Sulphation heterogeneity in the trisaccharide (GalNAcS beta 1,4GlcA beta 1,3GalNAcS) isolated from the non-reducing terminal of human aggrecan chondroitin sulphate

We report here the isolation and sulphation isomer analyses of trisaccharid es GalNAc(beta 1, 4)GlcA(beta 1, 3)GalNAcS (in which S indicates sulphate) derived from the non-reducing termini of aggrecan chondroitin sulphate. Rat chondrosarcoma and human aggrecans were digested for Ih at 37 degrees C wi th 30 mu-units of endo-chondroitinase ABC per mu g of chondroitin sulphate, and trisaccharides were isolated from the digests by ToyoPearl HW40S gel-f iltration chromatography. Four trisaccharide species were identified; their sulphation isomer compositions, as determined by digestion with chondroiti nase ACII and fluorescence-based ion-exchange HPLC, were GalNAc4S beta 1,4 GlcA beta 1,3GalNAc4S, GalNAc4S beta 1,4GlcA beta 1, 3GalNAc6S, GalNAc4,6S beta 1,4GlcA beta 1,3GalNAc4S and GalNAc4,6S beta 1,4GlcA beta 1,3GalNAc6S. The abundances of such sequences in chondroitin sulphate on aggrecan from normal (foetal to 72 years of age) and from osteoarthritic human knee carti lages were also established. The results showed that non-reducing terminal GalNAc4S or GalNAc4,6S can be linked to either a 4-sulphated or a 6-sulphat ed disaccharide, suggesting that the sulphation of the last disaccharide mi ght not have a direct effect on the specificity of chondroitin sulphate ter minal GalNAc sulphotransferases. Furthermore, for each aggrecan preparation examined, the 4S-to-6S ratio of all chain interior disaccharides was equiv alent to that in the last repeating disaccharides at the nonreducing termin us, suggesting that neither chondroitin 4-sulpho-transferase nor chondroiti n 6-sulphotransferase shows preferential activity near the chain terminus.