Cartilage proteoglycan binding region and link protein. Radioimmunoassays and the detection of masked determinants in aggregates.
Ontology highlight
ABSTRACT:
SUBMITTER: Ratcliffe A
PROVIDER: S-EPMC1152137 | biostudies-other | 1983 Aug
REPOSITORIES: biostudies-other
Similar Datasets
Project description: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.
| S-EPMC1144955 | biostudies-other
Project description:Proteoglycan fractions were prepared from pig laryngeal cartilage. The effect of link-protein on the properties of proteoglycan-hyaluronate aggregates was examined by viscometry and analytical ultracentrifugation. Aggregates containing link-protein were more stable than link-free aggregates at neutral pH, at temperatures up to 50 degrees C and in urea (up to 4.0M). Oligosaccharides of hyaluronate were able to displace proteoglycans from link-free aggregates, but not from the link-stabilized aggregates. Both types of aggregate were observed in the ultracentrifuge, but at the concentration investigated (less than 2 mg/ml) the link-free form was partially dissociated and the proportion aggregated varied with the pH and temperature and required more hyaluronate for saturation than did link-stabilized aggregate. The results showed that link-protein greatly strengthened the binding of proteoglycans to hyaluronate and suggest that under physiological conditions it 'locks' proteoglycans on to the hyaluronate chain.
| S-EPMC1186361 | biostudies-other
Project description:Pig articular cartilage was maintained in culture for 3 days with and without porcine interleukin 1. The proteoglycans remaining in the cartilage and those released into the medium were analysed by using radioimmunoassays for the hyaluronate-binding region, link protein and keratan sulphate. In interleukin 1-treated cultures after 3 days there was 38% release of total glycosaminoglycans into the medium, 18% release of binding region, 14% release of link protein and 20% release of keratan sulphate epitope, whereas in control cultures the proportions released were much less (16, 9, 10 and 7% respectively). Characterization of the proteoglycans in the media after 1.5 days and 3 days of culture showed that interleukin 1 promoted the release of proteoglycan of large average size and also the release of link protein and of low-Mr binding region which was unattached to proteoglycan. Both the link protein and binding region released were able to bind to exogenously added hyaluronate, whereas the proteoglycan in the medium was not. The proteoglycans extracted from cultured cartilage were similar to those from fresh cartilage: they contained a high proportion of aggregating proteoglycans and some low-Mr binding region. The proportion of this binding region extracted from the interleukin 1-treated cartilage was increased. The presence of interleukin 1 in the cultures therefore appeared to increase the rate of proteolytic degradation of proteoglycan in the matrix and to lead to a more rapid loss of intact binding region, of link protein and of large proteoglycan fragments into the medium.
| S-EPMC1147171 | biostudies-other
Project description:The hyaluronate-binding proteins from bovine nasal cartilage, i.e. the hyaluronate-binding region of the proteoglycan and the link protein, were labelled with 125I and separated from each other by gel chromatography. The proteins were characterized by molecular-weight determinations and their purity was established by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis and immunodiffusion. The binding properties of the two proteins by hyaluronate-substituted Sepharose gel were compared. It was found that both proteins behaved similarly. They bound with the same efficiency to the gel, they showed the same time course of binding, had slightly different pH optima for binding and both proteins had a decreasing affinity for the gel with increasing ionic strength. The binding to the gel could be inhibited by soluble hyaluronate, and the minimum size of a hyaluronate oligosaccharide required for inhibition was in both cases a decasaccharide (only even-numbered oligosaccharides were tested). The proteins did not show any co-operative binding in the system tested, which could be explained by the large number of binding sites in the hyaluronate-substituted gel. Binding constants for the protein-hyaluronate interaction were estimated. A value of 1.3 x 10(7) M-1 was obtained for the hyaluronate-binding region of the proteoglycan, in agreement with literature data. The corresponding value for the link protein was 0.7 x 10(7) M-1.
| S-EPMC1163372 | biostudies-other
Project description:Cartilage proteoglycan aggregate formation was studied by zonal rate centrifugation in sucrose gradients. Proteoglycan aggregates, monomers and proteins could be resolved. It was shown that the optimal proportion of hyaluronic acid for proteoglycan aggregate formation was about 1% of proteoglycan dry weight. The reaggregation of dissociated proteoglycan aggregate A1 fraction was markedly concentration-dependent and even at 9 mg/ml only about 90% of the aggregates were reformed. The lowest proportion of link protein required for maximal formation of link-stabilized proteoglycan aggregates was 1.5% of proteoglycan dry weight. It was separately shown that link protein co-sedimented with the proteoglycan monomer. By competition with isolated hyaluronic acid-binding-region fragments, a proportion of the link proteins was removed from the proteoglycan monomers, indicating that the link protein binds to the hyaluronic acid-binding region of the proteoglycan monomer.
| S-EPMC1163179 | biostudies-other
Project description:Proteoglycan aggregates free of non-aggregating proteoglycan have been prepared from the annuli fibrosi and nuclei pulposi of intervertebral discs of three human lumbar spines by extraction with 4M-guanidinium chloride, associative density gradient centrifugation, and chromatography on Sepharose CL-2B. The aggregate (A1-2B.V0) was subjected to dissociative density-gradient ultracentrifugation. Three proteins of Mr 38 900, 44 200 and 50 100 found in the fraction of low buoyant density (A1-2B.V0-D4) reacted with antibodies to link protein from newborn human articular cartilage. After reduction with mercaptoethanol, two proteins of Mr 43 000 and two of Mr 20 000 and 14 000 were seen. The A1-2B.V0-D4 fraction, labelled with 125I, coeluted with both hyaluronate and a hyaluronate oligosaccharide (HA14) on a Sepharose CL-2B column. HA10 and HA14 reduced the viscosity of A1 fractions; HA4, HA6 and HA8 did not. HA14 decreased the viscosity of disc proteoglycans less than it did that of bovine cartilage proteoglycans. Thus, although a link protein was present in human intervertebral disc, it stabilized proteoglycan aggregates less well than did the link protein from bovine nasal cartilage.
