Hyaluronic Acid-binding Region Research Articles

Chondrons from articular cartilage. (IV). Immunolocalization of proteoglycan epitopes in isolated canine tibial chondrons.

Chondrons have recently been extracted from adult articular cartilages and techniques developed to study their structure and composition in isolation. This study introduces methods to immobilize isolated canine chondrons in thin layers of agarose gel for immunohistochemistry and future in vitro studies. An antibody to Type VI collagen which stained the chondron in suspension was used to successfully validate the system and its feasibility for immunoelectron microscopy. Monoclonal and polyclonal antibodies to a variety of epitopes on the proteoglycan molecule were tested on fresh and fixed plugs cored from chondron-agarose gels. Plugs were immunolabeled with peroxidase-diaminobenzidine before or after digestion with testicular hyaluronidase or chondroitinase ABC. Trypsin/chymotrypsin were used to challenge epitopes of the core protein. The results indicate that epitopes to keratan sulfate, chondroitin sulfate, hyaluronate binding region, and core protein are localized in the chondron. Consistent staining was found in the tail and interconnecting segments between chondrons, whereas staining of the pericellular matrix and capsule adjacent to the chondrocyte varied according to the enzyme pre-treatment employed. We conclude that isolated chondrons are rich in proteoglycan monomer, which is particularly concentrated in the tail and interconnecting segments of the chondron where it could function to protect and stabilize the chondrocyte.

Proteoglycans of human articular cartilage. Identification of several populations of large and small proteoglycans and of hyaluronic acid-binding proteins in successive cartilage extracts

Two specimens of human articulage were successively extracted with solutions of phosphate-buffered saline (PBS), 7 M-urea and 4 M-guanidine hydrochloride (Gdn-HCl). Proteoglycans from individual extracts were fractionated by DEAE-Sephacel chromatography and gel chromatography on Sephacryl S-400. The presence of three populations of large proteoglycans was demonstrated in all three extracts by composite agarose/polyacrylamide-gel electrophoresis (CAPAGE). The population corresponding to the fastest CAPAGE band of aggregating proteoglycans was shown to be extremely polydisperse, having Mr (as estimated by SDS/PAGE) decreasing continuously from more than 300,000 to the size corresponding to 'free' hyaluronic acid-binding region (HABR) (about 70,000). A rather polydisperse set of HABR-containing fragments which spanned a broad range of sizes, and also differed in their keratan sulphate contents, was isolated from both 7 M-urea and 4 M-Gdn-HCl extracts. PBS and 7 M-urea extracts, but not the Gdn-HCl extract, further contained small proteoglycans, identified as fast-migrating bands on CAPAGE electrophoretograms. One of those small species was recognized with an antibody against the small proteoglycan PG II; the other two remain to be positively identified. However, the glycosaminoglycan of the small species which was present exclusively in the PBS extract was identified as keratan sulphate; this species may thus belong to the family of small keratan sulphate-containing proteolygans.

Spatial and temporal changes in the distribution of proteoglycans during avian neural crest development

In this study, we describe the distribution of various classes of proteoglycans and their potential matrix ligand, hyaluronan, during neural crest development in the trunk region of the chicken embryo. Different types of chondroitin and keratan sulfate proteoglycans were recognized using a panel of monoclonal antibodies produced against specific epitopes on their glycosaminoglycan chains. A heparan sulfate proteoglycan was identified by an antibody against its core protein. The distribution of hyaluronan was mapped using a biotinylated fragment that corresponds to the hyaluronan-binding region of cartilage proteoglycans. Four major patterns of proteoglycan immunoreactivity were observed. (1) Chondroitin-6-sulfate-rich proteoglycans and certain keratin sulfate proteoglycans were absent from regions containing migrating neural crest cells, but were present in interstitial matrices and basement membranes along prospective migratory pathways such as the ventral portion of the sclerotome. Although initially distributed uniformly along the rostrocaudal extent of the sclerotome, these proteoglycans became rearranged to the caudal portion of the sclerotome with progressive migration of neural crest cells through the rostral sclerotome and their aggregation into peripheral ganglia. (2) A subset of chondroitin/keratan sulfate proteoglycans bearing primarily unsulfated chondroitin chains was observed exclusively in regions where neural crest cells were absent or delayed from entering, such as the perinotochordal and subepidermal spaces. (3) A subset of chondroitin/keratan sulfate proteoglycans was restricted to the perinotochordal region and, following gangliogenesis, was arranged in a metameric pattern corresponding to the sites where presumptive vertebral arches form. (4) Certain keratan sulfate proteoglycans and a heparan sulfate proteoglycan were observed in basement membranes and in an interstitial matrix uniformly distributed along the rostrocaudal extent of the sclerotome. After gangliogenesis, the neural crest-derived dorsal root and sympathetic ganglia contained both these proteoglycan types, but were essentially free of other chondroitin/keratan-proteoglycan subsets. Hyaluronan generally colocalized with the first set of proteoglycans, but also was concentrated around migrating neural crest cells and was reduced in neural crest-derived ganglia. These observations demonstrate that proteoglycans have diverse and dynamic distributions during times of neural crest development and chondrogenesis of the presumptive vertebrae. In general, chondroitin/keratan sulfate proteoglycans are abundant in regions where neural crest cells are absent, and their segmental distribution inversely correlates with that of neural crest-derived ganglia.

