Monoclonal antibodies to different protein-related epitopes of human articular cartilage proteoglycans.

Monoclonal antibodies produced against chondroitinase-treated human adult cartilage proteoglycans were selected for their ability to recognize epitopes on native proteoglycans. Binding analyses revealed that four of these monoclonal antibodies (BCD-4, BCD-7, EFG-4 and KPC-190) each recognized a different epitope on the same proteoglycan molecule which represents a subpopulation of a high buoyant density (D1) fraction of human articular cartilage proteoglycans (10, 30, 50 and 60% in fetal-newborn, 1.5 years old, 15 years old and 52-56 years old cartilages, respectively). Analysis of epitope specificities revealed that BCD-7 and EFG-4 monoclonal antibodies recognized epitopes on proteoglycan monomer which are associated with the protein structure in that they are sensitive to cleavage by Pronase, papain and alkali treatment and do not include keratan sulphate, chondroitin sulphate or oligosaccharides. The BCD-4 and KPC-190 epitopes also proved to be sensitive to Pronase or papain digestion or to alkali treatment, but keratanase or endo-beta-galactosidase also reduced the immunoreactivity of these epitopes. These observations indicate that the BCD-4 and KPC-190 epitopes represent peptides substituted with keratan sulphate or keratan sulphate-like structures. The BCD-4 epitope is, however, absent from a keratan sulphate-rich fragment of human adult proteoglycan, while the other three epitopes were detected in this fragment. None of these four epitopes were detected in the link proteins of human cartilage, in the hyaluronic acid-binding region of human newborn cartilage proteoglycan, in Swarm rat chondrosarcoma proteoglycan, in chicken limb bud proteoglycan monomer and in the small dermatan sulphate-proteoglycan of bovine costal cartilage. EFG-4 and KPC-190 epitopes were not detected in human fetal cartilage proteoglycans, although fetal molecules contained trace amounts of epitopes reactive with BCD-4 and BCD-7 antibodies.     

Comparison of proteoglycans from bovine articular cartilage.

Four bovine articular cartilages have been compared with regard to the chemical composition of the whole cartilages, the amount of proteoglycan selectively extracted with 3 M MGCl2 or with 3 M guanidine-HCl, and the compositions and physical properties of the isolated proteoglycans. The whole cartilages differ but slightly in composition. Occipital condylar cartilage, a thin cartilage from the smallest joint, contains 4% more collagen and proportionately less proteoglycan than proximal humeral, the thickest cartilage from the largest joint. Each cartilage contains a pool of proteoglycan that resists extraction with 3 M MgCl2 but is extracted with 3 M guanidine-HCl. The proteoglycan extracted from each cartilage with 3 M guanidine-HCl contains a high molecular weight proteoglycan-collagen complex demonstrated by analytical ultracentrifugation and by the turbidity of its visible and ultra-violet spectra. The four cartilages appear to differ most remarkably in the fraction of total proteoglycan extracted from each as proteoglycan-collagen complex.     

The occurrence of low buoyant density proteoglycans in embryonic chick cartilage

Two proteoglycan fractions, PCS-H and PCS-L, have previously been isolated from 4 M guanidine HCl extract of embryonic chick cartilages. This communication reports further studies with PCS-L indicating that this fraction contains several different forms, of which one differs from hitherto known cartilage proteoglycans in 1) markedly lower buoyant density, 2) susceptibility to reduction with 2-mercaptoethanol, 3) aggregate-forming ability in 4 M guanidine HCl, and 4) presence of dermatan sulfate-chondroitin sulfate copolymer chains. Also isolated from the PCS-L fraction is a keratan sulfate-rich proteoglycan which represents the smallest molecular size species in cartilage proteoglycan populations.     

Ultrastructural localization and internalization of proteoglycan epitopes in a human non-Hodgkin (B) lymphoma

Summary In a human non-Hodgkin (B) lymphoma xenograft (HT-117) heparan sulphate (HS) proved to be the main cell surface glycosaminoglycan, in contrast to the chondroitin sulphate dominance in normal lymphoid cells. Using anti-proteoglycan (PG) antibodies and immunoelectronmicroscopy, two heparan sulphate proteoglycans (transferrin receptor (TfR) and fibroblast membrane type) and one chondroitin sulphate proteoglycan (articular cartilage type) molecule were co-localized as random clusters on the surface of these lymphoma cells. Double labelling revealed that during internalization, which occurred via endosomes avoiding the lysosomal system, the different proteoglycan (PG) antigens became separated. The TfR and fibroblast membrane type HSPG epitopes reappeared on plasmalemmal vesicles derived most probably from the multivesicular endosomes, representing a unique form of exocytosis. It is suggested that different cell membrane PGs are integrated into subunits of yet unknown function in these human non-Hodgkin (B) lymphoma cells.     

