13C-1H magnetic double-resonance study of fetal enamel matrix proteins

Recently developed ¹³C-¹H nuclear magnetic double-resonance techniques have been used to study proteins in the intact fetal enamel matrix. Enamel protein chains undergoing rapid, almost isotropic motion were detected in scalar decoupled ¹³C-nmr spectra, while motionally restricted enamel protein chains were principally observed in proton-enhanced spectra. The latter spectra were obtained using a matched Hartmann-Hahn contact to transfer polarization from protons to carbons (cross-polarization). Both mobile and motionally restricted enamel protein chains were observed in dipolar decoupled ¹³C-nmr spectra. A comparison of integrated intensities obtained from the scalar decoupled and dipolar decoupled spectra showed that 70% of the fetal enamel protein chains exhibit rapid, nearly isotropic molecular motion (τ ? 10^?6 sec), while the remaining 30% are rigid or undergo only anisotropic molecular motion.     

The isolation of collagen-associated proteoglycan from bovine nasal cartilage and its preferential interaction with alpha2 chains of type I collagen.

A collagen complex from bovine nasal cartilage was prepared by extraction of the tissue with 3M-MgCl2 solutions, by using two different procedures. When it was compared with calf skin acid-soluble tropocollagen by polyacrylamide-gel electrophoresis, the 3M-MgCl2-soluble cartilage collagen in the complex appeared to be predominantly type I in nature, consisting of both alpha1 and alpha2 chains. The soluble cartilage collagens were digested with purified bacterial collagenase, and the soluble digests were fractionated on Sepharose 4B. Hydroxyproline-free proteoglycan was isolated in the excluded volume of the column eluate, and this was found to be an aggregate which could be dissociated to link proteins and proteoglycan subunit by equilibrium-density-gradient centrifugation in a CsCl-4M-guanidinium chloride gradient. Interaction with calf skin-soluble tropocollagen was studied by CM-cellulose chromatography. The link-protein system did not interact, but proteoglycan from the bottom of the gradient did interact. In addition, when proteoglycan subunit was allowed to interact with collagen, there was a preferential binding to the alpha2 and beta12 components, and this effect was also observed with the proteoglycan material obtained from the collagenase digests of 3M-MgCl2-soluble cartilage collagen complexes. However, specificity for alpha2 and beta12 chains was not exhibited by chondroitin sulphate glycosaminoglycan, and it is therefore concluded that preference for alpha2 and beta12 chains is a function of the intact proteoglycan structure.     

Cartilage proteoglycan aggregates. Electronmicroscopic studies of native and fragmented molecules

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.     

Digestion of proteoglycan by Bacteroides thetaiotaomicron

It has been shown previously that Bacteroides thetaiotaomicron, a human colonic anaerobe, can utilize the tissue mucopolysaccharide chondroitin sulfate as a source of carbon and energy and that the enzymes involved in this utilization are all cell associated (A. A. Salyers and M. B. O'Brien, J. Bacteriol. 143:772-780, 1980). Since chondroitin sulfate does not generally occur in isolated form in tissue, but rather is bound covalently in proteoglycan, we investigated the extent to which chondroitin sulfate which is bound in such a sterically hindered complex can be utilized by intact bacteria. Intact cells of B. thetaiotaomicron were able to digest chondroitin sulfate in proteoglycan, although at a slightly slower rate than free chondroitin sulfate. Prior digestion of proteoglycan with trypsin to produce small fragments of protein with several chondroitin sulfate chains attached did not increase the rate at which the bound chondroitin sulfate was digested. Accordingly, the slower rate of digestion was probably due to attachment of chondroitin sulfate chains to the protein backbone rather than to steric hindrance by other components of the proteoglycan. When proteoglycan which had been incubated with intact bacteria was treated with sodium borohydride to release the undigested fragments of chondroitin sulfate from the protein backbone, the size and composition of the fragments indicated that intact bacteria were able to digest all but three monosaccharides of the chondroitin sulfate chains. Thus, despite steric hindrance due to attachment of the chondroitin sulfate chains to the protein backbone, digestion of bound chondroitin sulfate by intact bacteria was nearly complete.     