Collagen-binding proteins in collagenase-released matrix vesicles from cartilage. Interaction between matrix vesicle proteins and different types of collagen.

Recent evidence indicates that matrix vesicles (MV) interact with cartilage-specific collagens and other matrix proteins. Both type II and X collagens bind to and cosediment with MV. Our companion study shows that MV also are tightly coupled to proteoglycan link proteins (LP) and hyaluronic acid-binding region (HABR) in cartilage matrix. Here we sought to identify proteins responsible for the nexus between MV and matrix collagens using affinity chromatography with types I, II, and X collagen-Sepharose columns. Elution with NaCl step-gradients in the presence of nonionic detergent was used to assess the affinity between the MV proteins and the covalently attached collagens. Several MV proteins were found to bind to native type I, II, and X collagens but none bound to denatured type I collagen. Alkaline phosphatase, proteoglycan LP and HABR, and the 33- and 67-kDa annexins, bound with varying affinities to the native type I, II and X columns. In particular, LP and HABR, the 67-kDa annexin, and alkaline phosphatase bound with high affinity to the cartilage-specific collagens, although LP, HABR, and a 37-kDa protein also bound less tightly to native type I collagen. Thus, several MV proteins bind specifically to native type II and X collagens and should promote interaction between MV and the extracellular matrix. Such interactions may be important in MV formation, or in MV-mediated mineralization.

Association between proteoglycans and matrix vesicles in the extracellular matrix of growth plate cartilage.

Matrix vesicles (MV) are microstructures localized to the extracellular matrix of developing hard tissues that induce mineral formation. MV proteins are not well characterized, and little is known of how they interact with the surrounding matrix. However, recent electron microscopic studies indicate that MV interact with matrix proteins in growth plate cartilage. In the studies now reported, procedures developed for dissecting various components from isolated MV led to the discovery that two major vesicle proteins (38 and 46 kDa) are readily released from MV by low ionic strength solutions. These low ionic strength-soluble proteins (LISSP) were shown to be major fragments of the link protein (LP) and hyaluronic acid-binding region (HABR) of matrix proteoglycans: they react immunologically with highly specific monoclonal antibodies to LP and HABR, and the NH2-terminal sequence of the 38-kDa LISSP is essentially identical to residues 40-78 of chicken cartilage LP and that the 46-kDa LISSP represents HABR. Release of both LISSP is enhanced by hyaluronidase treatment, indicating anchorage by a hyaluronate-mediated mechanism. Both LP and HABR are firmly attached to MV in either isotonic or hypertonic solutions. In contrast, our other studies show that dissociation of type II collagen from MV occurs only with hypertonic salts which do not release the LISSP. Thus, strong interactions occur under physiological conditions between MV and both the proteoglycans and collagens, but these take place by different mechanisms.

