VARIATIONS IN THE CHONDROITIN SULFATE-PROTEIN LINKAGE REGION OF AGGRECANS FROM BOVINE NASAL AND HUMAN ARTICULAR CARTILAGES

Aggrecan-derived chondroitin sulfate (CS) chains, released by beta-eli mination, were derivatized with p-aminobenzoic acid or p-aminophenol; radioiodinated; and subjected to graded or complete degradations by ch ondroitin ABC lyase to generate linkage region fragments of the basic structure Delta GlyUA-GalNAc-GlcUA-Gal-Gal-Xyl-R (where Delta GlyUA re presents 4,5-unsaturated glycuronic acid, and R is the adduct), by cho ndroitin AC lyase to generate the shorter fragment Delta GlyUA-Gal-Gal -Xyl-R, or by chondroitin C lyase to generate the same fragment when i t was linked to a 6-O-sulfated or unsulfated GalNac at the nonreducing end. Fragments were separated by size using gel chromatography, by ch arge using ion-exchange chromatography, and by size/charge using elect rophoresis and then characterized by stepwise degradations from the no nreducing end by using mercuric acetate to remove all terminal Delta G lyUA, by bacterial glycuronidase to remove the same residue when linke d to unsulfated or 6-O-sulfated GalNAc/Gal, by mammalian 4-sulfatase t o remove sulfate from terminal GalNAc 4-O-sulfate, by chondro-4-sulfat ase to remove 4-O-sulfate from other GalNAc/Gal residues, and by beta- galactosidase to remove terminal Gal. Results with CS from bovine nasa l cartilage aggrecan show that, in nearly all chains, Xyl and probably also the first Gal are unsubstituted, whereas the second Gal is 4-O-s ulfated in one CS chain out of five. The first disaccharide repeat is sulfated at C-4 of GalNAc in one chain out of three and unsulfated in the other two. A sulfated first disaccharide is always joined to an un sulfated GlcUA-Gal-Gal sequence. In contrast, CS from human articular cartilage usually has a sulfated first disaccharide repeat. In CS from young human cartilage, sulfate groups are mostly at C-4 of GalNAc in the major part of the chain, but at C-6 in the nonreducing distal port ion. In CS from old cartilage, sulfation at C-6 of GalNAc is a major f eature from the nonreducing end down to approximately positions 4 and 5 from the linkage region, where GalNAc 4-O-sulfate is common.