scholarly journals Structure and interactions of cartilage proteoglycan binding region and link protein

1985 ◽  
Vol 228 (1) ◽  
pp. 77-85 ◽  
Author(s):  
F Bonnet ◽  
D G Dunham ◽  
T E Hardingham
Keyword(s):  
Polyclonal Antibodies ◽  
Sedimentation Equilibrium ◽  
Antigenic Determinants ◽  
Gel Chromatography ◽  
Binding Region ◽  
Keratan Sulphate ◽  
Link Protein ◽  
Lysine Residues ◽  
Proteoglycan Aggregates ◽  
Self Association

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.

scholarly journals Immunochemical analysis of cartilage proteoglycans. Antigenic determinants of substructures

1979 ◽  
Vol 179 (1) ◽  
pp. 35-45 ◽  
Author(s):  
J Wieslander ◽  
D Heinegård
Keyword(s):  
Hyaluronic Acid ◽  
Chondroitin Sulphate ◽  
Antigenic Site ◽  
Trypsin Digestion ◽  
Antigenic Determinants ◽  
Binding Region ◽  
Keratan Sulphate ◽  
Link Protein ◽  
Before And After ◽  
Hyaluronic Acid Binding

Antibodies were raised in rabbits by injection of cartilage proteoglycan monomers, isolated hyaluronic acid-binding region, polysaccharide-peptides prepared by trypsin digestion of proteoglycans and link-protein. The rabbits injected with the proteoglycan monomers made antibodies reacting with the intact proteoglycan. The antiserum contained antibodies specific for, and also reacting with, the isolated hyaluronic acid-binding region and the keratan sulphate-rich region. In addition there were probably antibodies reacting with other structures of the proteoglycan monomer. When isolated hyaluronic acid-binding region was used for immunization the antibodies obtained reacted specifically with the hyaluronic acid-binding region. The antibodies obtained from rabbits immunized with the polysaccharide-peptides reacted with the proteoglycan monomers and showed a reaction identical with that of the chondroitin sulphate-peptides isolated after trypsin digestion of proteoglycans. The antibodies prepared with the link-protein as the antigen reacted only with the link-protein and not with any preparation from the proteoglycan monomer. Neither did any of the antisera raised against the proteoglycan monomer or its substructures react with the link-protein. Separately it was shown that the peptide ‘maps’ prepared from trypsin digests of the link-protein and the hyaluronic acid-binding region were different. Therefore it appears that the link-protein is not structurally related to the proteoglycan or the hyaluronic acid-binding region. Digestion of proteoglycan monomers or isolated hyaluronic acid-binding region with trypsin did not destroy the antigenic sites of the hyaluronic acid-binding region. In contrast trypsin digests of previously reduced and alkylated preparations did not react with the anti-(hyaluronic acid-binding region). The trypsin digests, however, reacted with both the antibodies directed against the chondroitin sulphate-peptides and those against the keratan sulphate-peptides. Trypsin digestion of the link-proteins destroyed the antigenic site and the reactivity with the antibodies. By combining immunoassay of proteoglycan preparations before and after trypsin digestion it is feasible to quantitatively determine its substructures by using the antisera described above.

scholarly journals Studies on the antigenic determinants in the self-association of IgG rheumatoid factor

1981 ◽  
Vol 154 (1) ◽  
pp. 112-125 ◽  
Author(s):  
FA Nardella ◽  
DC Teller ◽  
M Mannik
Keyword(s):  
Sedimentation Equilibrium ◽  
Antigenic Determinants ◽  
Fab Fragment ◽  
Fab Fragments ◽  
Polymer Formation ◽  
Igg Rheumatoid Factor ◽  
Normal Human ◽  
Self Association ◽  
Equilibrium Ultracentrifugation ◽  
Antigen Antibody