Articular cartilage cultured with catabolin (pig interleukin 1) synthesizes a decreased number of normal proteoglycan molecules.

A homogeneous preparation of catabolin from pig leucocytes caused a reversible dose-dependent (0.01-1 nM) decrease in the synthesis of proteoglycan in slices of pig articular cartilage cultured in serum-free medium. The monomers that were synthesized and secreted in the presence of catabolin had the same average hydrodynamic size and ability to aggregate as the controls, and the core protein was substituted with the same number of glycosaminoglycan chains. The chains were the same average length and charge as normal and were sulphated to the same extent as the controls. Newly synthesized extracellular proteoglycan was not preferentially degraded. A 2-3-fold increase in glycosaminoglycan synthesis occurred in control and catabolin-treated cartilage in the presence of beta-D-xyloside (1 mM), more than 80% being secreted into the medium as free chains. Decreased incorporation of sulphate was not reversed in the presence of lysosomal-enzyme inhibitors, and there was no evidence in pulse-chase experiments of increased intracellular degradation of glycosaminoglycan chains before secretion. It is concluded that catabolin-treated cartilage synthesizes a smaller number of normal proteoglycan molecules.     

Association of the aggrecan keratan sulfate-rich region with collagen in bovine articular cartilage

Aggrecan, the predominant large proteoglycan of cartilage, is a multidomain macromolecule with each domain contributing specific functional properties. One of the domains contains the majority of the keratan sulfate (KS) chain substituents and a protein segment with a proline-rich hexapeptide repeat sequence. The function of this domain is unknown but the primary structure suggests a potential for binding to collagen fibrils. We have examined binding of aggrecan fragments encompassing the KS-rich region in a solid-phase assay. A moderate affinity (apparent Kd = 1.1 microM) for isolated collagen II, as well as collagen I, was demonstrated. Enzymatic digestion of the KS chains did not alter the capacity of the peptide to bind to collagen, whereas cleavage of the protein core abolished the interaction. The distribution of the aggrecan KS-rich region in bovine tarsometatarsal joint cartilage was investigated using immunoelectron microscopy. Immunoreactivity was relatively low in the superficial zone and higher in the intermediate and deep zones of the uncalcified cartilage. Within the pericellular and territorial matrix compartments the epitopes representing the aggrecan KS-rich region were detected preferentially near or at collagen fibrils. Along the fibrils, epitope reactivity was non-randomly distributed, showing preference for the gap region within the D-period. Our data suggest that collagen fibrils interact with the KS-rich regions of several aggrecan monomers aligned within a proteoglycan aggregate. The fibril could therefore serve as a backbone in at least some of the aggrecan complexes.     

Mechanism for the decreased biosynthesis of cartilage proteoglycan in the scorbutic guinea pig

Our previous work showed that vitamin C deficiency caused about a 70-80% decrease in the incorporation of [35S]sulfate into proteoglycan of guinea pig costal cartilage, coordinately with a decrease in collagen synthesis (Bird, T. A., Spanheimer, R. G., and Peterkofsky, B. (1986) Arch. Biochem. Biophys. 246, 42-51). We examined the mechanism for decreased proteoglycan synthesis by labeling normal and scorbutic cartilage in vitro with radioactive precursors. Proteoglycan monomers from scorbutic tissue were of a slightly smaller average hydrodynamic size than normal but there was no difference in the size of the glycosaminoglycan chains isolated after papain digestion. The type of glycosaminoglycans synthesized and the degree of sulfation were unaffected as determined by chondroitinase ABC digestion and duel labeling with [35S]sulfate and [3H]glucosamine. Conversion of [3H]glucosamine to [3H]galactosamine also was unimpaired. There was about a 40% decrease in core protein synthesis, measured by [14C]serine incorporation and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Nevertheless, decreased incorporation of [35S]sulfate into scorbutic tissue persisted in the presence of p-nitrophenyl-beta-D-xyloside and cycloheximide, which indicated that the site of the scorbutic defect was beyond core protein synthesis and xylosylation. Galactosyltransferase activity in scorbutic cartilage decreased to about one-third the levels in control samples in parallel with the decreases in proteoglycan and collagen synthesis. Our results suggest that the step catalyzed by this enzyme activity, the addition of galactose to xylose prior to chondroitin sulfate chain elongation, is the major site of the scorbutic defect in proteoglycan synthesis. Decreased enzyme activity may be related to increased cortisol levels in scorbutic serum.     

