Interaction of cartilage proteoglycans with hyaluronic acid

Abstract Most proteoglycans are present in hyaline cartilage matrices as aggregates with as many as 100 molecules, each with average molecular weight of about 2 × 10 6 , bound through specific, noncovalent interactions to individual strands of hyaluronic acid (HA). The interactions with HA are mediated by the HA‐binding region of the core protein, which is located at one end of each of the interactive proteoglycans. A fragment of the core protein, average molecular weight of about 6 × 10 4 , which contains the HA‐binding site, can be isolated in an active form from trypsin digests of proteoglycan aggregates. The “active” HA‐binding site in this preparation interacts strongly with HA‐10 but weakly with HA‐8, (oligomers of HA derived from partial digests of HA with testicular hyaluronidase); HA‐9 derived from β‐glucuronidase digestion of HA‐10 also interacts strongly. No polysaccharide other than HA has been found to interact. Christner, Brown, and Dziewiatkowski (personal communication) modified the carboxyls on glucuronic acid groups in mixture of HA‐10 to HA‐30, and they found that the interaction with proteoglycan no longer occurred if about 60% of the total carboxyls were (a) methyl esterified, (b) reduced to glucose, or (c) substituted with glycine in amide linkage. Saponification of the methyl esters restored activity. Dansylation of lysine residues in the HA‐binding region preparation abolished binding activity. However, when the dansylation reaction was done in the presence of HA, the HA‐binding activity was protected. Acetylation of the same residues did not abolish binding activity but did prevent subsequent inactivation by dansylation. Hardingham, Ewins, and Muir (Biochem J 157:127–143, 1976) studied the effect of various amino acid modifiers on the interaction of intact proteoglycans with HA and showed that reaction of arginine residues with low concentrations of 2,3‐butanedione was particularly effective in destroying binding. In sum, the data above suggests that the HA‐binding region (a) contains accessible arginine residues necessary for activity, (b) contains lysine residues near the binding site which, when substituted with bulky groups such as dansyl, but not acetyl, sterically block interaction, and (c) requires a length of HA with at least 4.5 repeat disaccharides containing 3, and possibly 4, unmodified glucuronic acid carboxyls for interaction. The possible relevance of proteoglycan‐hyaluronic acid interaction to the observations that hyaluronic acid specifically inhibits proteogly can synthesis by cultured chondrocytes is discussed.