The oxidant hypochlorite (OCl −), a product of the myeloperoxidase system, degrades articular cartilage proteoglycan aggregate

The myeloperoxidase-derived oxidant, hyprochlorite (OCL −) was shown to be able to degrade proteogylcan aggregate prepared from bovine articular cartilage. Exposure of proteoglycan aggregate to OCl − concentrations less than 10 −4 resulted in a decrease in the size of the constituent proteoglycan monomers, which were unable to reaggregate with hyaluronate due to the loss of the hyaluronic acid binding region as indicated by immunoblotting using the monoclonal 1-C-6 antibody. Analysis of the [ 35S]-labeled core proteins by SDS / polyacrylamide electrophoresis and flurography indicated a decrease in the size of the core protein. These data suggest that concentrations of OCl − below 10 −3 M results in the cleavage of the proteoglycan core protein in or near the hyaluronic acid binding region. The physiological consequences of these data are discussed. Exposure to higher concentrations (>10 −3) of OCl − caused more extensive degradation of the core protein; however, there was no evidence to suggest that OCl − cleaves glycosaminoglycan (GAG) chains.

Cartilage proteoglycan‐induced arthritis in BALB/c mice. Antibodies that recognize human and mouse cartilage proteoglycan and can cause depletion of cartilage proteoglycan with little or no synovitis

Human fetal cartilage proteoglycan (PG) induces the development of an erosive polyarthritis and spondylitis in BALB/c mice. We have examined the properties of 3 monoclonal antibodies (MAb) to human fetal cartilage PG isolated from immunized mice that cross-react with mouse cartilage PG. Compared with sera from arthritic mice, which contain antibodies reactive with keratan sulfate, MAb 202 (IgG1) reacted only with a protein-related epitope that is distributed on both hyaluronic acid-binding and chondroitin sulfate-attachment regions. MAb 813 (IgG1) reacted with the same fragments and recognized an epitope with the immunologic characteristics of keratan sulfate. MAb 945 (IgM) remains to be further characterized. Introduction of hybridomas secreting MAb 202 and MAb 945 into irradiated mice resulted in the loss of PG from articular cartilage and from growth plate cartilage (with MAb 202 only), as revealed by a loss of staining with toluidine blue. There was no synovial hyperplasia with MAb 202, but some hyperplasia and mononuclear cell infiltration was seen with MAb 945. This was accompanied by the binding of immunoglobulins to articular cartilage, as demonstrated by immunofluorescence. The hybridoma secreting MAb 813 produced no cartilage changes or synovitis, and there was no immunoglobulin binding to cartilage. Polymorphonuclear leukocyte infiltration was never observed with these antibodies. These studies indicate that MAb reactive with mouse cartilage PG can cause the depletion of PG from hyaline cartilage by mechanisms that may be both complement dependent and complement independent. Antibodies may serve to release and expose PG antigen to immune cells, as well as causing a loss of the mechanical properties of cartilage that are PG dependent.

Chick cartilage chondroitin sulfate proteoglycan core protein. I. Generation and characterization of peptides and specificity for glycosaminoglycan attachment.

Peptides were derived from the large chondroitin sulfate proteoglycan from chick cartilage by clostripain digestion. Using differential chondroitinase ABC and keratanase treatment and direct carbohydrate analysis, three major peptides of 86, 75, and 27 kDa were shown to bear only chondroitin sulfate chains. Another major peptide of 65 kDa was shown to contain both chondroitin sulfate and keratan sulfate chains, allowing it to be separated from the peptides derived from the chondroitin sulfate domain by DEAE-cellulose chromatography. An additional new peptide (100 kDa) containing keratan sulfate chains was found only in clostripain digests of proteoglycan-hyaluronate-link protein aggregates. Unlike any of the other peptides derived from clostripain digestion of proteoglycan monomer or aggregate, this peptide had the properties of a functional hyaluronate binding region. All of these peptides were purified to apparent homogeneity by preparative electroelution from sodium dodecyl sulfate-polyacrylamide gel electrophoresis and deglycosylated with anhydrous hydrogen fluoride. Automated Edman degradation of the two largest chondroitin sulfate peptides revealed that they had unique N termini and several unrecognized residues, which were all subsequently revealed to be modified serine residues following deglycosylation. The keratan sulfate-bearing peptide also had a unique N terminus, which contained a single unrecognized residue, even after HF deglycosylation. Finally, the N terminus of the hyaluronate binding region was blocked. These studies allow estimates of core peptide masses in the absence of carbohydrate as well as provide primary amino acid sequence for O-xylosylated serine residues in the multiply substituted proteoglycans.

