Electron microscopic studies of proteoglycan aggregates from bovine articular cartilage.

Proteoglycan aggregates from bovine articular cartilage have been visualized by electron microscopy of mixed proteoglycan-cytochrome c monolayers. The proteoglycan aggregates consist of proteoglycan subunits arising laterally at fairly regular intervals (20 to 30 nm) from the opposite sides of an elongated filamentous structure. The filamentous backbone in individual aggregates varies in length from 400 to 4000 nm. The individual proteoglycan subunits in the aggregate vary in length from 100 to 400 nm. However, there is no difference in the average size of the proteoglycan subunits associated with the largest or smallest aggregates. The sizes of the individual aggregates are determined mainly by the lengths of their filamentous backbones. The stoichiometry of binding of subunits to filament, calculated from the data reported here, is close to that for the binding of subunits to hyaluronic acid reported by others.     

Antigenic determinants of two components of proteoglycan complex from bovine cartilage

The immunological properties of a glycoprotein fraction and of proteoglycan subunits obtained from bovine nasal cartilage by nondisruptive methods of isolation have been studied. Using the techniques of hemagglutination and hemagglutination inhibition, we found that the glycoprotein contains most of the species-specific determinants, whereas the proteoglycan subunits contain most of the cross-reacting ones.     

The role of link protein in mediating the interaction between hyaluronic acid and newly secreted proteoglycan subunits from adult human articular cartilage

Normal adult human articular cartilage in organ culture secretes proteoglycan subunits that cannot initially interact in a normal manner with hyaluronic acid unless the latter is present at high concentrations and a neutral pH is employed. However, if the newly secreted subunit is allowed to mature in the cartilage matrix for up to 12 h, then its ability to interact is indistinguishable from that of its more mature counterparts. This conversion does not take place if the proteoglycan subunits are incubated in dilute solutions in the absence of the cartilage, and it is prevented by culturing at low temperature. The newly secreted proteoglycan subunits can, however, be induced to interact with hyaluronic acid by the presence of link proteins. The complex formed by these three components cannot be dissociated in the presence of hyaluronic acid oligosaccharides, suggesting a normal aggregate configuration. It is thus possible that proteoglycan aggregate formation within the cartilage is initially mediated by the presence of link proteins, which induce a conformational change with the hyaluronic acid-binding region of the proteoglycan subunits, although additional modification may be necessary to render any such change irreversible.     

Molecular size distribution of proteoglycan subunits and glycosaminoglycans synthesized by chondrocytes under conditions of reduced proteoglycan synthesis

The rate of proteoglycan synthesis by chondrocytes in vitro was depressed by either omitting l-glutamine from the incubation medium or by addition of proteoglycan subunit to the medium. The molecular size distribution on Sepharose 2B of the proteoglycan subunits synthesized by the chondrocytes under these conditions of reduced proteoglycan synthesis was found to be the same as those synthesized by the control cells. Likewise, the molecular size distribution on Sepharose 6B CL of the glycosaminoglycan chains synthesized by the depressed cells was found to be similar to that observed in untreated chondrocytes. This work demonstrates that, under conditions of reduced proteoglycan synthesis, fewer proteoglycan subunits are synthesized by chondrocytes and that the molecular size distribution of these macromolecules is similar to those synthesized by untreated cells.     

The action of human articular-cartilage metalloproteinase on proteoglycan and link protein. Similarities between products of degradation in situ and in vitro.

Interleukin 1 stimulation of human articular cartilage in organ culture produced the concomitant release of proteoglycan fragments and latent metalloproteinase. The released fragments ranged in size from that of almost intact proteoglycan subunits to the product of limiting digestion generated by the activated metalloproteinase. None of the fragments possessed the ability to interact with hyaluronic acid. Analysis of proteoglycan aggregate digested with the activated metalloproteinase showed that isolated hyaluronic acid-binding regions were produced from the proteoglycan subunits, and that the two higher-Mr link-protein components (Mr 48,000 and 44,000) were converted into the lowest-Mr component (Mr 41,000). Link protein extracted from cartilage under stimulation with interleukin 1 showed a similar conversion. These results suggest that interleukin 1 stimulates the release of latent metalloproteinase from chondrocytes and that a proportion of the enzyme is activated in situ in the cartilage matrix. The mode of action of the activated enzyme is compatible with a role in the changes in proteoglycan structure seen in aging.     

Identification of a hyaluronic acid-binding protein that interferes with the preparation of high-buoyant-density proteoglycan aggregates from adult human articular cartilage.

