Keratan sulphate in the tumour environment

Keratan sulphate (KS) is a bioactive glycosaminoglycan (GAG) of some complexity composed of the repeat disaccharide D-galactose β1→4 glycosidically linked to N-acetyl glucosamine. During the biosynthesis of KS, a family of glycosyltransferase and sulphotransferase enzymes act sequentially and in a coordinated fashion to add D-galactose (D-Gal) then N-acetyl glucosamine (GlcNAc) to a GlcNAc acceptor residue at the reducing terminus of a nascent KS chain to effect chain elongation. D-Gal and GlcNAc can both undergo sulphation at C6 but this occurs more frequently on GlcNAc than D-Gal. Sulphation along the developing KS chain is not uniform and contains regions of variable length where no sulphation occurs, regions which are monosulphated mainly on GlcNAc and further regions of high sulphation where both of the repeat disaccharides are sulphated. Each of these respective regions in the KS chain can be of variable length leading to KS complexity in terms of chain length and charge localization along the KS chain. Like other GAGs, it is these variably sulphated regions in KS which define its interactive properties with ligands such as growth factors, morphogens and cytokines and which determine the functional properties of tissues containing KS. Further adding to KS complexity is the identification of three different linkage structures in KS to asparagine (N-linked) or to threonine or serine residues (O-linked) in proteoglycan core proteins which has allowed the categorization of KS into three types, namely KS-I (corneal KS, N-linked), KS-II (skeletal KS, O-linked) or KS-III (brain KS, O-linked). KS-I to -III are also subject to variable addition of L-fucose and sialic acid groups. Furthermore, the GlcNAc residues of some members of the mucin-like glycoprotein family can also act as acceptor molecules for the addition of D-Gal and GlcNAc residues which can also be sulphated leading to small low sulphation glycoforms of KS. These differ from the more heavily sulphated KS chains found on proteoglycans. Like other GAGs, KS has evolved molecular recognition and information transfer properties over hundreds of millions of years of vertebrate and invertebrate evolution which equips them with cell mediatory properties in normal cellular processes and in aberrant pathological situations such as in tumourogenesis. Two KS-proteoglycans in particular, podocalyxin and lumican, are cell membrane, intracellular or stromal tissue–associated components with roles in the promotion or regulation of tumour development, mucin-like KS glycoproteins may also contribute to tumourogenesis. A greater understanding of the biology of KS may allow better methodology to be developed to more effectively combat tumourogenic processes

Constant and variable domains of different disaccharide structure in corneal keratan sulphate chains.

Four peptidokeratan sulphate fractions of different Mr and degree of sulphation were cut from the pig corneal keratan sulphate distribution spectrum. After exhaustive digestion with keratanase, the fragments were separated on DEAE-Sephacel and Bio-Gel P-10 and analysed for their Mr, degree of sulphation and amino sugar and neutral sugar content. It was found that every glycosaminoglycan chain is constructed of a constant domain of non-sulphated and monosulphated disaccharide units and a variable domain of disulphated disaccharide units. Total neuraminic acid of the four peptidokeratan sulphates was recovered from their isolated linkage-region oligosaccharides. In kinetic studies, the four peptidokeratan sulphates were investigated for Mr distribution after various incubation times with keratanase. There was a continuous shift towards lower Mr and no appearance of a distinct intermediate-sized product at any degradation time. The linkage-region oligosaccharide was already being liberated after a very short incubation period. From the results of these kinetic investigations in connection with the results of neuraminic acid analyses it is suggested that there exists only one disaccharide chain per peptidokeratan sulphate molecule. A model of corneal keratan sulphate is postulated. One of the alpha-mannose residues in the linkage region is bound to an oligosaccharide consisting of a lactosamine and a terminal sialic acid. The other alpha-mannose residue is attached to the disaccharide chain. This chain contains one or two non-sulphated disaccharide units at the reducing end, followed by 10-12 monosulphated disaccharide units. The disulphated disaccharide moiety of variable length is positioned at the non-reducing end of the chain

Structure of the keratan sulphate chains attached to fibromodulin isolated from bovine tracheal cartilage. Oligosaccharides generated by keratanase digestion.

The structure of the repeat region and chain caps of the N-linked keratan sulphate chains attached to bovine tracheal cartilage fibromodulin has been examined. The chains were fragmented by keratanase digestion, the resultant oligosaccharides isolated by strong anion-exchange chromatography, and their structures determined using high-field 1H-n.m.r. spectroscopy. The chains were found to possess the following general structure: [formula: see text] All of the capping oligosaccharides isolated terminate with alpha(2-3)-linked N-acetylneuraminic acid. No alpha(2-6)-linked N-acetylneuraminic acid chain terminators, nor any fucose, alpha (1-3)-linked to N-acetylglucosamine along the repeat region, were detected. This work demonstrates that the structure of the repeat region and chain caps of N-linked keratan sulphate attached to fibromodulin isolated from bovine tracheal cartilage is identical with that of O-linked keratan sulphate chains attached to aggrecan derived from non-articular cartilage