Visualization of hyaluronic acid in the anterior segment of rabbit and monkey eyes

Hyaluronic acid was visualized using a new histochemical technique: the hyaluronic acid-binding region of the cartilage proteoglycan was isolated, linked with biotin and used in histological sections as a ligand for hyaluronic acid. Staining was performed using the avidin-biotin-peroxidase technique. The presence of hyaluronic acid in the anterior segments of rabbit and monkey eyes was studied in fresh-frozen, as well as in fixed paraffin sections; addition of cetyl-pyridinium chloride to the fixative was essential. In both species intense staining was seen in the stroma of the conjunctiva, the connective tissue of the ciliary processes, and on the apical membranes of the corneal endothelium. Species differences were observed in both conventional and unconventional outflow pathways. In rabbits, the tissue located directly adjacent to the aqueous plexus and the connective tissue surrounding the poorly developed ciliary muscle were stained. In monkeys, only the anterior, non-filtering part of the trabecular meshwork and the outer wall of Schlemm's canal showed intense staining in paraffin sections. In frozen sections, the inner wall (cribriform region of the trabecular meshwork) was also stained. The spaces between the anterior part of the ciliary muscle bundles remained almost unstained.

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 allow stronger interaction with hyaluronate. In a second step, which is pH- and temperature-dependent, the change becomes inrreversible. 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.

Monoclonal Antibodies Reacting with Tryptic Hyaluronic Acid-Binding Region and Link Protein Fragments of Bovine Nasal Cartilage Proteoglycan

BALB/c mice were immunized with isolated trypsin-produced hyaluronic acid-binding region (HABR) or HABRIlink protein complex from bovine nasal cartilage proteoglycan (PG) aggregates conjugated to keyhole limpet hemocyanin.The monoclonal antibodies (Mabs) raised were characterized by solid-phase ELISA inhibition and SDS-polyacrylamide gel electrophoresis immunoblotting.Nine Mabs react with intact PG monomer, HABR and HABRIlink complex.Two of the anti-HABR Mabs appear to be directed to epitopes in or near the HA-binding site of PG monomer; one of these epitopes is insensitive to reduction and alkylation and pronase treatment and is likely to consist of carbohydrate.The remaining anti-HABR Mabs react with PG aggregate, are non-reactive with pronase-treated PG monomer and vary in their reactivity with reduced and alkylated PG monomer.Three Mabs react with link protein-related epitopes.One of these Mabs reacts only with the tryptic link protein fragment, the others also react with PG aggregates and the two native link proteins.Immunological studies of cartilage PG should be facilitated by the availability of Mabs specific for these functionally significant components in native PG aggregates.

Purification and characterization of a hyaluronan-binding protein from rat chondrosarcoma

Swarm rat chondrosarcoma contains a hyaluronan-binding protein of molecular mass 102 kDa (HABP102). The protein is present in 4 M-guanidinium chloride extracts of the chondrosarcoma and can be incorporated into reconstituted proteoglycan aggregates, but it is not present in native proteoglycan aggregates or in 0.5 M-guanidinium chloride extracts. HABP102 is unlikely to be an integral membrane protein, as it does not require detergent for extraction, is not enriched in hydrophobic amino acids and does not bind avidly to octyl-Sepharose. The protein stains poorly with Coomassie Blue and is only visible on PAGE gels after staining with silver. Disulphide bonds are essential for the binding of HABP102 to hyaluronan, and bivalent cations are not required for this interaction. HABP102 can be purified from dissociative chondrosarcoma extracts by sequential density-gradient centrifugation, hyaluronan-Sepharose affinity chromatography and hydrophobic-interaction chromatography. The amino acid composition is similar to that of domains 1-4 of the chondrosarcoma proteoglycan core protein, but peptide analysis after digestion with Staphylococcus aureus V8 proteinase and chymotrypsin and different immunoreactivity suggest that HABP102 is not closely related to proteoglycan hyaluronan-binding region. HABP102 is a glycoprotein containing N-acetylgalactosamine, N-acetylglucosamine, mannose and galactose.