The number, location, and other characteristics of the antigenic determinants for self-association of IgG-rheumatoid factors (IgG-RF) were examined using the IgG-RF isolated from the plasma of one patient as a model system. Affinity chromatography was employed for isolation of the IgG-RF. Sedimentation equilibrium ultracentrifugation was used to study the various interactions. The antigenic valence of IgG-RF Fc, normal human Fc, and rabbit Fc fragments was two for the interaction with Fab fragments from IgG-RF, as might be expected from the molecular symmetry of IgG. The antigenic valence of intact normal IgG, however, was only one, indicating that when one of the available antigenic determinants interacted with the Fab fragment of IgG-RF, the other determinant becomes sterically inaccessible. Reduction and alkylation, known to increase the flexibility of the hinge region, did not alter the antigenic valence of IgG for Fab fragments of IgG-RF. The antigenic valence of IgG-RF in self-association could not be experimentally determined but must be two to permit the observed concentration-dependent further polymer formation of IgG-RF dimers. Unique antigenic determinants on the Fc fragments of IgG-RF were sought and not found, thus reaffirming the formation of two antigen-antibody bonds as the basis for dimerization of IgG-RF molecules. The pFc' and Fc' fragments, representing Cγ3 domains of IgG, failed to show significant interaction with Fab fragments of IgG-RF, indicating that the antigenic determinants were not expressed by the Cγ3 regions but are located either on Cγ2 region or require intact Cγ2 and Cγ3 regions for expression. These conclusions were corroborated by the antigenic valence of one for the Fc(i) fragment, a new papain-generated intermediate fragment of Fc, composed of two intact Cγ3 domains and one intact Cγ2 domain. Normal IgG, because of its valence of one for interaction with IgG-RF, would effectively terminate further polymerization of IgG-RF dimers. This may well in part explain the finding of smaller IgG-RF complexes in the serum than in synovial fluid of patients with rheumatoid arthritis.

scholarly journals Hyaluronic acid in cartilage and proteoglycan aggregation

1974 ◽  
Vol 139 (3) ◽  
pp. 565-581 ◽  
Author(s):  
Timothy E. Hardingham ◽  
Helen Muir
Keyword(s):  
Hyaluronic Acid ◽  
Buoyant Density ◽  
Ion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Gel Chromatography ◽  
Guanidinium Chloride ◽  
Keratan Sulphate ◽  
Sulphate Content ◽  
Lower Protein ◽  
Proteoglycan Aggregates

1. Dissociation of purified proteoglycan aggregates was shown to release an interacting component of buoyant density higher than that of the glycoprotein-link fraction of Hascall & Sajdera (1969). 2. This component, which produced an increase in hydrodynamic size of proteoglycans on gel chromatography, was isolated by ECTEOLA-cellulose ion-exchange chromatography and identified as hyaluronic acid. 3. The effect of pH of extraction showed that the proportion of proteoglycan aggregates isolated from cartilage was greatest at pH4.5. 4. The proportion of proteoglycans able to interact with hyaluronic acid decreased when extracted above or below pH4.5, whereas the amount of hyaluronic acid extracted appeared constant from pH3.0 to 8.5. 5. Sequential extraction of cartilage with 0.15m-NaCl at neutral pH followed by 4m-guanidinium chloride at pH4.5 was shown to yield predominantly non-aggregated and aggregated proteoglycans respectively. 6. Most of the hyaluronic acid in cartilage, representing about 0.7% of the total uronic acid, was associated with proteoglycan aggregates. 7. The non-aggregated proteoglycans were unable to interact with hyaluronic acid and were of smaller size, lower protein content and lower keratan sulphate content than the disaggregated proteoglycans. Together with differences in amino acid composition this suggested that each type of proteoglycan contained different protein cores.

scholarly journals Articular cartilage cultured with interleukin 1. Increased release of link protein, hyaluronate-binding region and other proteoglycan fragments