Application of 2-aminopyridine fluorescence labeling to glycosaminoglycans

A pyridylamination method was applied to glycosaminoglycans and the characteristics of the resulting pyridylamino glycosaminoglycans were examined. First, glycosaminoglycan chains, which uniformly possess a xylose residue at their reducing termini, were liberated from proteoglycan by successive digestion with protease and endo-beta-xylosidase. Then the glycosaminoglycan chains were coupled with 2-aminopyridine by reductive amination with sodium cyanoborohydride for 15 h according to the method of Hase, S. et al. [J. Biochem. 95, 197-203 (1984)]. The pyridylamination reaction caused neither depolymerization, de-N-acetylation, nor de-N- or de-O-sulfation. The pyridylamino glycosaminoglycan chains had an intact linkage region (GlcA-Gal-Gal-Xyl) between the carbohydrate chain and the peptide core of the proteoglycan. These pyridylamino glycosaminoglycans should be useful as substrates for endo-type glycosidases that act on glycosaminoglycan chains and as markers for studies of glycosaminoglycan metabolism.     

A monoclonal antibody that specifically recognizes a glucuronic acid 2-sulfate-containing determinant in intact chondroitin sulfate chain

Monoclonal antibodies produced against chick embryo limb bud proteoglycan (PG-M) were selected for their ability to recognize determinants on intact chondroitin sulfate chains. One of these monoclonal antibodies (IgM; designated MO-225) reacts with PG-M, chick embryo cartilage proteoglycans (PG-H, PG-Lb, and PG-Lt), and bovine nasal cartilage proteoglycan, but not with Swarm rat chondrosarcoma proteoglycan. The reactivity of PG-H to MO-225 is not affected by keratanase digestion but is completely abolished after chondroitinase digestion. Competitive binding analyses with various glycosaminoglycan samples indicate that the determinant recognized by MO-225 resides in a D-glucuronic acid 2-sulfate(beta 1----3)N-acetylgalactosamine 6-sulfate disaccharide unit (D-unit) common to antigenic chondroitin sulfates. A tetrasaccharide trisulfate containing D-unit at the reducing end is the smallest chondroitin sulfate fragment that can inhibit the binding of the antibody to PG-H. Decreasing the size of a D-unit-rich chondroitin sulfate by hyaluronidase digestion results in progressive reduction in its inhibitory activity. The results suggest that the epitope has a requirement for a long stretch of a disaccharide-repeating structure for a better fit to the antibody.     

On the anisotropy and inhomogeneity of permeability in articular cartilage

Articular cartilage is known to be anisotropic and inhomogeneous because of its microstructure. In particular, its elastic properties are influenced by the arrangement of the collagen fibres, which are orthogonal to the bone-cartilage interface in the deep zone, randomly oriented in the middle zone, and parallel to the surface in the superficial zone. In past studies, cartilage permeability has been related directly to the orientation of the glycosaminoglycan chains attached to the proteoglycans which constitute the tissue matrix. These studies predicted permeability to be isotropic in the undeformed configuration, and anisotropic under compression. They neglected tissue anisotropy caused by the collagen network. However, magnetic resonance studies suggest that fluid flow is "directed" by collagen fibres in biological tissues. Therefore, the aim of this study was to express the permeability of cartilage accounting for the microstructural anisotropy and inhomogeneity caused by the collagen fibres. Permeability is predicted to be anisotropic and inhomogeneous, independent of the state of strain, which is consistent with the morphology of the tissue. Looking at the local anisotropy of permeability, we may infer that the arrangement of the collagen fibre network plays an important role in directing fluid flow to optimise tissue functioning.     