Inhibition of neural crest cell migration by aggregating chondroitin sulfate proteoglycans is mediated by their hyaluronan-binding region

We have recently shown that the large hyaluronan-aggregating chondroitin sulfate proteoglycan from cartilage (PG-LA) is unfavorable as a substrate for neural crest cell migration in vitro and that this macromolecule inhibits cell dispersion on fibronectin substrates when included in the medium ( R. Perris and S. Johansson, 1987, J. Cell Biol. 105, 2511–2521). In this study we present data on the specificity of the migration-repressing activity of PG-LA and data on the molecular mechanisms by which the proteoglycan might impair neural crest cell motility. Soluble PG-LA potently impaired cell migration on substrates of laminin/laminin-nidogen, vitronectin, and collagen types I, III, IV, and VI. When tested in solid-phase binding assays, PG-LA bound avidly to substrates of collagen types I–III and V. Conversely, minimal amounts of the proteoglycan bound to substrates of laminin-nidogen, vitronectin, collagen types IV and VI, and fibronectin or to a proteolytic fragment encompassing its cell-binding domain (105 kDa). Preincubation of these substrates with soluble PG-LA prior to plating of the cells had no effect on their locomotory behavior. These results indicate that PG-LA affects neural crest cell movement primarily through an interaction with the cell surface, rather than by association with the cell motility-promoting substrate molecules. The molecular interaction of soluble PG-LA with neural crest cells was further examined by analyzing the effects of isolated domains of the proteoglycan on cell migration on fibronectin. Addition of chondroitin sulfate chains, the core protein free of glycosaminoglycans, the isolated hyaluronan-binding region (HABr), or a proteolytic fragment corresponding to the keratan sulfate-enriched domain of the PG-LA to neural crest cells migrating on fibronectin or the 105-kDa fibronectin fragment had no significant effect on their motility. After reduction and alkylation, PG-LA was considerably less efficient in perturbing cell movement on fibronectin substrates and virtually ineffective in altering migration on the 105-kDa fragment. In the presence of hyaluronan fragments of 16–30 monosaccharides in length, or an antiserum against the HABr, the migration repressing activity of PG-LA was reduced in a dose-dependent fashion. Furthermore, the inhibitory action of PG-LA was significantly reduced by treatment of the cells with Streptomyces hyaluronidase. These findings indicate that hyaluronan-aggregating proteoglycans inhibit neural crest cell movement by interacting with the cell surface, at which the molecules binds via its HABr to cell membrane-anchored hyaluronan. We propose that by this mechanism, PG-LA-related proteoglycans may regulate neural crest cell migration by altering the function of cell surface receptors for motility-promoting molecules.

Histochemical localization of hyaluronate in human oral epithelium using a specific hyaluronate-binding probe

Biochemical data suggest that gingival epithelium contains hyaluronate, but there is little histochemical information about its localization. Hyaluronate was here visualized in gingival and buccal mucosa using a specific probe derived from the hyaluronate binding region of cartilage proteoglycan. Hyaluronate was found both in the gingival and buccal epithelium, but its localization was correlated with the type of keratinization. In the keratinized epithelium of gingiva, whether ortho- or parakeratotic, the intercellular spaces from basal to upper spinous layers displayed strong staining, most intense in the middle spinous cell layer. The uppermost vital cell layers as well as the cornified cell layer remained unstained. In the non-keratinized epithelium of buccal mucosa and the local non-keratinized areas of gingiva, only the basal cells and the lowermost spinous cell layers stained for hyaluronate, whereas the majority of the upper epithelium was negative. Electron microscopic examination of the basal and spinous cell layers displayed hyaluronate, both associated with the cell surface and free in the intercellular space. The subepithelial connective tissue showed positive but diffuse staining in all specimens.

