Sulfation and Cation Effects on the Conformational Properties of the Glycan Backbone of Chondroitin Sulfate Disaccharides

Abstract

Chondroitin sulfate (CS) is one of several glycosaminoglycans that are major components of proteoglycans. A linear polymer consisting of repeats of the disaccharide -4GlcAβ1-3GalNAcβ1-, CS undergoes differential sulfation resulting in five unique sulfation patterns. Because of the dimer repeat, the CS glycosidic “backbone” has two distinct sets of conformational degrees of freedom defined by pairs of dihedral angles: (ϕ1, ψ1) about the β1-3 glycosidic linkage and (ϕ2, ψ2) about the β1-4 glycosidic linkage. Differential sulfation and the possibility of cation binding, combined with the conformational flexibility and biological diversity of CS, complicate experimental efforts to understand CS three-dimensional structures at atomic resolution. Therefore, all-atom explicit-solvent molecular dynamics simulations with Adaptive Biasing Force sampling of the CS backbone were applied to obtain high resolution, high precision free energies of CS disaccharides as a function of all possible backbone geometries. All ten disaccharides (β1-3 vs. β1-4 linkage x five different sulfation patterns) were studied; additionally, ion effects were investigated by considering each disaccharide in the presence of either neutralizing sodium or calcium cations. GlcAβ1-3GalNAc disaccharides have a single, broad, thermodynamically important free-energy minimum whereas GalNAcβ1-4GlcA disaccharides have two such minima. Calcium cations but not sodium cations bind to the disaccharides, and binding is primarily to the GlcA –COO− moiety as opposed to sulfate groups. This binding alters the glycan backbone thermodynamics in instances where a calcium cation bound to –COO− can act to bridge and stabilize an interaction with an adjacent sulfate group, whereas, in the absence of this cation, the proximity of a sulfate group to –COO− results in two like charges being both desolvated and placed adjacent to each other and is found to be destabilizing. In addition to providing information on sulfation and cation effects, the present results can be applied to building models of CS polymers and as a point of comparison in studies of CS polymer backbone dynamics and thermodynamics.

The dual role of the glycosaminoglycan chondroitin‐6‐sulfate in the development, progression and metastasis of cancer

“…By contrast, all of the disulfated CS disaccharides revealed the greatest conformational rigidity . Moreover, it has been reported that in contrast to the 6‐ O ‐sulfation, the addition of a sulfate moiety to the position at the 4 C of the GalNAc residue in the monosulfated disaccharide significantly reduced the rotation of the disaccharide backbone compared to the geometry of the unsulfated disaccharide . These restrictions affected the flexibility of the backbone at the reducing side, but especially at the non‐reducing side of the 4‐ O ‐sulfated GalNAc residue .…”

“…GlcA‐GalNAc and GalNAc‐GlcA) were found to display the greatest backbone flexibility compared to the corresponding sulfated analogues . By contrast, all of the disulfated CS disaccharides revealed the greatest conformational rigidity . Moreover, it has been reported that in contrast to the 6‐ O ‐sulfation, the addition of a sulfate moiety to the position at the 4 C of the GalNAc residue in the monosulfated disaccharide significantly reduced the rotation of the disaccharide backbone compared to the geometry of the unsulfated disaccharide .…”

“…However, some flexibility of this backbone that improves fitting the CS chain to a bound molecule can be regulated by rotation around the glycosidic linkages, which is defined by the pair of dihedral angles ψ and φ. To assess the conformational flexibility of various CS disaccharides, free energies have recently been determined to be a function of the disaccharide backbone geometry using molecular dynamics simulations with all‐atom explicit‐solvent force field technology . Using this method, two sequential variants of the unsulfated CS disaccharide (i.e.…”

“…Using this method, two sequential variants of the unsulfated CS disaccharide (i.e. GlcA‐GalNAc and GalNAc‐GlcA) were found to display the greatest backbone flexibility compared to the corresponding sulfated analogues . By contrast, all of the disulfated CS disaccharides revealed the greatest conformational rigidity .…”

“…Moreover, it has been reported that in contrast to the 6‐ O ‐sulfation, the addition of a sulfate moiety to the position at the 4 C of the GalNAc residue in the monosulfated disaccharide significantly reduced the rotation of the disaccharide backbone compared to the geometry of the unsulfated disaccharide . These restrictions affected the flexibility of the backbone at the reducing side, but especially at the non‐reducing side of the 4‐ O ‐sulfated GalNAc residue . Thus, because the monosulfated disaccharides are predominant elements in the CS composition, any marked alterations in the proportion between the 6‐ O ‐sulfated and 4‐ O ‐sulfated units can significantly modulate the binding potential of this GAG through an influence on the conformational flexibility of the backbone of its chain and/or the proper positioning of the disulfated disaccharides.…”