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.     

Isolation of dermatan sulfate proteoglycans from mature bovine articular cartilages

Two species of dermatan sulfate proteoglycans, called DS-PGI and DS-PGII, have been isolated from mature bovine articular cartilages. On sodium dodecyl sulfate-polyacrylamide gel electrophoresis at low ionic strength in 0.01 M phosphate the dermatan sulfate proteoglycans appeared as a single polydisperse species whose molecular weight ranged from 80,000 to 140,000. The dermatan sulfate proteoglycans eluted as a single peak on Sepharose CL-4B chromatography in 4 M guanidine hydrochloride and showed no tendency to separate into two components. Following chondroitinase AC and ABC digestion, a core protein was obtained whose molecular weight was 45,000. However, what appeared to be a single dermatan sulfate proteoglycan was consistently separated into two species of distinctly different mobilities by sodium dodecyl sulfate-polyacrylamide gel electrophoresis at high ionic strength in 0.375 M Tris. The molecular weight of the smaller species (DS-PGII) ranged from 87,000 to 120,000. The molecular weight of the larger species (DS-PGI) ranged from 165,000 to 285,000. DS-PGI self-associates in 0.375 M Tris, while DS-PGII does not. This phenomenon was exploited to separate DS-PGI and DS-PGII by preparative electrophoresis on 5 to 20% gradient slab gels. The immunological identities of the individual species, DS-PGI and DS-PGII, were examined by enzyme-linked immunosorbent assay using polyclonal antiserum to cartilage-specific proteoglycan monomer from bovine articular cartilage and polyclonal and monoclonal antibodies to DS-PGII. The polyclonal antiserum to cartilage-specific proteoglycan monomer did not react with DS-PGI or DS-PGII, indicating that DS-PGI and DS-PGII possess different core proteins from cartilage-specific proteoglycan monomer. Polyclonal and monoclonal antibodies raised against the mixture of DS-PGI and DS-PGII reacted strongly with DS-PGII, but weakly or not at all with DS-PGI. These results suggest that DS-PGI and DS-PGII possess different core proteins and may represent two different species of dermatan sulfate proteoglycans.     

Mammalian eyes and associated tissues contain molecules that are immunologically related to cartilage proteoglycan and link protein

Monospecific antibodies to bovine nasal cartilage proteoglycan monomer and link protein were used to demonstrate that immunologically related molecules are present in the bovine eye and associated tissues. With immunofluorescence microscopy, reactions for both proteoglycan and link protein were observed in the sclera, the anterior uveal tract, and the endoneurium of the optic nerve of the central nervous system. Antibody to bovine nasal cartilage proteoglycan also reacted with some connective tissue sheaths of rectus muscle and the perineurium of the optic nerve of the central nervous system. Antibody to proteoglycan purified from rat brain cross-reacted with bovine nasal cartilage proteoglycan, indicating structural similarities between these proteoglycans. ELISA studies and crossed immunoelectrophoresis demonstrated that purified dermatan sulphate proteoglycans isolated from bovine sclera did not react with these antibodies but that the antibody to cartilage proteoglycan reacted with other molecules extracted from sclera. Two molecular species resembling bovine nasal link protein in size and reactivity with antibody were also demonstrated in scleral extracts: the larger molecule was more common. Antibody to link protein reacted with the media of arterial vessels demonstrating the localization of arterial link protein described earlier. Tissues that were unstained for either molecule included the connective tissue stroma of the iris, retina, vitreous body, cornea, and the remainder of the uveal tract. These observations clearly demonstrate that tissues other than cartilage contain molecules that are immunologically related to cartilage-derived proteoglycans and link proteins.     

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.     

Characterization of proteoglycans from the calcified matrix of bovine bone.

