Aggregation of Cartilage Proteoglycans

TLDR

The authors isolated a protein‑keratan sulfate core from bovine nasal proteoglycan by chondroitinase digestion and Sepharose 2B chromatography, and purified hyaluronic acid oligosaccharides using Sephadex G‑50 chromatography of hyaluronidase digests. The core binds strongly to hyaluronic acid oligomers of ten or more disaccharide units, occupying 8–10 units per core, while smaller HA fragments or chondroitin fail to inhibit binding and only low concentrations of HA10 reduce the viscosity of HA–proteoglycan mixtures.

Abstract

A protein-keratan sulfate core preparation isolated from a chondroitinase digest of bovine nasal proteoglycan chromatographed as a single peak (Kav = 0.65) on a Sepharose 2B column. Sepharose 2B elution patterns for mixtures of the core preparation with different proportions of hyaluronic acid indicated that slightly more than 70% of the core molecules could bind to hyaluronic acid. When the hyaluronic acid was saturated with core molecules, each core molecule occupied a minimum length of 8 to 10 hyaluronic acid disaccharides. Hyaluronic acid oligosaccharides were isolated by Sephadex G-50 chromatography of partial digests of hyaluronic acid with testicular hyaluronidase. Sepharose 2B chromatographic analyses showed that the interaction of reactive core molecules with larger hyaluronic acid molecules was strongly inhibited by HA10 (5 disaccharide repeat units) and by HA9 derived from a β-glucuronidase digest of HA10. HA8, smaller hyaluronic acid oligosaccharides and chondroitin were essentially ineffective in inhibiting the core-hyaluronic acid interaction. Low concentrations of HA10 effectively decreased the viscosity of a mixture of hyaluronic acid with intact proteoglycan, whereas higher concentrations of HA8, HA6, or HA4 caused much smaller decreases. Thus the binding site for hyaluronic acid in reactive core (or intact proteoglycan) molecules interacts strongly with HA10 or larger oligomers, while it only interacts weakly with HA4-HA8 oligomers. Chondroitin did not decrease the viscosity of such a mixture at all, showing that there is specificity for the configuration of the 4-hydroxyl in the glucosamine residues in the hyaluronic acid oligosaccharides.