Abstract
Samples of aggrecan chondroitin sulfate, isolated from normal human knee cartilages of individuals from fetal to 72 years of age, were digested with chondroitin lyases. The products were analyzed by fluorescence-based anion exchange high performance liquid chromatography to separate and quantitate nonreducing terminal structures, in addition to internal unsaturated disaccharide products. The predominant terminal structures were the monosaccharides, GalNAc4S and GalNAc4,6S as they were present on 85-90% of all chains. The remaining chains terminated with the disaccharides GlcAbeta1,3GalNAc4S and GlcAbeta1,3GalNAc6S. Marked changes in the relative abundance of these terminals were identified in the transition from growth cartilage to adult articular cartilage. First, terminal GalNAc residues were almost exclusively 4-sulfated in aggrecan from fetal through 15 years of age, but were approximately 50% 4,6-disulfated in aggrecans from adults (22-72 years of age). Second, the terminal disaccharide GlcAbeta1,3GalNAc4S was on approximately 7% of chains on aggrecan from fetal through 15 years of age, but on only approximately 3% of chains on adult aggrecan. In contrast, the proportion of chains terminating in GlcAbeta1,3GalNAc6S, approximately 9%, was unchanged 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 phase 3B3(-) immunoassay of fetal and adult aggrecans showed that the content of the terminal disaccharide does not necessarily correlate with immunoreactivity of the proteoglycan, as chain density and presentation on the solid phase are critical factors for recognition of chain terminals by 3B3. The quantitative results obtained from chemical analyses of all nonreducing termini of aggrecan chondroitin sulfate chains revealed important changes in chain termination that occur when cellular 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 chains in the biosynthesis pathway of chondroitin sulfate proteoglycans and their modulation in tissue development and pathology.
Highlights
Proteoglycans (PGs)1 are important components of extracellular matrices
We examined the possibility that the marked change in the average hydrodynamic size of CS on aggrecan deposited into the cartilages at different stages of development and maturation may be accompanied by changes in the composition of the nonreducing chain terminals
At skeletal maturity (.15 years) as growth cartilage is removed and replaced by adult cartilage, the average aggrecan CS chain size was decreased from 20 kDa to about 8 kDa, and the ratio of 6- to 4-sulfation on interior disaccharides is increased from ;0.77 to ;23 in the adult
Summary
B-Glucuronidase (bovine liver), monoclonal antibody 3B3 ascites fluid, and rat anti-mouse IgM were from ICN. CS contents of purified aggrecan were determined by digesting 10-mg portions with 5 milliunits each of chondroitinase ABC and ACII, and quantitating unsaturated disaccharides (DDi0S, DDi4S, and DDi6S) by capillary zone electrophoresis (5) From such analyses, the values for micrograms of CS Ddisaccharides/mg of dimethylmethylene blue reactivity were 0.91 6 0.01 in the fetal and young juvenile preparations (,15 yr) and 0.68 6 0.02 in the adult preparations (.15 yr). CS Mapping of Membrane Immobilized Macromolecules—Four dots for each concentration of immobilized 35S-labeled rat chondrosarcoma aggrecan (nitrocellulose and nylon N1) or papain generated 35S-labeled CS peptides (nylon N1) were incubated for 6 h at 37 °C in 200 ml of 50 mM sodium acetate, pH 7.5, with 1 milliunit each of chondroitinase ABC and ACII. The 30-s exposure time was chosen to give the optimum range for assaying aggrecan populations of low and high 3B3(2) reactivity
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