Passive loss of proteoglycan from articular cartilage explants

The addition of proteinase inhibitors (1 mM phenylmethylsulfonyl fluoride, 10 mM N-ethylmaleimide, 0.25 mM benzamidine hydrochloride, 6.25 mM EDTA, 12.5 mM 6-aminohexanoic acid and 2 mM iodoacetic acid) to explant cultures of adult bovine articular cartilage inhibits proteoglycan synthesis as well as the loss of the macromolecule from the tissue. Those proteoglycans lost to the medium of explant cultures treated with proteinase inhibitors were either aggregates or monomers with functional hyaluronic acid-binding regions, whereas proteoglycans lost from metabolically active tissue also included a population of monomers that were unable to aggregate with hyaluronate. Analysis of the core protein from proteoglycans lost into the medium of inhibitor-treated cultures showed the same size distribution as the core proteins of proteoglycans present in the extracellular matrix of metabolically active cultures. The core proteins of proteoglycans appearing in the medium of metabolically active cultures showed that proteolytic cleavage of these macromolecules occurred as a result of their loss from the tissue. Explant cultures of articular cartilage maintained in medium with proteinase inhibitors were used to investigate the passive loss of proteoglycan from the tissue. The rate of passive loss of proteoglycan from the tissue was dependent on surface area, but no difference in the proportion of proteoglycan aggregate to monomer appearing in the medium was observed. Furthermore, proteoglycans were lost at the same rate from the articular and cut surfaces of cartilage. Proteoglycan aggregates and monomer were lost from articular cartilage over a period of time, which indicates that proteoglycans are free to move through the extracellular matrix of cartilage. The movement of proteoglycans out of the tissue was shown to be temperature dependent, but was different from the change of the viscosity of water with temperature, which indicates that the loss of proteoglycan was not solely due to diffusion. The activation energy for the loss of proteoglycans from articular cartilage was found to be similar to the binding energies for electrostatic and hydrogen bonds.

Xylosyl transfer to the core protein precursor of the rat chondrosarcoma proteoglycan

Rat chondrosarcoma chondrocytes were labeled with [3H]serine or [3H]mannose as a precursor. Intracellular proteoglycan core protein precursor was purified from cell lysates by immunoprecipitation with polyclonal antibodies against the hyaluronic acid-binding region, followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The core precursor was eluted from the gels and treated with alkaline borohydride in order to convert serine residues substituted with xylose or N-acetylgalactosamine to alanine (or with alkaline sulfite to convert them to cysteic acid). After acid hydrolysis, the proportions of labeled serine and alanine (or cysteic acid) were determined by high performance liquid chromatography, and the results were compared with those obtained for the completed proteoglycan molecules isolated from the same cultures. In the completed proteoglycans, about 55% of the serine residues were substituted with xylose or N-acetylgalactosamine, while the corresponding figure for the intracellular precursor molecules was less than 5%. These results indicate, in agreement with our previous kinetic data, that the major part of the xylosyl transfer to the chondrosarcoma proteoglycan core protein precursor must occur late in the processing sequence, i.e. after about 85% of its intracellular lifetime and no more than 7 min before the addition of the rest of the chondroitin sulfate chain. The ratio of [3H]mannose to [3H]fucose in the core precursor was about 19, while that for the complete proteoglycan was about 2. This indicates the presence of high mannose, N-linked oligosaccharides on the core protein precursor which are converted to the complex forms on the completed proteoglycan. These data provide further support that the core precursor resides mainly in the pre-Golgi compartment and that xylosylation occurs mainly in a Golgi compartment.

Membrane‐associated hyaluronate‐binding activity of chondrosarcoma chondrocytes

The association of hyaluronate with the surface of chondrocytes was examined by several approaches using primary cultures of chondrocytes derived from the Swarm rat chondrosarcoma. In culture, chondrosarcoma chondrocytes produced large pericellular coats, which can be visualized by particle exclusion, and which can be removed by Streptomyces hyaluronidase. Exposure of chondrocytes, which had been metabolically labelled with 3H-acetate, to exogenous hyaluronate or to Streptomyces hyaluronidase resulted in the release of 36-38% of the endogenous, labelled chondroitin sulfate from the cell layer into the incubation solution. These results imply that at least 37% of the cell layer chondroitin sulfate proteoglycan is retained there by an interaction with hyaluronate. Thus membranes were prepared from cultured chondrocytes and examined for sites which bind 3H-hyaluronate. Binding was observed and found to be saturable, specific for hyaluronate, of high affinity (Kd = approximately 10(-10) M), and destroyed by treating the membranes with trypsin. The 3H-hyaluronate-binding activity was inhibited competitively by hyaluronate decasaccharides but not by hexasaccharides or octasaccharides, indicating that the binding sites recognize a sequence of hyaluronate composed of five disaccharide repeats. The binding activity was partially purified from a detergent extract of chondrocyte membranes by ion exchange chromatography on DEAE-cellulose, followed by affinity chromatography on wheat germ agglutinin-agarose. Analysis of the partially purified binding activity by SDS-PAGE revealed five protein bands of 48,000-66,000 daltons in silver-stained gels. SDS-PAGE followed by Western blotting and exposure to monoclonal antibodies which recognize epitopes present in link protein and in the hyaluronate-binding region of cartilage proteoglycan revealed no immunoreactive protein bands in the partially purified material. We conclude that one mechanism by which hyaluronate associates with the chondrocyte surface may be via interaction with a membrane-bound hyaluronate-binding protein which is distinct from link protein and proteoglycan.

