General composition and structure Proteoglycans are macromolecules which consist of a protein backbone to which GAG chain(s) and Nand/or 0-linked oligosaccharides are covalently attached (Fig. 1). The composition of GAG chains of PGs and their linkages to core proteins are described in Table 1. In the corneal stroma, KS is attached to protein by a linkage of the kind used to attach the complex-type and highmannose oligosaccharides of glycoproteins and PGs to protein [118]. To the core proteins of all PGs are also bound 0-linked oligosaccharides and/or complex-type N-linked oligosaccharides [81]. The core proteins of PGs have been the least well studied components of these molecules: this is discussed below. The complete core protein sequence has so far been established for only one PG [20]. In view of the considerable complexity ofPG structure and composition, for us to organize a logical classification scheme for PGs we need to know not only the identities of the constituent GAGs but also, and primarily, the core-protein sequences. We will now discuss the different types of PGs and their known or suspected functions.

Proteoglycans in health and disease: structures and functions.

Chondroitin sulphate proteoglycans are synthesised by different tissues and cell types, and the chondroitin sulphate chains are variably sulphated. Three monoclonal antibodies 3B3, 7D4 and 6C3 that recognise different native chondroitin sulphate epitopes have been used to investigate changes in structure during embryonic tissue development in the chick and in the response of mature canine articular cartilage during experimental osteoarthritis. Strong focal expression of the epitopes was seen during development of chick bursa, which was different for the three epitopes and which changed during 5 days of development. In embryonic chick limb, although chondroitin sulphate is present throughout the cartilage, the 3B3 epitope, which is at the non-reducing terminus of chains, was only expressed on chondroitin sulphate within one region of the sub-articular cartilage. In mature canine articular cartilage the expression of this epitope on proteoglycans was very low, but when determined 3 or 6 months after induction of experimental osteoarthritis the level was greatly increased in all joints tested (23/23). The abundance of the other two native chondroitin sulphate epitopes was also increased in this experimental disease. The results show that expression of the chondroitin sulphate epitopes detected by the monoclonal antibodies changes during cellular differentiation and development and suggests that it is closely controlled by the cells synthesising chondroitin sulphate chains.

Modulation of native chondroitin sulphate structure in tissue development and in disease.

Hyaluronan and chondroitin/dermatan sulfate are glycosaminoglycans that play major roles in the biomechanical properties of a wide variety of tissues, including cartilage. A chondroitin/dermatan sulfate chain can be divided into three regions: (1) a single linkage region oligosaccharide, through which the chain is attached to its proteoglycan core protein, (2) numerous internal repeat disaccharides, which comprise the bulk of the chain, and (3) a single nonreducing terminal saccharide structure. Each of these regions of a chondroitin/dermatan sulfate chain has its own level of microheterogeneity of structure, which varies with proteoglycan class, tissue source, species, and pathology. We have developed rapid, simple, and sensitive protocols for detection, characterization and quantitation of the saccharide structures from the internal disaccharide and nonreducing terminal regions of hyaluronan and chondroitin/dermatan sulfate chains. These protocols rely on the generation of saccharide structures with free reducing groups by specific enzymatic treatments (hyaluronidase/chondroitinase) which are then quantitatively tagged though their free reducing groups with the fluorescent reporter, 2-aminoacridone. These saccharide structures are further characterized by modification through additional enzymatic (sulfatase) or chemical (mercuric ion) treatments. After separation by fluorophore-assisted carbohydrate electrophoresis, the relative fluorescence in each band is quantitated with a cooled, charge-coupled device camera for analysis. Specifically, the digestion products identified are (1) unsaturated internal Deltadisaccharides including DeltaDiHA, DeltaDi0S, DeltaDi2S, DeltaDi4S, DeltaDi6S, DeltaDi2,4S, DeltaDi2,6S, DeltaDi4,6S, and DeltaDi2,4,6S; (2) saturated nonreducing terminal disaccharides including DiHA, Di0S, Di4S and Di6S; and (3) nonreducing terminal hexosamines including glcNAc, galNAc, 4S-galNAc, 6S-galNAc, and 4, 6S-galNAc.

Microanalysis of enzyme digests of hyaluronan and chondroitin/dermatan sulfate by fluorophore-assisted carbohydrate electrophoresis (FACE).

Microanalysis of glycosaminoglycan-derived oligosaccharides labeled with a fluorophore 2-aminobenzamide by high-performance liquid chromatography: application to disaccharide composition analysis and exosequencing of oligosaccharides.

Two Distinct Chondroitin Sulfate ABC Lyases AN ENDOELIMINASE YIELDING TETRASACCHARIDES AND AN EXOELIMINASE PREFERENTIALLY ACTING ON OLIGOSACCHARIDES

A method was developed for the analysis of non-reducing terminal structure of radiolabelled chondroitin sulphate chains with the aid of N-acetylgalactosamine 4-sulphatase ('terminal 4-sulphatase'), N-acetylgalactosamine 6-sulphatase ('terminal 6-sulphatase'), beta-glucuronidase and beta-N-acetylhexosaminidase Studies with this method on the non-reducing terminal structure of [35S]sulphate- and [3H]glucose-labelled chondroitin sulphate chains from rat and chick-embryo cartilages showed that the presence of a high proportion of 4-sulphated hexosamine residues is a common feature of the termini of newly synthesized chondroitin sulphate chains Of the non-reducing terminal 4-sulphated hexosamine residues, about 14% (chick embryo) or 46% (rat) contained an additional sulphate group at position 6 The internal portion of the chondroitin sulphate chains, in contrast, contained little or no 4,6-bis-sulphated hexosamine residue, suggesting that 4,6-bis-sulphated structure may play a role in biosynthetic control at the level of chain termination

A distinct terminal structure in newly synthesized chondroitin sulphate chains.

Glycosaminoglycan sulfotransferases in human and animal sera.

Difference between N-acetylgalactosamine 4-sulfate 6-O-sulfotransferases from human serum and squid cartilage in specificity toward the terminal and interior portion of chondroitin sulfate.

H Inoue

Papers

A distinct terminal structure in newly synthesized chondroitin sulphate chains.

Glycosaminoglycan sulfotransferases in human and animal sera.

Difference between N-acetylgalactosamine 4-sulfate 6-O-sulfotransferases from human serum and squid cartilage in specificity toward the terminal and interior portion of chondroitin sulfate.

A sulfotransferase-sulfatase system in avian oviduct which catalyzes a conversion of UDP-N-acetylgalactosamine 4-sulfate to the 6-sulfate isomer.