Adult human articular cartilage contains a hyaluronic acid-binding protein of Mr 60 000-75 000, which contains disulphide bonds essential for this interaction. The molecule can compete with proteoglycan subunits for binding sites on hyaluronic acid, and can also displace proteoglycan subunits from hyaluronic acid if their interaction is not stabilized by the presence of link proteins. The abundance of this protein in the adult accounts for the reported inability to prepare high-buoyant-density proteoglycan aggregates from extracts of adult human cartilage [Roughley, White, Poole & Mort (1984) Biochem. J. 221, 637-644], whereas the deficiency of the protein in newborn human cartilage allows the normal recovery of proteoglycan aggregates from this tissue. The protein shares many common features with a hyaluronic acid-binding region derived by proteolytic treatment of a proteoglycan aggregate preparation, and this may also represent its origin in the cartilage, with its production increasing during tissue maturation.     

Studies on the interaction of newly secreted proteoglycan subunits with hyaluronate in human articular cartilage

Newly secreted proteoglycans from adult human cartilage do not interact well with hyaluronate, but attain this ability with time in the extracellular matrix. The conversion process occurs in all types of cartilagenous matrix, as newborn cartilage cultures, chondrosarcoma cultures and adult chondrocyte cultures each secreted proteoglycan subunits which exhibited the delayed aggregation phenomenon. However, the rate of conversion is probably dependent upon the structure of the surrounding matrix and the cell type. In vitro, link protein appears to enhance an initial change in the hyaluronate-binding region of the newly secreted proteoglycan subunits to allows stronger interaction with hyaluronate. In a second step, which is pH- and temperature-dependent, the change becomes irreversible. Thus, in addition to its role in stabilizing the interaction of mature proteoglycan subunits with hyaluronate, link protein may also aid in promoting the conversion of the newly synthesized proteoglycan subunit to a form that is capable of strong interaction with hyaluronate.     

Structural analysis of glycopeptides by polyacrylamide gel electrophoresis

Three different well-characterized preparations of proteoglycan subunits were analyzed by high-performance liquid chromatography on a silica-based material bonded with an amide phase. The biochemical integrity of the proteoglycan subunits was retained during this procedure. The high sensitivity coupled with the increased speed of high-performance liquid chromatography will permit rapid analysis and comparisons of very small specimens.     

Resolution of cartilage proteoglycan and its proteolytic degradation products by high-performance liquid chromatography using a gel filtration system

Cartilage proteoglycan subunits are resolved from their various-size proteolytic degradation products by a gel filtration high-performance liquid chromatography system using a Bio-Gel TSK-60 column in tandem with a Bio-Gel TSK-50 column. Molecules ranging in size from the intact proteoglycan to single chondroitin sulfate chains are eluted in the included volume. Each analysis takes less than 30 min to complete, and with purified samples as little as 20 micrograms of proteoglycan is required. The method can be applied to the measurement of proteoglycan in mixtures, such as tissue culture media, by monitoring effluent fractions using the dimethylmethylene blue dye-binding assay.     

Treatment of cartilage proteoglycan aggregate with hydrogen peroxide. Relationship between observed degradation products and those that occur naturally during aging.

The effects of treatment of purified neonatal human articular-cartilage proteoglycan aggregate with H2O2 were studied. (1) Exposure of proteoglycan aggregate to H2O2 resulted in depolymerization of the aggregate and modification of the core protein of both the proteoglycan subunits and the link proteins. (2) Treatment of the proteoglycan aggregate with H2O2 rendered the proteoglycan subunits unable to interact with hyaluronic acid, with minimal change in their hydrodynamic size. (3) Specific cleavages of the neonatal link proteins occurred. The order in which the major products were generated and their electrophoretic mobilities resembled the pattern observed during human aging. (4) The proteolytic changes in the link proteins were inhibited in the presence of transition-metal-ion chelators, thiourea or tetramethylurea, suggesting that generation of hydroxyl radicals from H2O2 by trace transition-metal ions via a site-specific Fenton reaction may be responsible for the selective cleavages observed. (5) Cleavage of the link proteins in proteoglycan aggregates by H2O2 was shown to have a limited effect on the susceptibility of these proteins to cleavage by trypsin. (6) The relationship between these changes and those observed in cartilage during human aging suggests that some of the age-related changes in the structure of human cartilage proteoglycan aggregate may be the result of radical-mediated damage.     

Degradation of human proteoglycan aggregate induced by hydrogen peroxide. Protein fragmentation, amino acid modification and hyaluronic acid cleavage.

