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The chondroitin sulphate (CS) linkage regions have been isolated from human articular cartilage aggrecan (from 10- to 72-year-olds) by chondroitin ABC endolyase digestion and size-exclusion chromatography. Linkage region hexasaccharides have been characterized and their abundance estimated by high-pH anion-exchange chromatography. The basic structure for the CS linkage region oligosaccharides identified from human aggrecan is as follows: ΔUA(β1–3)GalNAc[0S/4S/6S](β1–4)GlcA(β1–3)Gal[0S/6S](β1–3)Gal(β1–4)Xyl, where ΔUA represents 4,5-unsaturated hexuronic acid, 4S and 6S represent an O-ester sulphate group on C-4 and C-6 respectively, and 0S represents zero sulphation. There are significant age-related changes in the abundance of the various N-acetylgalactosamine (GalNAc) sulphation forms identified, occurring up to approx. 20 years old. During the period from 10 to 20 years old the level of GalNAc 6-sulphation at the linkage region increases from approx. 43% to approx. 75%, while there is a corresponding reduction in unsulphated (approx. 30% to approx. 20%) and 4-sulphated (approx. 25% to approx. 6%) GalNAc residues. There is also an increase in the incidence of linkage region galactose 6-sulphation (approx. 2% to approx. 10%) which was only observed in linkage regions with GalNAc 6-sulphation. Beyond 20 years old there are few changes in the relative abundance of these GalNAc sulphation variants; however, there is a slight increase in the abundance of 6-sulphation between approx. 20 years old and approx. 40 years old and a slight decrease in its abundance beyond approx. 40 years old. Our data show that in the majority of chains from tissues of all ages the GalNAc residue closest to the linkage region is 6-sulphated, but the level of GalNAc 6-sulphation within the linkage region is lower than the average level observed within the repeat region.

Fried, M., Lauder, R. M., and Duffy, P. E. 2000. Plasmodium falciparum: Adhesion of placental isolates modulated by the sulfation characteristics of the glycosaminoglycan receptor. Experimental Parasitology95, 75–78.

We report the isolation, characterization and quantification of five octasaccharides, four hexasaccharides and two tetrasaccharides, derived from the chondroitin sulphate (CS) linkage region of 6-8-year-old bovine articular cartilage aggrecan, following digestion with chondroitin ABC endolyase. Using a novel high-pH anion-exchange chromatography (HPAEC) method, in conjunction with one- and two-dimensional 1H-NMR spectroscopy, we have identified the following basic structure for the CS linkage region of aggrecan: ∆UA(β1-3)GalNAc[0S/4S/6S](β1-4)GlcA(β1-3)GalNAc[0S/4S/6S](β1-4)GlcA(β1-3)Gal[0S/6S](β1-3)Gal(β1-4)Xyl, where ∆UA represents 4,5-unsaturated hexuronic acid, and 4S and 6S represent an O-ester sulphate group on C-4 and C-6 respectively. The octa-, hexa- and tetra-saccharide linkage region fragments were used to develop a HPAEC fingerprinting method, with detection at A232 nm, and a linear response to approx. 0.1 nmol of substance. The sulphation patterns of CS linkage regions, of up to octasaccharide in size, from articular and tracheal cartilage aggrecan were examined. The results show that in articular cartilage, for the majority (53%) of octasaccharides the 2-deoxy-2-N-acetyl amino-D-galactose (GalNAc) residues closest to the linkage region are both 6-sulphated; however, in a significant portion (34%), one or more of these GalNAc residues are unsulphated, and in 8% both are unsulphated. Approximately 10-18% of the chains have a 4-sulphated GalNAc in the first disaccharide, and 12% have a sulphated linkage region Gal residue. No evidence was found for uronic acid sulphation. These data show that there is a significant increase in the incidence of unsulphated and 4-sulphated GalNAc residues adjacent to the linkage region compared with the rest of the chain. Bovine tracheal cartilage linkage regions displayed very similar sulphation profiles to those from articular cartilage, despite the presence of a higher level of GalNAc 4-sulphation within the repeat region of the main CS chain.

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