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Biomedical subjects

T E Hardingham

Publications and source records attributed to T E Hardingham.

At least 55 records · Page 3Linked to original sources
PubMed1988

Cartilage proteoglycans. Assembly with hyaluronate and link protein as studied by electron microscopy.

Aggregates formed by the interaction of cartilage proteoglycan monomers and fragments thereof with hyaluronate were studied by electron microscopy by use of rotary shadowing [Wiedemann, Paulsson, Timpl, Engel & Heinegård (1984) Biochem. J. 224, 331-333]. The differences in shape and packing of the proteins bound along the hyaluronate strand in aggregates formed in the presence and in the absence of link protein were examined in detail. The high resolution of the method allowed examination of the involvement in hyaluronate binding of the globular core-protein domains G1, G2 and G3 [Wiedemann, Paulsson, Timpl, Engel & Heinegård (1984) Biochem. J. 224, 331-333; Paulsson, Mörgelin, Wiedemann, Beardmore-Gray, Dunham, Hardingham, Heinegård, Timpl & Engel (1987) Biochem. J. 245, 763-772]. Fragments comprising the globular hyaluronate-binding region G1 form complexes with hyaluronate with an appearance of necklace-like structures, statistically interspaced by free hyaluronate strands. The closest centre-to-centre distance found between adjacent G1 domains was 12 nm. Another fragment comprising the binding region G1 and the adjacent second globular domain G2 attaches to hyaluronate only by one globule. Also, the core protein obtained by chondroitinase digestion of proteoglycan monomer binds only by domain G1, with domain G3 furthest removed from the hyaluronate. Globule G1 shows a statistical distribution along the hyaluronate strands. In contrast, when link protein is added, binding is no longer random, but instead uninterrupted densely packed aggregates are formed.

Animals↗
PubMed1988

Increased concentrations of proteoglycan components in the synovial fluids of patients with acute but not chronic joint disease.

Synovial fluid samples (139) from 121 patients with rheumatoid arthritis, osteoarthritis, pseudogout, chronic pyrophosphate arthritis, gout, and reactive arthritis were analysed for cartilage proteoglycan components. Keratan sulphate (KS) epitope was determined by a competitive radioimmunoassay, and total sulphated glycosaminoglycans (S-GAG) were determined after papain digestion by a specific dye binding assay. Increased concentration of both KS epitope and S-GAG were found in synovial fluid from joints with acute inflammatory arthropathy (gout, pseudogout, and reactive arthritis). Analysis of consecutive samples from the same joint at different stages showed that the concentration of KS epitope or total S-GAG varied with acute inflammatory activity. In samples from patients with chronic conditions during active and inactive inflammatory phases concentrations were much lower and not distinguishable among these disease groups. The detection of raised concentration of proteoglycan components may reflect the rapid depletion or greatly increased turnover of proteoglycan in the articular cartilage during acute inflammation in the joint. This did not appear to be sustained in most patients with chronic joint diseases.

Acute Disease↗
PubMed1987

The detection of substructures within proteoglycan molecules. Electron-microscopic immuno-localization with the use of Protein A-gold.

Proteoglycan monomers from pig laryngeal cartilage were examined by electron microscopy with benzyldimethylalkylammonium chloride as the spreading agent. The proteoglycans appeared as extended molecules with a beaded structure, representing the chondroitin sulphate chains collapsed around the protein core. Often a fine filamentous tail was present at one end. Substructures within proteoglycan molecules were localized by incubation with specific antibodies followed by Protein A-gold (diameter 4 nm). After the use of an anti-(binding region) serum the Protein A-gold (typically one to three particles) bound at the extreme end of the filamentous region. A small proportion of the labelled molecules (10-15%) showed the presence of gold particles at both ends. A monoclonal antibody specific for a keratan sulphate epitope (MZ15) localized a keratan sulphate-rich region at one end of the proteoglycan, but gold particles were not observed along the extended part of the protein core. This distribution was not changed by prior chondroitin AC lyase digestion of the proteoglycan. Localization with a different monoclonal antibody to keratan sulphate (5-D-4) caused a change in the spreading behaviour of a proportion (approx. 20%) of the proteoglycan monomers that lost their beaded structure and appeared with the chondroitin sulphate chains projecting from the protein core. In these molecules the Protein A-gold localized antibody (5-D-4) along the length of the protein core whereas in those molecules with a beaded appearance it labelled only at one end. Labelling with either of the monoclonal antibodies was specific, as it was inhibited by exogenously added keratan sulphate. The differential localization achieved may reflect structural differences within the proteoglycan population involving keratan sulphate and the protein core to which it is attached. The results showed that by this technique substructures within proteoglycan molecules can be identified by Protein A-gold labelling after the use of specific monoclonal or polyclonal antibodies.

