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 Mr (sedimentation equilibrium) of the modified link protein was 41 700. Analysis of binding region showed it to contain 25% (w/w) carbohydrate, mainly in galactose, glucosamine, mannose and galactosamine. It contained some keratan sulphate, as digestion with endo-beta-D-galactosidase (keratanase) removed 28% galactose and 25% glucosamine and the Mr (sedimentation equilibrium) 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 sulphate was decreased by 47%. Preparation of a complex between binding region, link protein and HA approximately 34 showed a single component (5.5S) of Mr (sedimentation equilibrium) 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 were thus shown to retain properties comparable with those involved in the structure and organization of proteoglycan aggregates.

TSG‐6 interacts with hyaluronan and aggrecan in a pH‐dependent manner via a common functional element: implications for its regulation in inflamed cartilage

“…Lp, G1 and biotinylated G1 (b-G1) were prepared from porcine laryngeal cartilage as described before [4,22]; Lp was stored in 4 M guanidine-HCl, 50 mM Na-acetate, 1 mM Na P -EDTA, pH 5.8, due to its low solubility in water. Both G1 and b-G1, following biotinylation in the presence of HA, were puri¢ed by gel ¢ltration under dissociative conditions (i.e.…”

“…The interaction of the proteoglycan aggrecan with HA is mediated by its N-terminal G1 domain [4,5] which is comprised of an immunoglobulin module followed by two contiguous Link modules. The same organisation of modules is found in Lp, where the Link modules are both involved in binding to HA and the immunoglobulin module mediates the interaction with G1 [6].…”

Evidence of a defined spatial arrangement of hyaluronate in the central filament of cartilage proteoglycan aggregates

“…The binding is through a specialized portion of their core protein, the hyaluronate-binding region [6], to a decasaccharide segment of the hyaluronate chain [9]. This interaction is stabilized by the binding of a small glycoprotein, the link protein [6,8], with affinity for both hyaluronate [9,10] and the hyaluronate-binding region of the aggrecan monomer, leading to the formation of a very stable ternary complex [11]. This model has been extended by a combination of electron microscopy [12][13][14] and sequence analysis at the protein [15] and cDNA [16][17][18][19][20] level.…”

Release of hyaluronan and hyaladherins (aggrecan G1 domain and link proteins) from articular cartilage exposed to ADAMTS‐4 (aggrecanase 1) or ADAMTS‐5 (aggrecanase 2)

“…The aggrecan core protein comprises 3 globular domains: at the N-terminus, the G1 and G2 domains are separated by a protease-susceptible interglobular domain (IGD); between the G2 domain and the C-terminal G3 domain, hydrophilic sulfated glycosaminoglycan (sGAG)-bearing regions are present. Functionally, the aggrecan G1 domain binds with high affinity to hyaluronan (HA) and link protein to form stable ternary complexes in the extracellular matrix (2)(3)(4)(5).…”