The proteoglycans characterized were those isolated from the calcified matrix of mature bovine bone [Franzén & Heinegård (1984) Biochem. J. 224, 47-58]. The average molecular mass of the bone proteoglycan is 74 600 Da, determined by sedimentation-equilibrium centrifugation in 4M-guanidinium chloride. Its sedimentation coefficient (s0(20),w) is 3.04 S. The apparent Mr of its core protein is 46 000, estimated by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis of the chondroitinase ABC-digested proteoglycan. A more likely molecular mass of the core protein is 30 000 Da, as calculated from the molecular mass and the protein content (40%) of the proteoglycan. The bone proteoglycan contains one or probably two chondroitin sulphate chains each with a molecular mass (weight-average) of 33 700 Da and several oligosaccharides both of the N-glycosidically and the O-glycosidically linked type. Antibodies against the homogeneous bone proteoglycans were raised in rabbits. An e.l.i.s.a. (enzyme-linked immunosorbent assay) method was developed that allowed specific quantification of bone proteoglycans at nanogram levels. The specificity of the antibodies was tested by using the e.l.i.s.a. method. The bone proteoglycan showed partial cross-reactivity with the small proteoglycan of cartilage. The antibodies were used to localize immunoreactivity of bone proteoglycans by indirect immunofluorescence in frozen sections of foetal bovine epiphysial growth plate. The fluorescence was entirely found in the primary spongiosa, and no fluorescence was found among the hypertrophied chondrocytes or in the region of provisional calcification.     

The dermatan sulfate proteoglycans of bovine sclera and their relationship to those of articular cartilage. An immunological and biochemical study

Dermatan sulfate proteoglycans were isolated from adult bovine sclera and adult bovine articular cartilage. Their immunological relationships were studied by enzyme-linked immunosorbent assays using polyclonal antibodies raised against the large and small dermatan sulfate proteoglycans from sclera and a polyclonal and monoclonal antibody directed against the small dermatan sulfate proteoglycans from cartilage. The small dermatan sulfate proteoglycans from sclera and cartilage displayed immunological cross-reactivity while there was no convincing evidence of shared epitope(s) with the larger dermatan sulfate proteoglycans, nor did these larger proteoglycans share any common epitopes with each other. A hyaluronic acid binding region was detected immunologically on the larger scleral dermatan sulfate proteoglycan but was absent from the larger dermatan sulfate proteoglycan of cartilage and both the small dermatan sulfate proteoglycans. These antibodies were used in immunofluorescence microscopy to localize the scleral proteoglycans and molecules containing these epitopes in the eye. The large scleral dermatan sulfate proteoglycan was restricted to sclera while molecules related to the small scleral and cartilage proteoglycans were found in the sclera, anterior uveal tract, iris, and cornea. Amino acid sequencing of the amino-terminal regions of the core proteins of the small dermatan sulfate proteoglycans from sclera and articular cartilage showed that all the first 14 amino acids analyzed were identical and the same as reported earlier for the small bovine skin and tendon dermatan sulfate proteoglycans. These studies demonstrate that the larger dermatan sulfate proteoglycans of sclera and cartilage are chemically unrelated to each other and to the smaller dermatan sulfate proteoglycans isolated from these tissues. The latter have closely related core proteins and probably represent a molecule with a widespread distribution in which the degree of epimerization of glucuronic acid and iduronic acid varies between tissues.     

A Monoclonal Antibody which Recognizes a Glycosaminoglycan Epitope in Both Dermatan Sulfate and Chondroitin Sulfate Proteoglycans of Human Skin

Studies have been initiated to identify various cell surface and matrix components of normal human skin through the production and characterization of murine monoclonal antibodies. One such antibody, termed PG-4, identifies both cell surface and matrix antigens in extracts of human foetal and adult skin as the dermatan sulfate proteoglycans, decorin and biglycan, and the chondroitin sulfate proteoglycan versican. Treatment of proteoglycans with chondroitinases completely abolishes immunoreactivity for all of these antigens which suggests that the epitope resides within their glycosaminoglycan chains. Further evidence for the carbohydrate nature of the epitope derives from competition studies where protein-free chondroitin sulfate chains from shark cartilage react strongly; however, chondroitin sulfate chains from bovine tracheal cartilage fail to exhibit a significant reactivity, an indication that the epitope, although present in some chondroitin sulfate chains, does not consist of random chondroitin 4- or 6-sulfate disaccharides. The presence of the epitope on dermatan sulfate chains and on decorin was also demonstrated using competition assays. Thus, PG-4 belongs to a class of antibodies that recognize native epitopes located within glycosaminoglycan chains. It differs from previously described antibodies in this class in that it identifies both chondroitin sulfate and dermatan sulfate proteoglycans. These characteristics make PG-4 a useful monoclonal antibody probe to identify the total population of proteoglycans in human skin.     

Ultrastructural localization and internalization of proteoglycan epitopes in a human non-Hodgkin (B) lymphoma.

