CHEMICAL AND IMMUNOLOGICAL ASSAY OF THE NONREDUCING TERMINAL RESIDUESOF CHONDROITIN SULFATE FROM HUMAN AGGRECAN

Samples of aggrecan chondroitin sulfate, isolated from normal human kn ee cartilages of individuals from fetal to 72 years of age, were diges ted with chondroitin lyases, The products were analyzed by fluorescenc e-based anion exchange high performance liquid chromatography to separ ate and quantitate nonreducing terminal structures, in addition to int ernal unsaturated disaccharide products, The predominant terminal stru ctures mere the monosaccharides, GalNAc4S and GalNAc4,6S as they were present on 85-90% of all chains. The remaining chains terminated with the disaccharides GlcA beta 1,3GalNAc4S and GlcA beta 1,3GalNAc6S. Mar ked changes in the relative abundance of these terminals were identifi ed in the transition from growth cartilage to adult articular cartilag e, First, terminal GalNAc residues were almost exclusively 4-sulfated in aggrecan from fetal through 15 years of age, but were similar to 50 % 4,6-disulfated in aggrecans from adults (22-72 years of age). Second , the terminal disaccharide GlcA beta 1,3GalNAc4S was on similar to 7% of chains on aggrecan from fetal through 15 years of age, but on only similar to 3% of chains on adult aggrecan. In contrast, the proportio n of chains terminating in GlcA beta 1,3GalNAc6S, similar to 9%, was u nchanged from fetal to 72 years of age, This terminal disaccharide is proposed to be recognized by the widely used monoclonal antibody 3B3, However, chemical quantitation of the structure together with solid ph ase 3B3(-) immunoassay of fetal and adult aggrecans showed that the co ntent of the terminal disaccharide does not necessarily correlate with immunoreactivity of the proteoglycan, as chain density and presentati on on the solid phase are critical factors for recognition of chain te rminals by 3B3. The quantitative results obtained from chemical analys es of all nonreducing termini of aggrecan chondroitin sulfate chains r evealed important changes in chain termination that occur when cellula r activities are altered as adult articular cartilage is formed after removal of growth cartilage, These findings are discussed in relation to specific enzymatic steps that generate the nonreducing termini of c hains in the biosynthesis pathway of chondroitin sulfate proteoglycans and their modulation in tissue development and pathology.