Chondroitin/dermatan sulfate proteoglycan in human fetal membranes. Demonstration of an antigenically similar proteoglycan in fibroblasts

A proteoglycan was isolated from fetal membranes which had been separated from human postpartum placenta. The glycosaminoglycan side chains (Mr = 55,000) were found to be composed of 75% chondroitin sulfate and 23% dermatan sulfate as determined by chondroitinase ABC or AC II digestion. NH2-terminal microsequencing of the intact proteoglycan revealed a single amino acid sequence of (sequence; see text) A rabbit antiserum raised against the intact proteoglycan reacted in sodium dodecyl sulfate-polyacrylamide gel electrophoresis immunoblotting with Mr = 45,000 and 43,000 core polypeptides from chondroitinase-treated proteoglycan. Affinity-purified antibodies from this antiserum precipitated from human embryonic fibroblast culture fluid a proteoglycan which has an approximate Mr = 120,000 in sodium dodecyl sulfate-polyacrylamide gel electrophoresis. This proteoglycan has on the average two polysaccharide side chains. As defined by chondroitinase digestion, these chains consist of 66% dermatan sulfate and 20% chondroitin sulfate. Digestion of the glycosaminoglycan with chondroitinase ABC converted the proteoglycan to a Mr = 45,000 major and a Mr = 43,000 minor core polypeptide. Tissue immunofluorescence localized the proteoglycan to interstitial matrices, suggesting that it is a product of mesenchymal cells. The methods we have devised for the purification of the fetal membrane proteoglycan in chemical amounts and the antibodies we have prepared against it will allow studies on the structural and functional properties of the proteoglycan and on the expression of immunologically cross-reactive proteoglycans by various cells and tissues.     

Interaction of proteoglycans with the pericellular (1 alpha, 2 alpha, 3 alpha) collagens of cartilage

Interaction between cartilage proteoglycan and the collagen(s) composed of 1 alpha, 2 alpha, and 3 alpha chains was studied in vitro. Most of the collagen was insoluble under the conditions of assay (0.15 M NaCl, 0.008 M phosphate buffer, pH 7.4; 4 degrees C) and was in the form of fibrils 20 nm in diameter or thinner. The larger fibrils had 60-70 nm periodicity, characteristic of native collagens. Proteoglycan monomers which had been labeled by incubating cartilage slices in vitro with Na2 35SO4 were used to assay the interaction. The insoluble collagen fraction bound proteoglycan from solution. At proteoglycan:collagen ratios lower than 1:2, binding was rapid and linear, and the dissociation constant was 1.7 X 10(-9) M. At higher proteoglycan:collagen ratios, more proteoglycan was bound, but at a slower rate. Binding of proteoglycan to collagen did not require fibrils, since soluble 1 alpha, 2 alpha, and 3 alpha containing collagen also bound to proteoglycan and formed an insoluble complex. Denatured collagens did not bind proteoglycan or compete for binding with normal collagen. Optimum binding occurred with intact proteoglycan, but proteoglycan which had been treated with protease was also bound at low levels. Both protease-treated proteoglycan and free chondroitin sulfate competed with intact proteoglycan in the binding assays, but neither chondroitinase ABC-treated proteoglycan nor the oligosaccharides produced by digestion of chondroitin sulfate with testicular hyaluronidase altered the binding of proteoglycan to collagen. Hyaluronic acid did not compete with radioactive proteoglycan, but heparin and dextran sulfate were extremely effective inhibitors of binding. These data suggest a relatively nonspecific interaction between sulfated polyanions and 1 alpha, 2 alpha, and 3 alpha containing collagens. However, given the location of these collagens near the chondrocyte surface, the interaction of fibrillar 1 alpha, 2 alpha, 3 alpha collagen with proteoglycan is likely to occur and to be of biological importance.     

Interactions between bovine cornea proteoglycans and collagen.

Two types of proteoglycan subunits were obtained from bovine cornea, the first mainly composed of proteochondroitin sulphate and the second of proteokeratan sulphate. These two fractions can be obtained from the tissue as an aggregate, and are able to recombine each other after separation, to re-form the original structure. In order to investigate collagen-proteoglycan interactions, type-I collagen was isolated from bovine cornea by pepsin digestion followed by 3.5% (w/v) NaCl precipitation, and was then linked to CNBr-activated Sepharose 4B. Two identical columns were prepared, the first filled with collagen coupled to Sepharose 4B, the second with free Sepharose 4B. The two proteoglycan subunits and the aggregate were chromatographed on the two gels under the same conditions; the elution profiles showed that both the aggregate and the proteochondroitin sulphate subunit are retarded by the collagen coupled to Sepharose. No interaction, however, occurred when proteokeratan sulphate subunit was run through the columns. Chondroitinase digestion of the proteoglycan samples confirmed that chondroitin sulphate chains are mainly responsible for the interaction with collagen; their removal, in fact, completely abolishes any differences between the chromatographic behaviour on the collagen-Sepharose and the control columns.     

