Structure and interactions of cartilage proteoglycan binding region and link protein

Abstract

Binding region and link protein were prepared from pig laryngeal cartilage proteoglycans after chondroitinase ABC and trypsin digestion. Experiments on gel chromatography showed the purified binding region to interact reversibly with hyaluronate (HA), and this binding was also shown to be stabilized by native link protein. The trypsin-prepared link protein showed properties of self-association in solution that were partially inhibited by oligosaccharides (HA10-16) and abolished by modification of free amino groups (lysine residues) with 2-methylmaleic anhydride. The MW (sedimenation equilibrium) of the modified link protein was 41,700. Analysis of binding region showed it to contain 25% (wt/wt) carbohydrate, mainly in galactose, glucosamine, mannose and galactosamine. It contained some keratan sulfate, as digestion with endo-.beta.-D-galactosidase (keratanase) removed 28% galactose and 25% glucosamine and the MW decreased from 66,500 to 60,800. After keratanase digestion the interaction with polyclonal antibodies specific for binding region was unaffected, but the response in a radioimmunoassay with a monoclonal antibody to keratan sulfate was decreased by 47%. Preparation of a complex between binding region, link protein and .**GRAPHIC**. showed a single component (5.5S) of MW 133,500. In this complex the antigenic determinants of link protein appeared masked, as previously found with proteoglycan aggregates. The isolated binding region and link protein retained properties comparable with those involved in the structure and organization of proteoglycan aggregates.