Animals↗
PubMed1987

Viscoelastic properties of proteoglycan solutions with varying proportions present as aggregates.

Monomer and aggregated proteoglycans were prepared from pig laryngeal cartilage. Vascoelastic flow properties, comprising linear complex dynamic shear modulus, nonlinear steady-state shear-rate dependent viscosity, and primary normal stress difference, were measured in proteoglycan solutions containing varying proportions of aggregate (0-80%) and at different concentrations (10-50 mg/ml). Results were analyzed using the simple Oldroyd four-parameter nonlinear rate-type rheological equation. All solution properties were strongly dependent on proteoglycan concentration and on the proportion of aggregates present. Aggregation was found to have a great effect on the zero shear-rate viscosity at 50 mg/ml, which increased fivefold from 0-100% aggregate. The results showed that network formation in proteoglycan solutions increased with concentration from 10-50 mg/ml and also increased with aggregation. All proteoglycan solutions showed shear thinning, which was most marked with aggregated proteoglycan at high concentration (50 mg/ml), where the viscosity decreased tenfold from the zero shear-rate limit to the infinite shear-rate limit. The intermolecular interactions in the network were therefore increasingly disrupted by increasing shear rate, but repeated measurements showed that these were reversible changes and that testing did not induce disaggregation or degradation of proteoglycan. These rheological properties show that aggregation is likely to immobilize proteoglycan at high concentration within cartilage and to contribute to the material properties of the porous solid matrix of articular cartilage that are important for its load-bearing function.

Animals↗
PubMed1986

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

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.

Animals↗
PubMed1986

Cartilage proteoglycans.

The structure of the protein core of the high molecular weight aggregating proteoglycan from pig laryngeal cartilage has been investigated. Mild trypsin digestion of proteoglycan aggregates released a large (Mr approximately equal to 150K) protein-rich fragment that contained the hyaluronate-binding region (Mr 66K). Rotary-shadowing electron microscopy of this preparation showed it to contain 'double globe' structures, similar to those seen with intact proteoglycans. Interaction studies and immunochemical evidence showed that one of the globular domains was the binding region. The second globular domain did not interact with hyaluronate or share any major antigenic determinants with the binding region and its function remains unknown. Further evidence from rotary shadowing also suggested that the protein core contained a third globular domain at the C-terminal end. The complete protein core sequence thus contains long folded globular protein regions, in addition to the extended regions bearing glycosaminoglycan chains. Studies of proteoglycan turnover in explants of pig articular cartilage showed that proteoglycan fragments were continuously released into the medium during culture. These included large non-aggregating proteoglycan fragments, free binding region and also link protein. Proteoglycans retained within the cartilage matrix remained intact and able to aggregate. Only in the presence of interleukin 1 was there evidence of more extensive proteolytic digestion. The results suggest normal turnover to be a conservative mechanism involving the selective cleavage of proteoglycan close to the hyaluronate-binding region. This releases the major glycosaminoglycan-bearing domain and enables it to diffuse out of the matrix. The site of the initial cleavage appears to be in the region of the N-terminal globular domains.

Animals↗
PubMed1985

Structure and interactions of cartilage proteoglycan binding region and link protein.

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.

Animals↗
PubMed1985

The intracellular localisation of proteoglycans and their accumulation in chondrocytes treated with monensin.

Pig laryngeal chondrocytes incubated in the presence of monensin showed inhibition of [35S]sulphate incorporation and decreased secretion of proteoglycan into the culture medium, but no large decrease in protein synthesis. This lead to the intracellular accumulation of proteoglycan protein core, which was detected in immunoprecipitates of cell extracts. Using the same antiserum protein core was localised by electron microscopy with protein A-coated gold. In control chondrocytes, it was detected only in elements of the Golgi and in secretory vesicles, but following monensin treatment labelling was more intense in the Golgi and extended into the distended cisternae of the rough endoplasmic reticulum. The results suggest that monensin blocks proteoglycan protein core translocation between different elements of the Golgi and that this occurs prior to the major site of chondroitin sulphate synthesis on proteoglycan.