We have previously shown that treatment of neonatal human articular-cartilage proteoglycan aggregates with H2O2 results in loss of the ability of the proteoglycan subunits to interact with hyaluronic acid and in fragmentation of the link proteins [Roberts, Mort & Roughley (1987) Biochem. J. 247, 349-357]. We now show the following. (1) Hyaluronic acid in proteoglycan aggregates is also fragmented by treatment with H2O2. (2) Although H2O2 treatment results in loss of the ability of the proteoglycan subunits to interact with hyaluronic acid, the loss of this function is not attributable to substantial cleavage of the hyaluronic acid-binding region of the proteoglycan subunits. (3) In contrast, link proteins retain the ability to bind to hyaluronic acid following treatment with H2O2. (4) The interaction between the proteoglycan subunit and link protein is, however, abolished. (5) N-Terminal sequence analysis of the first eight residues of the major product of link protein resulting from H2O2 treatment revealed that cleavage occurred between residues 13 and 14, so that the new N-terminal amino acid is alanine. (6) In addition, a histidine (residue 16) is converted into alanine and an asparagine (residue 21) is converted into aspartate by the action of H2O2. (7) Rat link protein showed no cleavage or modifications in similar positions under identical conditions. (8) This species variation may be related to the different availability of histidine residues required for the co-ordination of the transition metal ion involved in hydroxyl-radical generation from H2O2. (9) Changes in function of these structural macromolecules as a result of the action of H2O2 may be consequences of both fragmentation and chemical modification.     

Physicochemical properties of cartilage proteoglycans extracted by lanthanum chloride.

Bovine nasal cartilage was extracted with 0.5 M LaCl3 and the extract then diluted with nine volumes of water. The resulting precipitated (PLaCl3) contained the proteoglycan subunits, together with minor protein components, but was essentially free from hyaluronic acid. The properties of PLaCl3 were investigated by chemical analysis, electrophoresis, viscometry and analytical ultracentrifugation, and the results compared with those for proteoglycan obtained by caesium chloride density gradient centrifugation of 2 M CaCl2 cartilage extracts. Proteoglycan subunits (A1D1) prepared from PLaCl3 showed identical properties to those obtained from other high ionic strength cartilage extracts.     

Isolation and characterisation of a neutral proteinase from the canine intervertebral disc

A neutral proteinase of 94 kDa capable of degrading gelatin, canine disc proteoglycan, and L-lysine and L-arginine peptide substrates has been isolated from the greyhound intervertebral disc. Strong inhibition of this proteinase with class-specific inhibitors, such as APMSF, TLCK and benzamidine indicated a 'serine'-type specificity. Metallo, aspartyl- and cysteine proteinase inhibitors were devoid of significant action. Degradation of the resident canine disc proteoglycan monomer by the disc proteinase was shown to occur at the hyaluronic acid binding region, thereby diminishing its ability to aggregate with hyaluronic acid. The hydrodynamic size of the proteoglycan degradation products was only slightly less than that of the intact disc proteoglycan subunits.     

Immunochemistry of cartilage proteoglycan. Immunodiffusion and gel-electrophoretic studies

Cartilage proteoglycan is thought to be composed of subunits, core proteins with covalently attached sulphated polysaccharide side chains, which form aggregates by non-covalent association with a link protein. The new technique of non-disruptive extraction followed by fractionation in caesium chloride gradients provides a useful means of preparing relatively pure proteoglycan aggregate, subunit and link fractions. Immunological studies of these fractions led to the identification of an antigen associated with the proteoglycan subunit which was common to several species and to the demonstration of additional species-specific antigens in aggregate and link fractions derived from bovine nasal cartilage. Polyacrylamide-gel electrophoresis with sodium dodecyl sulphate of bovine proteoglycan aggregate and link fractions gave two protein bands in the gels and a protein-polysaccharide band at the origin; subunit fractions gave only the band at the origin. These results are consistent with the current concept of cartilage proteoglycan structure.     

The inability to prepare high-buoyant-density proteoglycan aggregates from extracts of normal adult human articular cartilage.

High-buoyant-density proteoglycan aggregates could not be prepared from extracts of adult human cartilage by associative CsCl-density-gradient centrifugation with a starting density of 1.68 g/ml, even though proteoglycan subunits, hyaluronic acid and link proteins were all present. In contrast, aggregates could be prepared when extracts of neonatal human cartilage or bovine nasal cartilage were subjected to the same procedure. This phenomenon did not appear to be due to a defect within the hyaluronic acid-binding region of the adult proteoglycan subunit, but rather to an interference in the stability of the interaction between the proteoglycan subunit and hyaluronic acid towards centrifugation. The factor responsible for this instability was shown to reside within the low-density cartilage protein preparation obtained by direct dissociative CsCl-density-gradient centrifugation of the adult cartilage extract.     

Rounding up: Engineering 12-membered rings from the cyclic 11-mer TRAP

The protein TRAP (trp RNA binding attenuation protein) forms a highly thermostable ring-shaped 11-mer. By linking in tandem two, three, or four DNA sequences encoding TRAP monomers, we have engineered new rings that consist of 12 TRAP subunits and bind 12 ligand molecules. The hydrogen bonding pattern and buried surface area within and between subunits are essentially identical between the 11-mer and 12-mer crystal structures. Why do the artificial proteins choose to make single 12-mer rings? The 12-mer rings are highly sterically strained by their peptide linkers and far from thermostable. That proteins choose to adopt a strained conformation of few subunits rather than an unstrained one with 11 subunits demonstrates the importance of entropic factors in controlling protein-protein interactions in general.     