1986 ◽  
Vol 238 (2) ◽  
pp. 571-580 ◽  
Author(s):  
A Ratcliffe ◽  
J A Tyler ◽  
T E Hardingham
Keyword(s):  
Articular Cartilage ◽  
Interleukin 1 ◽  
Binding Region ◽  
Rapid Loss ◽  
Keratan Sulphate ◽  
Link Protein ◽  
The Matrix ◽  
The Media ◽  
Average Size

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.

scholarly journals Immunodiffusion studies of the tryptic fragments of bovine nasal-cartilage proteoglycan monomer of high buoyant density

1982 ◽  
Vol 203 (3) ◽  
pp. 691-698 ◽  
Author(s):  
Harold D. Keiser
Keyword(s):  
Chondroitin Sulphate ◽  
Buoyant Density ◽  
Antigenic Determinants ◽  
Density Gradient Ultracentrifugation ◽  
Binding Region ◽  
Keratan Sulphate ◽  
Nasal Cartilage ◽  
Cartilage Proteoglycan ◽  
Immunoprecipitation Reaction ◽  
Hyaluronic Acid Binding

Tryptic fragments of bovine nasal-cartilage proteoglycan, fractionated by dissociative density-gradient ultracentrifugation, were made to react by immunodiffusion against antiserum to a hyaluronidase-digest subfraction of cartilage proteoglycan monomer. This reaction produced two families of partly superimposed precipitin lines. One family was restricted to gradient fractions of medium or low buoyant density and included the immunoprecipitation reaction attributed to the hyaluronic acid-binding region of the cartilage proteoglycan monomer. The second family of precipitin lines was present alone in gradient fractions of high buoyant density. Immunodiffusion studies with antisera to relatively homogeneous keratan sulphate-rich and chondroitin sulphate-bearing fragment subfractions isolated from the gradient fraction of highest density indicated that both subfractions contained the antigenic determinants responsible for the second family of precipitin lines. Additional immunodiffusion studies, with the use of multispecific antisera to chondroitinase ABC digest and hyaluronidase digest of proteoglycan monomer, confirmed that the two subfractions shared antigenic determinants, and, in addition, indicated that these determinants were on one molecular species in the keratan sulphate-rich fragment subfraction and divided among at least three in the chondroitin sulphate-bearing fragment subfraction. Although an unprecedentedly large number of cartilage proteoglycan antigens could be recognized with the antisera employed in this cartilage proteoglycan antigens could be recognized with the antisera employed in this study, it was not possible to identify antigenic determinants unambiguously specific for the three structurally and functionally distinct regions of the cartilage proteoglycan monomer.

scholarly journals Cartilage proteoglycan aggregate formation Role of link protein

1981 ◽  
Vol 197 (3) ◽  
pp. 669-674 ◽  
Author(s):  
A Franzén ◽  
S Björnsson ◽  
D Heinegård
Keyword(s):  
Hyaluronic Acid ◽  
Dry Weight ◽  
Aggregate Formation ◽  
Binding Region ◽  
Link Protein ◽  
Cartilage Proteoglycan ◽  
Proteoglycan Aggregates ◽  
Hyaluronic Acid Binding

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.

scholarly journals A comparative study of the binding of cartilage link protein and the hyaluronate-binding region of the cartilage proteoglycan to hyaluronate-substituted Sepharose gel

1981 ◽  
Vol 199 (2) ◽  
pp. 297-305 ◽  
Author(s):  
A Tengblad
Keyword(s):  
Time Course ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Binding Constants ◽  
Minimum Size ◽  
Gel Chromatography ◽  
Binding Region ◽  
Link Protein ◽  
Nasal Cartilage ◽  
Number Of Binding Sites

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.

scholarly journals Metalloproteinase digestion of cartilage proteoglycan. Pattern of cleavage by stromelysin and susceptibility to collagenase

1991 ◽  
Vol 279 (3) ◽  
pp. 733-739 ◽  
Author(s):  
C Hughes ◽  
G Murphy ◽  
T E Hardingham
Keyword(s):  
Core Protein ◽  
Chondroitin Sulphate ◽  
Relative Viscosity ◽  
Gel Chromatography ◽  
High Concentration ◽  
Link Protein ◽  
Rapid Fall ◽  
Sds Page ◽  
Proteoglycan Aggregates ◽  
Rabbit Bone