Identification of L-selectin binding heparan sulfates attached to collagen type XVIII

L-selectin is a C-type lectin expressed on leukocytes that is involved in both lymphocyte homing to the lymph node and leukocyte extravasation during inflammation. Known L-selectin ligands include sulfated Lewis-type carbohydrates, glycolipids, and proteoglycans. Previously, we have shown that in situ detection of different types of L-selectin ligands is highly dependent on the tissue fixation protocol used. Here we use this knowledge to specifically examine the expression of L-selectin binding proteoglycans in normal mouse tissues. We show that L-selectin binding chondroitin/dermatan sulfate proteoglycans are present in cartilage, whereas L-selectin binding heparan sulfate proteoglycans are present in spleen and kidney. Furthermore, we show that L-selectin only binds a subset of renal heparan sulfates, attached to a collagen type XVIII protein backbone and predominantly present in medullary tubular and vascular basement membranes. As L-selectin does not bind other renal heparan sulfate proteoglycans such as perlecan, agrin, and syndecan-4, and not all collagen type XVIII expressed in the kidney binds L-selectin, this indicates that there is a specific L-selectin binding domain on heparan sulfate glycosaminoglycan chains. Using an in vitro L-selectin binding assay, we studied the contribution of N-sulfation, O-sulfation, C5-epimerization, unsubstituted glucosamine residues, and chain length in L-selectin binding to heparan sulfate/heparin glycosaminoglycan chains. Based on our results and the accepted model of heparan sulfate domain organization, we propose a model for the interaction of L-selectin with heparan sulfate glycosaminoglycan chains. Interestingly, this opens the possibility of active regulation of L-selectin binding to heparan sulfate proteoglycans, e.g. under inflammatory conditions.     

Effects of glutathione depletion on the synthesis of proteoglycan and collagen in cultured chondrocytes

We studied the effect of the depletion of glutathione on the synthesis of proteoglycan and collagen in cultured chick chondrocytes. When the cultured chondrocytes were incubated with 1 mM buthionine sulfoximine (BSO), a specific inhibitor of gamma-glutamyl-cysteine synthetase, the intracellular glutathione level markedly dropped within 12 h with no loss of cell viability. Incorporation of 35SO2-4 into proteoglycan was lowered in the presence of BSO. When the 35S-labeled proteoglycans were separated into two fractions by glycerol density gradient centrifugation, the inhibitory effect of BSO on the synthesis of proteoglycan was greater in the fast-sedimenting proteoglycan fraction, which consisted mainly of cartilage specific large proteoglycan (PG-H), than in the slowly sedimenting proteoglycan fraction. The inhibition by BSO of the synthesis of core protein-free glycosaminoglycan chains primed by p-nitrophenyl-beta-D-xyloside was smaller than the inhibition of the synthesis of proteoglycan. Analysis of glycosaminoglycans labeled with [3H]glucosamine indicated that the treatment of chondrocytes with BSO resulted in a small increase in the proportion of synthesis of hyaluronic acid to the synthesis of total glycosaminoglycan. The incorporation of [3H]proline into collagen was also inhibited by BSO. Sodium dodecyl sulfate polyacrylamide gel electrophoresis of the 3H-labeled collagen showed that, in the presence of BSO, processing of Type II collagen appeared to slow down and the proportion of Type X collagen synthesis was reduced.     

