Structure of the complex between hyaluronic acid, the hyaluronic acid-binding region, and the link protein of proteoglycan aggregates from the swarm rat chondrosarcoma.

A complex consisting of the hyaluronic acid-binding region of proteoglycan molecules, the link protein, and hyaluronic acid was purified from trypsin digests of aggregates isolated from the Swarm rat chondrosarcoma. Sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis showed that the molecular weights for the hyaluronic acid-binding region and the link protein were about 67,000 and 42,000, respectively. The complex contained an average of about 97 monosaccharide units of hyaluronic acid for each hyaluronic acidbinding region plus link protein pair. After treatment of the complex with chondroitinase ABC, a single protein peak eluted on Sepharose 6B at a position characteristic of a globular protein of molecular weight 112,000. This peak contained about 40% of the hyaluronic acid originally in the complex as well as both proteins. This suggests that the peak consisted of a ternary complex of 1 hyaluronic acid-binding region molecule, one link protein, and a hyaluronic acid oligosaccharide of about 41 monosaccharides. Aggregates were reconstituted in the presence of 3Hlabeled hyaluronic acid and subsequently digested with a protease-free hyaluronidase isolated from Streptomyces. The hyaluronic acid oligosaccharides protected from digestion were subsequently eluted on Sephacryl S-200 with 4 M guanidine hydrochloride as the eluent. The major 3H-labeled peak (about 70% of the total) had an average size of about 50 monosaccharides while the remainder had an average size of about 82 monosaccharides. This suggests that the hyaluronidase was able to digest most, but not all, regions between adjacent monomer proteoglycan and link protein pairs and that oligosaccharides of about 50 monosaccharides were protected. An aggregate sample was digested with chondroitinase and then either thrombin, trypsin, chymotrypsin, papain under mild conditions, or kallikrein. SDSpolyacrylamide gels indicated that thrombin left a larger fragment (II&. = 115,000) from the hyaluronic acid-binding region of the proteoglycan core protein than trypsin (M= = 67,000) or the remaining proteases (Mr = 55,000). These results indicate that the core protein of proteoglycans from the Swarm rat chondrosarcoma can be selectively degraded into fragments of distinct sizes.