O-linked oligosaccharides of human articular cartilage proteoglycan

O-linked oligosaccharides and keratan sulphate chains have been isolated from the proteoglycan subunits of human articular cartilage. The oligosaccharides possessed a size and chemical composition similar to the equivalent moieties present in the proteoglycan submits of the Swarm rat chondrosarcoma. Futhermore, the size and chemical composition of th oligosaccharides showed little change with the age of the individual from whom the proteoglycan was obtained. In contrast, the keratan sulphate chains appeared to increase in chain lenght with increased age of the individual. The total number of keratan sulphate and oligosaccharide chains per core protien decreased with age, but it was not clear whether there was any change in the ration of the two components with respect to one another.     

Isolation and characterization of a third proteoglycan (PG-Lt) from chick embryo cartilage which contains disulfide-bonded collagenous polypeptide

Chick embryo epiphyseal cartilage has been shown to contain three different proteoglycan species (PG-H, PG-Lb, and PG-Lt). This report is concerned with the purification and characterization of the third proteoglycan, PG-Lt. The proteoglycan can be separated from the other two by virtue of its low buoyant density in a CsCl density gradient and further purified by consecutive ion exchange and gel chromatography. The final preparation is composed of PG-Lt monomer and PG-Lt oligomer. The amino acid composition of PG-Lt is quite different from that of PG-H and PG-Lb and rather resembles that of collagens with respect to high content of glycine and high degrees of hydroxylation of proline and lysine. PG-Lt monomer is composed of disulfide-bonded subunits of Mr congruent to 120,000 and 190,000 as demonstrated by its gel electrophoretic behavior after reduction with 2-mercaptoethanol. The latter, but not the former, contains dermatan sulfate chains with glucuronic acid/iduronic acid residues and yields a protein-enriched core molecule of Mr congruent to 100,000 after digestion with chondroitinase ABC. Both of the protein subunits are completely digestible with bacterial collagenase. Immunofluorescence microscopic examination of cartilage tissues, using an antibody against PG-Lt, shows that this proteoglycan exists in both the cartilage matrix and perichondrial noncartilagenous region. When chondrocytes are plated onto tissue culture dishes, the antibody stains strands found on the cell surfaces and in the intercellular space of substrate-attached cell layers, suggesting that PG-Lt mediates cell-to-cell and cell-to-substrate contacts.     

Age-related changes in protein-related epitopes of human articular-cartilage proteoglycans.

Monoclonal antibodies were prepared that recognize different age-related epitopes on proteoglycan subunits of high buoyant density isolated from human epiphysial and articular cartilages. Antibody EFG-4 (IgG1) recognizes a proteinase-sensitive segment associated with the core protein. Antibody BCD-4 (IgG1) reacts with keratan sulphate bound to core protein. Both epitopes are minimally expressed in foetal cartilage and increase with age after birth to become maximally expressed in adult cartilage by about 30 years of age. In contrast, monoclonal antibody alpha HFPG-846 (IgM) recognizes a core-protein-related epitope that is maximally expressed in young foetal cartilage, declines up to birth and thereafter and is almost absent after about 30 years of age. Antibody alpha HFPG-846 was used to isolate by immuno-affinity chromatography two subpopulations of proteoglycan subunits from a 16-year-old-human cartilage proteoglycan subunit preparation. Only the antibody-unbound population showed a significant reaction with antibodies EGF-4 and BCD-4. The amino acid and carbohydrate compositions of these proteoglycan fractions were different, and one (antibody-bound) resembled those of foetal and the other (antibody-unbound) resembled those of adult proteoglycans isolated from 24-27-week-old-foetal and 52-56-year-old-adult cartilage respectively. These observations demonstrate that human cartilages contain at least two chemically and immunochemically distinct populations of proteoglycans, the proportions and content of which are age-dependent. It is likely that these populations represent the products of different genes, though their heterogeneity may be compounded by the result of different post-translation modifications.     

Age-related changes of proteoglycan subunits from sheep nasal cartilage

Proteoglycan subunits of sheep nasal cartilage from animals of five different ages were studied. There is a continuous reduction in the size and chondroitin sulphate content of the aggregable and non-aggregable subunits with ageing. For each age group, the non-aggregable are poorer in protein and keratan sulphate than the corresponding aggregable molecules. Irrespective of age, the amount of proteoglycan protein extracted from each gramme wet cartilage is the same. The amino acid composition and the proportion of the aggregable proteoglycans are also the same.