Fibronectin-mediated adhesion of fibroblasts: inhibition by dermatan sulfate proteoglycan and evidence for a cryptic glycosaminoglycan-binding domain

Dermatan sulfate proteoglycans (DS-PGs) isolated from bovine articular cartilage have been examined for their effects on the adhesive responses of BALB/c 3T3 cells and bovine dermal fibroblasts on plasma fibronectin (pFN) and/or type I collagen matrices, and compared to the effects of the chondroitin sulfate/keratan sulfate proteoglycan monomers (CS/KS-PGs) from cartilage. DS-PGs inhibited the attachment and spreading of 3T3 cells on pFN-coated tissue culture substrata much more effectively than the cartilage CS/KS-PGs reported previously; in contrast, dermal fibroblasts were much less sensitive to either proteoglycan class unless they were pretreated with cycloheximide. Both cell types failed to adhere to substrata coated only with the proteoglycans; binding of the proteoglycans to various substrata has also been quantitated. While a strong inhibitory effect was obtained with the native intact DS-PGs, little inhibitory effect was obtained with isolated DS chains (liberated by alkaline-borohydride cleavage) or with core protein preparations (liberated by chondroitinase ABC digestion). In marked contrast, DS-PGs did not inhibit attachment or spreading responses of either 3T3 or dermal fibroblasts on type I collagen-coated substrata when the collagen was absorbed with pFN alone, DS-PGs alone, or the two in combination. These results support evidence for (a) collagen-dependent, fibronectin-independent mechanisms of adhesion of fibroblasts, and (b) different sites on the collagen fibrils where DS-PGs bind and where cell surface "receptors" for collagen bind. Experiments were developed to determine the mechanism(s) of inhibition. All evidence indicated that the mechanism using the intact pFN molecule involved the binding of the DS-PGs to the glycosaminoglycan (GAG)-binding sites of substratum-bound pFN, thereby inhibiting the interaction of the fibronectin with receptors on the cell surface. This was supported by affinity chromatography studies demonstrating that DS-PGs bind completely and effectively to pFN-Sepharose columns whereas only a subset of the cartilage CS/KS-PG binds weakly to these columns. In contrast, when a 120-kD chymotrypsin-generated cell-binding fragment of pFN (CBF which has no detectable GAG-binding activity as a soluble ligand) was tested in adhesion assays, DS-PGs inhibited 3T3 adherence on CBF more effectively than on intact pFN. A variety of experiments indicated that the mechanism of this inhibition also involved the binding of DS-PGs to only substratum-bound CBF due to the presence of a cryptic GAG-binding domain not observed in the soluble CBF.(ABSTRACT TRUNCATED AT 400 WORDS)     

The interactions of cartilage proteoglycans with collagens are determined by their structures.

In the present work, the interaction of aggrecan, decorin and biglycan isolated from pig laryngeal cartilage and of the three squid cartilage proteoglycans with collagen type I and II was studied. The interaction was examined under conditions allowing the formation of collagen fibrils. It was found that biglycan interacted strongly with collagen type II and not with type I and the interaction seemed to proceed exclusively through its core proteins. Decorin interacted with collagen type I but not with type II. Aggrecan interacted very poorly with both collagen types. The two squid proteoglycans of large size, D1D1A and D1D2, interacted only with collagen type I through both glycosaminoglycans and core proteins. The third squid proteoglycan of small size, D1D1B, interacted poorly only with collagen type I. The results suggested that the interactions of cartilage proteoglycans with collagen were mainly due to the primary structure of both molecules, and would contribute to the maintenance of the integrity of the tissue. The biochemical significance of these interactions might be more critical in aged vertebrate cartilage, where loss of aggrecan and increase of the small proteoglycans was observed, a large proportion of which is found in the extracellular matrix free of glycosaminoglycan chains.     

Production and characterization of monoclonal antibodies directed against connective tissue proteoglycans