The action of purified rabbit bone stromelysin was investigated on proteoglycan aggregates from pig laryngeal cartilage. The enzyme caused a rapid fall in viscosity of proteoglycan aggregate solution (6 mg/ml), and the products of a partial digest (60% loss of relative viscosity) and a complete digest (95% loss of relative viscosity) were characterized. Analysis by gel chromatography on Sepharose 2B under associative conditions showed that 95% of the glycosaminoglycans in the complete digest were in small-sized fragments, whereas most of the hyaluronan-binding G1 domain and link protein remained intact and bound to hyaluronan. In contrast, there was extensive digestion of the G2 domain which resulted in 76% loss in its detection by immunoassay. Analysis of the partial digest also showed considerable loss (40%) of detection of the G2 domain, but the glycosaminoglycan-rich fragments were much larger than in the complete digest. There was also much less cleavage to create small fragments containing the G1 domain. This was evident on SDS/PAGE analysis where a 58 kDa G1 domain fragment was abundant in the complete digest, but was only present in small amounts in the partial digest. There was also only very limited conversion of link protein from a 44 kDa form to a 40 kDa form. The digestion of proteoglycan aggregate (6 mg/ml) by stromelysin was unaffected by the addition of a high concentration of extra chondroitin sulphate chains (14 mg/ml), and the digestion of proteoglycan monomer showed that the G1 domain was resistant to stromelysin digestion even when not bound to hyaluronan and link protein. The results show that stromelysin degrades the proteoglycan protein core with major cleavages close to, but not within, the G1 domain, and extensive cleavage in other regions. Experiments with purified collagenase, a metalloproteinase structurally related to stromelysin, showed that it too cleaved proteoglycan at several sites within the glycosaminoglycan-rich region of the core protein. Metalloproteinase attack on proteoglycan thus not only occurs with stromelysin but also with collagenase.

scholarly journals Self-association of α-chymotrypsin at low ionic strength in the vicinity of its pH optimum

1977 ◽  
Vol 161 (3) ◽  
pp. 687-694 ◽  
Author(s):  
R Tellam ◽  
D J Winzor
Keyword(s):  
Ionic Strength ◽  
Electrostatic Interactions ◽  
Sedimentation Equilibrium ◽  
Velocity Sedimentation ◽  
Gel Chromatography ◽  
Ph Optimum ◽  
Equilibrium Sedimentation ◽  
Equilibrium Distributions ◽  
Self Association ◽  
Low Ionic Strength

The self-association of alpha-chymotrypsin and its di-isopropyl phosphoryl derivative in in I0.03 sodium phophate buffer, pH7,9, was investigated by velocity sedimentation, equilibrium sedimentation and difference gel chromatography. No differences between the native and chemically modified enzyme were observed in the ultracentrifuge studies, and only a marginal (0.6%) difference in weight-average elution volume was detected by difference gel chromatography of 5g/litre solutions on Sephadex G-75. From quantitative analyses of sedimentation velocity and sedimentation-equilibrium distributions obtained with iPr2P (di-isopropylphosphoryl)-chymotrypsin, the polymerizing system is postulated to involve an indefinite association of dimer (with an isodesmic association constant of 0.68 litre/g) that is formed by a discrete dimerization step with equilibrium constant 0.25 litre/g. In addition to providing the best fit of the experimental results, this model of chymotrypsin polymerization at low ionic strength is also consistent with an earlier observation that dimer formation is a symmetrical head-to-head phenomenon under conditions of higher ionic strength (I0.29, pH7.9) where association is restricted to a monomer-dimer equilibrium. It is proposed that the dimerization process is essentially unchanged by variation in ionic strength at pH7.9, and that higher polymers are formed by an entirely different mechanism involving largely electrostatic interactions between dimeric species.

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