Secondary and tertiary structures involving chondroitin and chondroitin sulphates in solution, investigated by rotary shadowing/electron microscopy and computer simulation

Abstract

Rotary shadowing/electron microscopy of chondroitin 6-sulphate (CS6) and 4-sulphate (CS4) showed that the former, but not the latter, aggregated to mesh works. Preparations made from salt (ammonium acetate) solutions showed enhanced aggregation. Computer modelling, using molecular mechanics and dynamics, was applied to secondary structures (twofold helices) derived from NMR studies, to determine geometric and energetic constraints on duplex and higher-aggregate formation. The calculations suggested that chondroitin, CS6 and undersulphated CS4 could form duplexes, while CS4 could not, thus bridging the gap between atomic dimensions (NMR) and high polymer scale (clcctron microscopy). Calculations suggested that water structure helped to stabilise the twofold helix. It is proposed that the twofold helical, flat, tape-like molecules aggregate via hydrophobic bonding between the very extensive hydrophobic patches (9 CH units) repeated on alternating sides of the polymers. The negative charge of the polyanions opposes aggregate formation. Calculations showed that duplexes were formed with decreasing stability as the charge density increased, and as the charge was concentrated towards the centre line of the polymer (i.e. in CS4). The unsulphated polymer chondroitin could form duplexes and higher aggregates as readily as hyaluronan. Hyaluronan was calculated to form stable heteroduplexes with CS6 and CS4. The frequency and positioning of the sulphate-ester group within the polymer thus determines whether the molecule participates in duplex formation.The extra-cellular matrix of mammalian connective tissues is based on two structural elements: (a) insoluble fibres, predominantly collagenous ; (b) soluble polyanions, of a largely carbohydrate nature. The former resist or transmit tensile stresses, whereas the latter resist and absorb compressive Forces [I]. The two elements interact with each other in a very specific manner [2], implying that there are complementary binding sites, with the corollary that the shapes of the interactants must be at least semipermanent. The tertiary structures of collagen molecules, and to some extent the quaternary structure of collagen fibrils, are established [3], but the soluble polyanions have not been investigated as extensively, partly because they function in aqueous solution. There was doubt that they had stable secondary structures, or formed tertiary structures based on glycan/glycan interactions.The soluble carbohydrate polyanions are of two types, the proteoglycans, consisting of a polypeptide core with long polyanionic glycosaminoglycan (GAG) side chains, and hyaluronan (HA), a GAG that is not covalently attached to protein. [2], corresponded to characteristic differences in appearance in electron microscopy, after rotary shadowing [4-61. HA and the proteoglycan GAG chondroitin sulphate (CS), keratan sulphatc and dermatan sulphate consist of repeating disaccharide units, comprising glucosamine or galactosamine, and a uronic acid (glucuronic or iduronic) except in the case of k...

Functional Hierarchy of Simultaneously Expressed Adhesion Receptors: Integrin α2β1 but Not CD44 Mediates MV3 Melanoma Cell Migration and Matrix Reorganization within Three-dimensional Hyaluronan-containing Collagen Matrices

“…Efficient interpenetration and immobilization of HA in collagen lattices are supported by ultracentrifugation data showing a considerably greater mechanical stability and resistance to compression in the presence of HA (Fessler, 1960). The polysaccharide CS additionally cross-links HA monomers to form stable heteroduplexes (Scott et al, 1992), thereby increasing HA polymer rigidity (Nishimura et al, 1998). CS at physiological concentrations (5-40 mg/ml), as detected in dermis or cartilage, does not self-assemble (Scott et al, 1991 and, but increases the viscosity of HA gels Figure 6.…”

“…On 2-D surfaces, HA forming long and branched strands sticks to the surface, providing a highly stable and presumably nondeformable anchoring structure (Scott et al, 1991). In 3-D tissues, however, HA forms thick fibrillar aggregates 10 -40 nm in diameter that multimerize to 3-D honeycomb-like networks starting at a low dilution of Ͻ1 g/ml (Scott et al, 1992;Scott, 1995) and a low molecular weight of 300 kDa (Scott et al, 1992). HA self-assembly results in elastic gel-like meshworks that can occur independent of other ECM components (Scott et al, 1991).…”

“…HA self-assembly results in elastic gel-like meshworks that can occur independent of other ECM components (Scott et al, 1991). HA also interacts with and branches other ECM components such as collagen fibers (Turley et al, 1985;Scott et al, 1992), thereby potentially serving as a proadhesive substrate (Scott, 1995). Efficient interpenetration and immobilization of HA in collagen lattices are supported by ultracentrifugation data showing a considerably greater mechanical stability and resistance to compression in the presence of HA (Fessler, 1960).…”

Upregulation of chondroitin 6-sulphotransferase-1 facilitates Schwann cell migration during axonal growth

“…Both electron microscopy and computer modelling of chondroitin sulphate isomers have shown that 6-sulphated chains assume a secondary structure that can electrostatically and sterically accommodate close packing of 6-sulphated and non-sulphated chondroitins but not 4-sulphated chondroitins (Scott et al, 1992). The capacity for aggregation among 6-sulphated chondroitins and nonsulphated glycosaminoglycans such as hyaluronans (Turley and Roth, 1980;Scott, 2003) suggests a matrix that is not readily penetrable by advancing terminals of growing axons.…”

The analysis of intermolecular interactions in concentrated hyaluronan solutions suggest no evidence for chain–chain association

“…In the present study the sensitivity of HA networks to counterion type and valency was determined, as these are known to greatly affect rheological and hydrodynamic properties [5]. The presence of inter-chain associations that might involve hydrophobic interactions were investigated under physiological conditions, in solvents of varying polarity and in the presence of chaotropic agents [9,[14][15][16][17]. Intermolecular chain-chain associations were also investigated using HA oligosaccharides as low-molecular-mass competitors of such interactions [3,18,19].…”