Animals↗
PubMed1985

Proteoglycan biosynthesis in chondrocytes: protein A-gold localization of proteoglycan protein core and chondroitin sulfate within Golgi subcompartments.

The intracellular pathway of cartilage proteoglycan biosynthesis was investigated in isolated chondrocytes using a protein A-gold electron microscopy immunolocalization procedure. Proteoglycans contain a protein core to which chondroitin sulfate and keratan sulfate chains and oligosaccharides are added in posttranslational processing. Specific antibodies have been used in this study to determine separately the distribution of the protein core and chondroitin sulfate components. In normal chondrocytes, proteoglycan protein core was readily localized only in smooth-membraned vesicles which co-labeled with ricin, indicating them to be galactose-rich medial/trans-Golgi cisternae, whereas there was only a low level of labeling in the rough endoplasmic reticulum. Chondroitin sulfate was also localized in medial/trans-Golgi cisternae of control chondrocytes but was not detected in other cellular compartments. In cells treated with monensin (up to 1.0 microM), which strongly inhibits proteoglycan secretion (Burditt, L.J., A. Ratcliffe, P. R. Fryer, and T. Hardingham, 1985, Biochim. Biophys. Acta., 844:247-255), there was greatly increased intracellular localization of proteoglycan protein core in both ricin-positive vesicles, and in ricin-negative vesicles (derived from cis-Golgi stacks) and in the distended rough endoplasmic reticulum. Chondroitin sulfate also increased in abundance after monensin treatment, but continued to be localized only in ricin-positive vesicles. The results suggested that the synthesis of chondroitin sulfate on proteoglycan only occurs in medial/trans-Golgi cisternae as a late event in proteoglycan biosynthesis. This also suggests that glycosaminoglycan synthesis on proteoglycans takes place in a compartment in common with events in the biosynthesis of both O-linked and N-linked oligosaccharides on other secretory glycoproteins.

Animals↗
PubMed1984

The distribution of aggregating proteoglycans in articular cartilage: comparison of quantitative immunoelectron microscopy with radioimmunoassay and biochemical analysis.

Electron microscopic immunolocalization and radioimmunoassay have been used to determine the variation with depth of the hyaluronate-binding region of proteoglycan in articular cartilage. The cartilage was cut into serial sections from the articular surface to the bony margin, the proteoglycans were extracted from each section and determined by radioimmunoassay using antibodies raised against proteoglycan binding region. Proteoglycans were found to be most abundant in the middle zone and least abundant near the articular surface. Biochemical analysis for hexuronate in the same extracts showed a distribution of proteoglycan in agreement with these and other published results. The binding region antiserum was used for electron microscopic immunolocalization of proteoglycan with ultrathin sections of cartilage embedded in Lowicryl K4M resin. After digestion of the sections with chondroitinase ABC, the proteoglycans were localized using the antiserum and protein A-coated gold particles as immunolabel. The density of labeling was quantified using a Magiscan image analysis system. Throughout the depth of the cartilage matrix labeling was higher in the pericellular regions compared to the intercellular regions, and variation of the amount of immunolabel with depth was found to show a good correlation with the results from radioimmunoassay. Intracellular labeling of proteoglycans was mainly found over the Golgi region and in membrane-bound (secretory) vesicles.

Acrylic Resins↗
PubMed1980

Assembly of newly synthesized proteoglycan and link protein into aggregates in cultures of chondrosarcoma chondrocytes.

The synthesis and subsequent assembly of proteoglycan monomer and link protein into aggregates were examined using cultured chondrocytes from the Swarm rat chondrosarcoma. Proteoglycans purified directly from culture medium after a 6-h label with [14C]serine contained up to 80% of the radioactivity in aggregates, 8% of which was in link protein. The addition of hyaluronic acid oligosaccharides (averaging 50 monosaccharides) to the culture medium during labeling inhibited aggregate formation by 94%. More than 90% of the labeled link molecules (about 7% of the total radioactivity in the proteoglycan fraction) was associated in ternary complexes with monomeric proteoglycan and HA approximately 50. The stoichiometry of labeled link protein to labeled proteoglycan core protein aggregated with either macromolecular hyaluronic acid or hyaluronic acid oligosaccharides was calculated to be 1:1. The effect of exogenous proteoglycan on aggregate formation was examined by incubating cultures for 4 h with up to 5 mg of exogenous monomer. Over this time, about 85 micrograms of newly synthesized proteoglycan accumulated in the culture medium. With increasing amounts of exogenous monomer, up to 80 micrograms of 35S-labeled exogenous monomer was incorporated into link-stabilized aggregate, while the aggregation of endogenous proteoglycan decreased from 73 to 44%. The total endogenous plus exogenous monomer in link-stabilized aggregate increased to about 130 micrograms, approximately 1 1/2 times the total amount of endogenous monomer in the culture medium, indicating that at least 1 1/2 times more link molecules than endogenous proteoglycan molecules were present. Further, the molar ratio of labeled link to labeled monomer molecules associated with the nonaggregated proteoglycan fraction remained constant at approximately 1:1, indicating that the exogenous proteoglycans were ineffective in displacing link molecules from endogenous monomer.link complexes. The data suggest that the formation of proteoglycan aggregates may be a sequential process in which a link.monomer complex forms and this complex subsequently binds to hyaluronic acid.