| S-EPMC1144147 | biostudies-other
Project description:The interaction between proteoglycan and link protein extracted from bovine articular cartilage (15-18-month-old animals) was investigated in 0.5 M-guanidinium chloride. The proteoglycans, radiolabelled as the aggregate (A1 fraction), were fractionated by two 'dissociative' density-gradient centrifugations (A1D1D1) followed by a rate-zonal centrifugation (S1) to yield an A1D1D1S1 preparation. At least 65% of these proteoglycans were able to bind to hyaluronate, but only 52% were able to bind to link protein as assessed by chromatography on Sepharose CL-2B. Over 80% of the [3H]link-protein preparation, radiolabelled as the aggregate, was able to interact with proteoglycan as assessed by chromatography on Sepharose CL-4B. Equilibrium-boundary-centrifugation studies performed at low link-protein concentrations (2.42 x 10(-9) M-5.93 x 10(-8) M) were analysed by Scatchard-type plots and indicated a Kd of 1.5 x 10(-8) M and a stoichiometry, n = 0.56, i.e. approx. 56% of those proteoglycans capable of binding to link protein had a strong site for link protein if a 1:1 stoichiometry were assumed. However, experiments performed at higher link-protein concentrations (3.5 x 10(-7) M and 8 x 10(-7) M) yielded stoichiometry values which were link-protein-concentration-dependent. Non-equilibrium binding studies using chromatography on Sepharose CL-2B and rate-zonal centrifugation yielded apparent stoichiometries between 0.6 and 7.5 link-protein molecules per proteoglycan monomer as a function of increasing link-protein concentration. It was concluded that a proportion of the proteoglycan molecules had a strong site for binding a single link protein (Kd 1.5 x 10(-8) M) and that at high link-protein concentrations a weaker, open-ended, process of link-protein self-association nucleated upon the strong link-protein-proteoglycan complex occurred. Hyaluronate oligosaccharides appeared to abolish a proportion of this self-association (as observed by Bonnet, Dunham & Hardingham [(1985) Biochem. J. 228, 77-85] in a study of link-protein-hyaluronate-oligosaccharide interactions) so as to leave a link protein:proteoglycan stoichiometry of 2. It is not clear whether this second link-protein molecule binds directly to the proteoglycan or to the first link protein.
| S-EPMC1148641 | biostudies-other
Project description:Human articular-cartilage link proteins are resolved into three components by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis, indicative of three different structures. The action of the proteinase clostripain yields a single link-protein component with electrophoretic properties analogous to those of the smallest (most mobile) native link protein, suggesting that this link protein may be derived naturally from one or both of the larger molecules by proteolytic cleavage in situ. Upon chemical deglycosylation of native link protein two components are resolved, suggesting that two of the link proteins differ only in their degree and/or type of oligosaccharide substitution. This pattern is compatible with a proteolytic origin for the smallest link protein. During aging further proteolytic fragmentation occurs, though it is only apparent on reduction of disulphide bonds. This fragmentation occurs at identical sites in all three native link proteins, indicating the existence of a large region common to all the link proteins, which appears to consist predominantly of the C-terminal half of the molecules. These observations are compatible with the variation in oligosaccharide and proteolytic heterogeneity occurring at the N-terminus of the link proteins.
| S-EPMC1152954 | biostudies-other
Project description:Ordered conformations of proteoglycan-hyaluronic acid aggregates in the intercellular matrix in cartilage were observed by X-ray diffraction. The sodium salt form of three samples, (a) aggregated proteoglycan, (b) disaggregated proteoglycan and (c) reconstituted disaggregated proteoglycan, give essentially similar X-ray fibre-type diffraction photographs. The patterns correlate with the chondroitin 4-sulphate component and can be interpreted as twofold helical conformations, similar to that observed previously for the free acid form of chondroitin 4-sulphate (Isaac & Atkins, 1973). The information takes us one step nearer the situation found in cartilage in vivo.
| S-EPMC1168199 | biostudies-other
Project description:1. Proteoglycan aggregates from bovine nasal cartilage were studied by using electron microscopy of proteoglycan/cytochrome c monolayers. 2. The aggregates contained a variably long central filament of hyaluronic acid with an average length of 1037nm. The proteoglycan monomers attached to the hyaluronic acid appeared as side chain filaments varying in length (averaging 249nm). They were distributed along the central filament at an average distance of about 36nm. 3. Chondroitin sulphate side chains were removed from the proteoglycan monomers of the aggregates by partial chondroitinase digestion. The molecules obtained had the same general appearance as intact aggregates. 4. Proteoglycan aggregates were treated with trypsin and the largest fragment, which contains the hyaluronic acid, link protein and hyaluronic acid-binding region, was recovered and studied with electron microscopy. Filaments that lacked the side chain extensions and had the same length as the central filament in the intact aggregate were observed. 5. Hyaluronic acid isolated after papain digestion of cartilage extracts gave filaments with similar length and size distribution as observed for the central filament both in the intact aggregate and in the trypsin digests. 6. Umbilical-cord hyaluronic acid was also studied and gave electron micrographs similar to those described for hyaluronic acid from cartilage. However, the length of the filament was somewhat shorter. 7. The electron micrographs of both intact and selectively degraded proteoglycans corroborate the current model of cartilage proteoglycan structure.
| S-EPMC1186153 | biostudies-other