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Fibromodulin from bovine articular cartilage has been subjected to lectin affinity chromatography by Sambucus nigra lectin which binds α(2-6)- linked N-acetylneuraminic acid, and the structure of the keratan sulphate in the binding and non-binding fractions examined by keratanase II digestion and subsequent high pH anion exchange chromatography. It has been confirmed that the keratan sulphate chains attached to fibromodulin isolated from bovine articular cartilage may have the chain terminating N-acetylneuraminic acid residue α(2-3)- or α(2-6)-linked to the adjacent galactose residue. Although the abundance of α(2-6)-linked N-acetylneuraminic acid (ca. 22%) is such that this could cap one of the four chains in almost all fibromodulin molecules, it was found that ca. 34% of the fibromodulin proteoglycan molecules from bovine articular cartilage were capped exclusively with α(2-3)-linked N-acetylneuraminic acid. The remainder of the fibromodulin proteoglycans, which bound to the lectin had a mixture of α(2-3)- and α(2-6)-linked N-acetylneuraminic acid capping structures. The keratan sulphates attached to fibromodulin molecules capped exclusively with α(2-3)- linked N-acetylneuraminic acid were found to have a higher level of galactose sulphation than those from fibromodulin with both α(2-3)- and α(2-6)-linked N-acetylneuraminic acid caps, which bound to the Sambucus nigra lectin. In addition, both pools contained chains of similar length (ca. 8-9 disaccharides). Both also contained α(1-3)-linked fucose, showing that this feature does not co-distribute with α(2-6)-linked N-acetylneuraminic acid, although these two features are present only in mature articular cartilage. These data show that there are discrete populations of fibromodulin within articular cartilage, which may have differing impacts upon tissue processes.

Chondroitin and dermatan sulfate (CS and DS) chains were isolated from bovine tracheal cartilage and pig intestinal mucosal preparations and fragmented by enzymatic methods. The oligosaccharides studied include a disaccharide and hexasaccharides from chondroitin ABC lyase digestion as well as trisaccharides already present in some commercial preparations. In addition, other trisaccharides were generated from tetrasaccharides by chemical removal of nonreducing terminal residues. Their structures were examined by high-field 1H and 13C NMR spectroscopy, after reduction using sodium borohydride. The main hexasaccharide isolated from pig intestinal mucosal DS was found to be fully 4-O-sulfated and have the structure: ΔUA(β1–3)GalNAc4 S(β1–4) l-IdoA(α1–3)GalNAc4 S(β1–4) l-IdoA(α1–3)GalNAc4 S-ol, whereas one from bovine tracheal cartilage CS comprised only 6-O-sulfated residues and had the structure: ΔUA(β1–3)GalNAc6 S(β1–4)GlcA(β1–3)GalNAc6 S(β1–4)GlcA(β1–3)GalNAc6 S-ol. No oligosaccharide showed any uronic acid 2-sulfation. One novel disaccharide was examined and found to have the structure: GalNAc6 S(β1–4)GlcA-ol. The trisaccharides isolated from the CS/DS chains were found to have the structures: ΔUA(β1–3)GalNAc4 S(β1–4)GlcA-ol and ΔUA(β1–3)GalNAc6 S(β1–4)GlcA-ol. Such oligosaccharides were found in commercial CS/DS preparations and may derive from endogenous glucuronidase and other enzymatic activity. Chemically generated trisaccharides were confirmed as models of the CS/DS chain caps and included: GalNAc6 S(β1–4)GlcA(β1–3)GalNAc4 S-ol and GalNAc6 S(β1–4)GlcA(β1–3)GalNAc6 S-ol. The full assignment of all signals in the NMR spectra are given, and these data permit the further characterization of CS/DS chains and their nonreducing capping structures. [ABSTRACT FROM AUTHOR]

Fibromodulin has been isolated from bovine and equine articular cartilage and the attached keratan sulphate chains subjected to digestion by keratanase II. The oligosaccharides generated have been reduced and subsequently isolated by strong anion-exchange chromatography. Their structures have been determined by high-field ¹H-NMR spectroscopy and high-pH anion-exchange chromatography. Both α(2-6)- and α(2-3)- linked N-acetylneuraminic acid have been found as a branch in both repeat region and capping oligosaccharides. These data demonstrate that there are fundamental differences between the structures present in the N-linked keratan sulphate chains attached to fibromodulin from articular cartilage and those from tracheal cartilage, which lack both α(2-6)-linked N-acetylneuraminic acid and α(1-3)-linked fucose. It has been confirmed that the keratan sulphate chains are short, being only eight or nine disaccharides in length. Very significant differences in the levels of galactose sulphation have been identified at the non-reducing end of the chain. The galactose residue adjacent to the non-reducing cap is sulphated in only 1-3% of chain, compared with a sulphation level of over 40% closer to the reducing end. This to the potential for functional importance. The repeat region and capping fragments of the N-linked keratan sulphates from bovine and equine articular cartilage fibromodulin have been found to have to the following general structure: NeuAc-9α2-3/6) Gal [6SO3-](β1-4) glcNAc6SO3-(β1-3)Gal [6SO3-] (β1-4) { [Fuc(α1-3)]0-1 GlcNAc6SO3- (β1-3) Gal-[6SO3-](β1-4)}6-7 GlcNAc6SO3-. [ABSTRACT FROM AUTHOR]