In a human non-Hodgkin (B) lymphoma xenograft (HT-117) heparan sulphate (HS) proved to be the main cell surface glycosaminoglycan, in contrast to the chondroitin sulphate dominance in normal lymphoid cells. Using anti-proteoglycan (PG) antibodies and immunoelectronmicroscopy, two heparan sulphate proteoglycans (transferrin receptor (TfR) and fibroblast membrane type) and one chondroitin sulphate proteoglycan (articular cartilage type) molecule were co-localized as random clusters on the surface of these lymphoma cells. Double labelling revealed that during internalization, which occurred via endosomes avoiding the lysosomal system, the different proteoglycan (PG) antigens became separated. The TfR and fibroblast membrane type HSPG epitopes reappeared on plasmalemmal vesicles derived most probably from the multivesicular endosomes, representing a unique form of exocytosis. It is suggested that different cell membrane PGs are integrated into subunits of yet unknown function in these human non-Hodgkin (B) lymphoma cells.     

Isolation and partial characterization of proteoglycans from rat incisors.

Newly synthesized proteoglycans of rat incisors were labelled in vivo for 6h with [35S]-sulphate in order to facilitate their detection during purification and characterization. Proteoglycans were extracted from non-mineralized portions (predentine) of rat incisors with 4M-guanidinium chloride and subsequently from dentine by demineralization with a 0.4M-EDTA solution containing 4M-guanidinium chloride. Both extractions were performed at 4 degrees C in the presence of proteinase inhibitors. Purification of proteoglycans was achieved with a procedure involving gel-filtration chromatography, selective precipitation of phosphoproteins, affinity chromatography and ion-exchange chromatography. Two proteoglycan populations were found in the initial extract (Pd-PG I and Pd-PG II), whereas only one fraction (D-PG) was obtained after demineralization. The minor proteoglycan fraction from the first extract, Pd-PG I, although not totally characterized, differed sharply from the other proteoglycans in that it had a larger molecular size with larger glycosaminoglycan chains composed of chondroitin 4- and 6-sulphate isomers. In contrast, the major proteoglycans Pd-PG II and D-PG had smaller hydrodynamic sizes with smaller glycosaminoglycan chains (but larger than those from bovine nasal cartilage proteoglycans) composed exclusively of chondroitin 4-sulphate. The major proteoglycans were incapable of interacting with hyaluronic acid. In general, the amino acid compositions of the major proteoglycans of rat incisors resembled that of bovine nasal cartilage proteoglycans, but the former had lower proline, valine, isoleucine, leucine, and higher aspartic acid, contents.     

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.     

Cartilage proteoglycans

The predominant proteoglycan present in cartilage is the large chondroitin sulfate proteoglycan 'aggrecan'. Following its secretion, aggrecan self-assembles into a supramolecular structure with as many as 50 monomers bound to a filament of hyaluronan. Aggrecan serves a direct, primary role providing the osmotic resistance necessary for cartilage to resist compressive loads. Other proteoglycans expressed during chondrogenesis and in cartilage include the cell surface syndecans and glypican, the small leucine-rich proteoglycans decorin, biglycan, fibromodulin, lumican and epiphycan and the basement membrane proteoglycan, perlecan. The emerging functions of these proteoglycans in cartilage will enhance our understanding of chondrogenesis and cartilage degeneration.     

Insulin modifies the properties of glucose transporters in rat brown adipose tissue.

The influence of cyclic AMP on cartilage degradation was investigated by using phosphodiesterase inhibitors [theophylline and 3-isobutyl-1-methylxanthine (IBMX)], forskolin (which activates the catalytic subunit of adenylate cyclase) and cyclic AMP analogues (dibutyryl and 8-bromo). Breakdown was assessed by quantification of proteoglycans released into the media of 8-day bovine nasal-septum cartilage cultures. Theophylline (1-20 mM), IBMX (0.01-2 mM) and dibutyryl cyclic AMP (0.1-2 mM) had little or no influence on the rate of proteoglycan release from unstimulated (no-endotoxin) cartilages. A small but detectable increase in breakdown was observed with 8-bromo cyclic AMP (0.5-2 mM) and forskolin (50-75 micrograms/ml). To examine potential inhibitory influences of these agents, the cyclic AMP modulators were added to cultures simultaneously treated with Salmonella typhosa endotoxin (12-25 micrograms/ml), a potent stimulator of cartilage degradation. The 3-4-fold stimulation of breakdown by endotoxin was strikingly inhibited by all three classes of cyclic AMP regulators. Optimal inhibition was found at 10-20 mM-theophylline, 1-2 mM-IBMX, 50-75 micrograms of forskolin/ml, 2 mM-dibutyryl cyclic AMP and 2 mM-8-bromo cyclic AMP. Inhibition was shown to be reversible, indicating that cartilages were viable after treatment. Sepharose CL-2B chromatography of proteoglycan products released from treated cartilages showed that the endotoxin-stimulated shift to lower average Mr was significantly prevented by cyclic AMP analogues and phosphodiesterase inhibitors. Together, these results show that agents which increase cyclic AMP inhibit both quantitative and qualitative aspects of endotoxin-mediated cartilage degradation.     