Proteoglycan profiles obtained by electrophoresis and triple immunoblotting

Using synovial fluid of individuals with osteoarthritis as a prototypic biologic fluid containing one part proteoglycan per 100 to 1000 parts of other protein, profiles of proteoglycan were produced without preliminary purification through agarose-acrylamide gel electrophoresis, transfer to nitrocellulose, and triple immunoblotting. Chondroitin sulfate, keratan sulfate, and a hyaluronic acid binding region appear to be present on individual synovial fluid proteoglycans in variable amounts, and consequently a triple immunoblot using monoclonal antibodies to these three epitopes has the potential for developing a proteoglycan profile. The profile is assembled by means of densitometric scans of autoradiograms obtained after use of 125I-labeled anti-mouse immunoglobulin. By contrast to the profile of a relatively homogeneous proteoglycan purified from articular cartilage extracts, the proteoglycans of synovial fluid appeared to be quite heterogeneous with the bulk of keratan sulfate epitopes migrating ahead of the bulk of the chondroitin sulfate epitopes. Most of the proteoglycans appeared to possess a hyaluronate binding region.

Immunoelectron microscopic localization of hyaluronic acid-binding region and link protein epitopes in brain.

The 1C6 monoclonal antibody to the hyaluronic acid-binding region weakly stained a 65-kD component in immunoblots of the chondroitin sulfate proteoglycans of brain, and the 8A4 monoclonal antibody, which recognizes two epitopes in the polypeptide portion of link protein, produced strong staining of a 45-kD component present in the brain proteoglycans. These antibodies were utilized to examine the localization of hyaluronic acid-binding region and link protein epitopes in rat cerebellum. Like the chondroitin sulfate proteoglycans themselves and hyaluronic acid, hyaluronic acid-binding region and link protein immunoreactivity changed from a predominantly extracellular to an intracellular (cytoplasmic and intra-axonal) location during the first postnatal month of brain development. The cell types which showed staining of hyaluronic acid-binding region and link protein, such as granule cells and their axons (the parallel fibers), astrocytes, and certain myelinated fibers, were generally the same as those previously found to contain chondroitin sulfate proteoglycans and hyaluronic acid. Prominent staining of some cell nuclei was also observed. In agreement with earlier conclusions concerning the localization of hyaluronic acid and chondroitin sulfate proteoglycans, there was no intracellular staining of Purkinje cells or nerve endings or staining of certain other structures, such as oligodendroglia and synaptic vesicles. The similar localizations and coordinate developmental changes of chondroitin sulfate proteoglycans, hyaluronic acid, hyaluronic acid-binding region, and link protein add further support to previous evidence for the unusual cytoplasmic localization of these proteoglycans in mature brain. Our results also suggest that much of the chondroitin sulfate proteoglycan of brain may exist in the form of aggregates with hyaluronic acid.

An enzyme-linked immunosorbent-inhibition assay for quantitation of hyaluronan (hyaluronic acid) in biological fluids

An enzyme-linked immunosorbent-inhibition assay for quantitation of hyaluronic acid (HA) is described. The principle of the method depends on the specific binding of HA to the hyaluronic acid-binding region (HABR) of proteoglycan (PG) monomers. The remaining uncomplexed PG monomers were determined by incubation with specific monoclonal antibodies to HABR followed by addition of polyclonal antibodies against PG monomers and enzyme-conjugated antibodies. The HA in samples was quantified by comparing their inhibitory capacity in the assay against a standard inhibition curve obtained using highly purified HA. This method was used to quantitate HA at nanogram levels in normal sera and synovial fluids. The level in normal human sera was found to be 28 ± 17 ng/ml which compared favorably with values obtained using a commercial radioassay kit on the same samples. The assay was used to measure HA in synovial fluid from patients with rheumatoid and osteoarthritis and the results obtained were comparable with data published by others.