Monoclonal antibodies have been raised against determinants present in cartilage proteoglycan. Characterization of the specificity of these antibodies indicated that they recognize determinants present in the keratan sulfate glycosaminoglycan chain and on chondroitin sulfate oligosaccharide stubs attached to the proteoglycan core protein after chondroitinase digestion of the proteoglycan (i.e., delta-unsaturated 4- and 6-sulfated and unsulfated chondroitin sulfate on the proteoglycan core). The antibody recognizing keratan sulfate has been used to demonstrate the presence of a keratan sulfate-rich proteoglycan subpopulation that increases with increasing age of animal compared with chondroitin sulfate-rich proteoglycans. Monoclonal antibodies recognizing determinants on chondroitinase-treated proteoglycan have been used in immunohistochemical localization studies determining the differential distribution of 4- and 6-sulfated and unsulfated proteoglycans in tissue sections of cartilage and other noncartilaginous tissues. Digestion with chondroitinase ABC or ACII can be used to differentiate between chondroitin sulfate and dermatan sulfate proteoglycan in different connective tissues. In addition, the presence of a 6-sulfated chondroitin sulfate proteoglycan that is associated with membranes surrounding nerve and muscle fiber bundles is described. Monoclonal antibodies were also raised against the link protein(s) of cartilage proteoglycan aggregate. They have been used in peptide map analyses of link protein and in demonstrating the presence of a high-mannose oligosaccharide chain of the link proteins. The presence of high-mannose oligosaccharide structures on the link protein(s) accounts for the microheterogeneity of the link proteins (link proteins 1, 2, or 3) that is observed on sodium dodecyl sulfate-polyacrylamide gels.     

Synthesis and structure of proteoglycan core protein

Studies of the structure and synthesis of cartilage proteoglycan core protein have been carried out. Deglycosylation of completed, secreted proteoglycan by HF-pyridine treatment yielded an intact homogeneous core protein of approximately 210,000 daltons, with a blocked amino-terminus. Greater than 95% of chondroitin sulfate chains and 80% of N- and O-linked oligosaccharides were removed by the procedure, which made the product an excellent xylosyltransferase acceptor. Little alteration of core protein structure occurred during the HF-pyridine treatment as shown by complete immunoreactivity with antiserums prepared against hyaluronidase-digested proteoglycan. In other studies, the initially synthesized precursor for proteoglycan core protein was found to be approximately 376,000 daltons and localized to the rough membrane fractions. This precursor already contained N-linked oligosaccharides, and was also able to accept xylose, thereby initiating chondroitin sulfate chains. The precursor was translocated intact in an energy-dependent manner to smooth membrane-Golgi fractions where further processing of high mannose type of oligosaccharides and addition of glycosaminoglycan chains occurred. The subcellular distribution pattern of the chondroitin sulfate-synthesizing enzymes corroborated the proposed topological modifications of the proteoglycan core protein precursor.     

Dermatan sulphate proteoglycan from human articular cartilage. Variation in its content with age and its structural comparison with a small chondroitin sulphate proteoglycan from pig laryngeal cartilage.

Low molecular mass proteoglycans (PG) were isolated from human articular cartilage and from pig laryngeal cartilage, which contained protein cores of similar size (Mr 40-44 kDa). However, the PG from human articular cartilage contained dermatan sulphate (DS) chains (50% chondroitinase AC resistant), whereas chains from pig laryngeal PG were longer and contained only chondroitin sulphate (CS). Disaccharide analysis after chondroitinase ABC digestion showed that the human DS-PG contained more 6-sulphated residues (34%) than the pig CS-PG (6%) and both contained fewer 6-sulphated residues than the corresponding high Mr aggregating CS-PGs from these tissues (86% and 20% from human and pig respectively). Cross-reaction of both proteoglycans with antibodies to bovine bone and skin DS-PG-II and human fibroblasts DS-PG suggested that the isolated proteoglycans were the humans DS-PG-II and pigs CS-PG-II homologues of the cloned and sequenced bovine proteoglycan. Polyclonal antibodies raised against the pig CS-PG-II were shown to cross-react with human DS-PG-II. SDS/polyacrylamide-gel analysis and immunoblotting of pig and human cartilage extracts showed that some free core protein was present in the tissues in addition to the intact proteoglycan. The antibodies were used in a competitive radioimmunoassay to determine the content of this low Mr proteoglycan in human cartilage extracts. Analysis of samples from 5-80 year-old humans showed highest content (approximately 4 mg/g wet wt.) in those from 15-25 year-olds and lower content (approximately 1 mg/g wet wt.) in older tissue (greater than 55 years). These changes in content may be related to the deposition and maintenance of the collagen fibre network with which this class of small proteoglycan has been shown to interact.