The distribution of proteoglycans of high electrophoretic mobility in cartilages from different species and of different ages

The distribution of small proteoglycans of high relative electrophoretic mobility in cartilage of various species and of different ages was studied. Proteoglycans extracted by 4 M guanidinium chloride were purified by ion-exchange chromatography and assessed by gel electrophoresis. Proteoglycans fractionated by equilibrium density gradient centrifugation under ‘dissociative’ conditions were similarly purified and assessed. A rapid migrating population was found in articular cartilages of young humans, baboons, calfs, pigs, rabbits, rats, chickens and in mandibular and vertebral cartilages of dog-fish. It was not detected in unfractionated proteoglycans extracted from fetal rat, pig, calf, baboon and human cartilages. In baboon and human fetal cartilages of advanced gestational age, however, small amounts of the rapid population were present being detected in the low density fractions of dissociative gradients. The rapid migrating population was not found either in unfractionated or in fractionated proteoglycans obtained from articular cartilages of humans aged over 40. It was absent from human osteoarthritic cartilages but was detected even at advanced age in cartilages covering osteophytes.     

Polydispersity and heterogeneity of squid cranial cartilage proteoglycans as assessed by immunochemical methods and electron microscopy

The three populations of squid cranial cartilage proteoglycans, D1D1A, D1D1B and D1D2 appeared to have a high degree of polydispersity. Gel electrophoresis and immunoblotting analysis showed that polydispersity was mainly due to the variable size of chondroitin sulphate E chains. This was further ascertained after rotary shadowing electron microscopy of proteoglycan core proteins and glycosaminoglycan side chains and statistical analysis of the sizes measured for both components. Enzymic treatment of the proteoglycan core proteins produced different peptides from each population, suggesting that the observed heterogeneity of the proteoglycans is due to their core proteins. Antibodies were raised in rabbits against all proteoglycans and enzyme-linked immunosorbent analysis of proteoglycan core proteins revealed that the proteoglycans, even heterogeneous, shared many common epitopes. Part of the common proteoglycan epitopes were found to be located in chondroitin sulphate E chains. Heterogeneity of squid proteoglycans was also investigated by studying their interactions with collagen and it was found that only the two populations of high molecular mass, D1D1A and D1D2, were able to interact with only collagen type I, the latter stronger than the former.     

Isolation and characterization of proteoglycans from growing antlers of wapiti (Cervus elaphus)

Proteoglycans were extracted with 4 M guanidine–HCl from the zone of maturing chondrocytes, the site of endochondral ossification of growing antlers of wapiti (Cervus elaphus). Proteoglycans were isolated by DEAE-Sephacel chromatography and separated by Sepharose CL-4B chromatography into three fractions. Fraction I contained a high molecular mass (>1000 kDa) chondroitin sulfate proteoglycan capable of interacting with hyaluronic acid. Its amino acid composition resembled that of the cartilage proteoglycan, aggrecan. Fraction II contained proteoglycans with intermediate molecular weight which were recognized by monoclonal antibodies specific to chondroitin sulfate and keratan sulfate. Fraction III contained a low molecular mass (<160 kDa) proteoglycan, decorin, with a glucuronate-rich glycosaminoglycan chain.     

Distribution of chondroitin sulfate in cartilage proteoglycans under associative conditions.

Proteoglycan aggregates and proteoglycan subunits were extracted from bovine articular cartilage with guanidine-HC1 folowed by fractionation by equilibrium centrifugation in cesium chloride density gradients. The distribution of chondroitin sulfates (CS) in the cartilage proteoglycans was studied at the disaccharide level by digestion with chondroitinases. In the proteoglycan aggregate fraction, it was observed that the proportion of 4-sulfated disaccharide units to total CS increased from the bottom to the top fractions, whereas that of 6-sulfated disaccharide units was in the reverse order. Thus, the ratio of 4-sulfated disaccharide units to 6-sulfated disaccharide units increased significantly with decreasing density. The proportion of non-sulfated disaccharide units to total CS tended to increase with increasing density. These data indicate a polydisperse distribution of CS chains, under the conditions used here, in proteoglycan aggregates from bovine articular cartilage.