Animals↗
PubMed1980

Equilibrium-binding studies of pig laryngeal cartilage proteoglycans with hyaluronate oligosaccharide fractions.

The binding of hyaluronate oligosaccharide fractions to proteoglycans from pig laryngeal cartilage has been studied by equilibrium dialysis in dilute solution. It has been shown that: (1) each proteoglycan monomer binds only one hyaluronate oligosaccharide molecule [containing about eighteen saccharide residues (HA approximately 18) and of number-average molecule weight (Mn) 37501]; (2) the dissociation constant, Kd, for interaction between proteoglycan monomer and oligosaccharide HA approximately 18 is 3 x 10(-8) M at 6 degrees C at I 0.15-0.5, pH 7.4; (3) the dissociation constant has little dependence on temperature, so that Kd at 54 degrees C is 3 x 10(-7) M under the same conditions; (4) the aggregatability is high at 6 degrees C, falls significantly at 54 degrees C, but much of it can be recovered on cooling to 6 degrees C again, demonstrating reversible denaturation; (5) a method for determining the proportion of the proteoglycan molecules capable of binding to hyaluronate by equilibrium dialysis was compared with gel-chromatographic and ultracentrifugal methods; (6) a hyaluronate oligosaccharide, HA approximately 56 (Mn 11 000), could bind more than one proteoglycan molecule; (7) consideration of ultracentrifugal data shows that when proteoglycans bind to a hyaluronate of larger size (mol..wt. 670 000), an average Kd of 12 x 10(7) M fits the data in 0.5 M-guanidine hydrochloride at 20 degrees C.

Animals↗
PubMed1979

Biosynthesis of proteoglycans and their assembly into aggregates in cultures of chondrocytes from the Swarm rat chondrosarcoma.

Cultured chondrocytes from the Swarm rat chondrosarcoma incorporate [35S]sulfate into proteoglycans typical of hyaline cartilage. The movement of newly synthesized proteoglycans from inside the cells into the extracellular matrix and, finally, into the culture medium was examined by measuring the distribution of 35S-labeled proteoglycans in the medium, a 4 M guanidine HCl extract of the cell layer, and in the remaining residue for a number of chase times following a 5-min pulse with [35S]sulfate. When hyaluronate oligosaccharides containing greater than or equal to 10 monosaccharides were included in the chase media, a proportion of newly synthesized proteoglycans were displaced from the matrix (4 M extract) into the culture medium. This displacement was greatest when oligomers were in the chase media between 10 and 20 min after the pulse, approximately the time when the molecules are being secreted from the cells. The proportion of link-stabilized aggregate in the medium was examined by Sepharose 2B chromatography after adding an excess of unlabeled monomer which displaces labeled monomer from complexes with hyaluronate which are not link-stabilized. The proportion of link-stabilized aggregate increased from 12% to about 70% between 12 and 120 min of chase. The presence of 40 micron hyaluronate oligosaccharides of 16 monosaccharides in the chase media retarded but did not prevent aggregate formation. Oligomers of about 50 monosaccharides, which are large enough to bind both a monomer proteoglycan and a link protein, almost completely prevented the formation of the large link-stabilized aggregates. The results suggest: (a) newly synthesized proteoglycans are not bound into link-stabilized aggregates at the time of secretion; (b) hyaluronic acid oligomers which are long enough to interact only with the hyaluronic acid-binding site of proteoglycans will retard but not prevent link-stabilized aggregation; and (c) hyaluronic acid oligomers long enough to accommodate additionally a link protein form a link-stabilized ternary complex and prevent aggregation with larger hyaluronic acid molecules.

Animals↗