformer and corresponded to the ¹H–¹H distance restraints.

Figure 6

Comparison of NMR-based β(1→3) linkage geometries for: (A) chondroitin-4-sulfate, (B) unsulfated chondroitin, and (C) hyaluronan. Geometries from: (A) Yu et al., (B) the lowest energy 3D-model of CN₆ in this work, and (C) Almond et al. For clarity, all hydrogen atoms are hidden except for OH-4. Indicated distances (Å) are between GalNAc/GlcNAc OH-4 and GlcA O5 atoms.