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.     

The Engelbreth-Holm-Swarm mouse tumor produces undersulfated heparan sulfate and oversulfated galactosaminoglycans

Glycosaminoglycans were prepared from the Engelbreth-Holm-Swarm mouse tumor. Enzymatic analysis demonstrated heparan sulfate (95.8%) and chondroitinase ABC-sensitive galactosaminoglycans (4.2%). HPLC analysis of the disaccharide units showed that heparan sulfate chains were undersulfated on average, comprising approximately 30% nonsulfated and 60% N-sulfated disaccharide units with small proportions of other monosulfated and disulfated disaccharide units. In contrast, galactosaminoglycan chains were oversulfated, containing an appreciable proportion (15%) of a 4,6-disulfated (so-called E-type) disaccharide unit in addition to 51% of a 4-sulfated, 22% of a 6-sulfated, and 11% of a nonsulfated disaccharide unit. The significance of the oversulfated disaccharide structure is discussed in relation to the possible regulation of functions of hybrid proteoglycans from which the galactosaminoglycan chains are derived.     

The low sulfated chondroitin sulfate proteoglycans of human placenta have sulfate group-clustered domains that can efficiently bind Plasmodium falciparum-infected erythrocytes

Plasmodium falciparum infection in pregnant women results in the chondroitin 4-sulfate-mediated adherence of the parasite-infected red blood cells (IRBCs) in the placenta, adversely affecting the health of the fetus and mother. We have previously shown that unusually low sulfated chondroitin sulfate proteoglycans (CSPGs) in the intervillous spaces of the placenta are the receptors for IRBC adhesion, which involves a chondroitin 4-sulfate motif consisting of six disaccharide moieties with approximately 30% 4-sulfated residues. However, it was puzzling how the placental CSPGs, which have only approximately 8% of the disaccharide 4-sulfated, could efficiently bind IRBCs. Thus, we undertook to determine the precise structural features of the CS chains of placental CSPGs that interact with IRBCs. We show that the placental CSPGs are a mixture of two major populations, which are similar by all criteria except differing in their sulfate contents; 2-3% and 9-14% of the disaccharide units of the CS chains are 4-sulfated, and the remainder are nonsulfated. The majority of the sulfate groups in the CSPGs are clustered in CS chain domains consisting of 6-14 repeating disaccharide units. While the sulfate-rich regions of the CS chains contain 20-28% 4-sulfated disaccharides, the other regions have little or no sulfate. Further, we find that the placental CSPGs are able to efficiently bind IRBCs due to the presence of 4-sulfated disaccharide clusters. The oligosaccharides corresponding to the sulfate-rich domains of the CS chains efficiently inhibited IRBC adhesion. Thus, our data demonstrate, for the first time, the unique distribution of sulfate groups in the CS chains of placental CSPGs and that these sulfate-clustered domains have the necessary structural elements for the efficient adhesion of IRBCs, although the CS chains have an overall low degree of sulfation.     

Maturation-related differences in the structure and composition of proteoglycans synthesized by chondrocytes from bovine articular cartilage

Calf (2-3-month-old) and steer (approximately 18-month-old) bovine articular chondrocytes were isolated and cultured as high density monolayers. The proteoglycans synthesized on day 5 during a 15-h period of labeling with [35S]sulfate or [3H]glucosamine were isolated and characterized. The majority (greater than 70%) of the newly synthesized proteoglycans were found in the medium. When viewed in the electron microscope, medium-derived proteoglycans of high buoyant density were longer in calf than in steer. The medium and extracts of the cell layer were pooled and the radiolabeled proteoglycans were fractionated by isopycnic density gradient centrifugation performed under dissociative conditions. The low buoyant density fraction contained, in both calf and steer, small-sized nonaggregating proteoglycans containing chondroitin sulfate. The high buoyant density fraction contained greater than 90% of the newly synthesized proteoglycans. The majority were able to interact with hyaluronic acid to form aggregates. Calf high buoyant density fraction proteoglycans were larger, had longer chondroitin sulfate chains and lower ratios of keratan sulfate chains/chondroitin sulfate chains than steer high buoyant density fraction proteoglycans. These maturation-related differences are typical of those present in the proteoglycans of the calf and steer cartilage matrix from which the chondrocytes were isolated. Experiments with beta-D-xylosides showed that steer cultures had the capacity to synthesize twice as many chondroitin sulfate chains/cell as calf cultures. At each xyloside concentration used, chondroitin sulfate chains were longer in calf than steer. At both ages, chain size decreased with increase in rate of synthesis; the relationship between chain size and rate of synthesis was, however, quite different at the two ages. The results of these studies suggest that articular chondrocytes have an inherent program that determines the quality of proteoglycans synthesized at different ages.     

Proteoglycans of mineralizing rib and epiphyseal cartilage

Rib cartilage from growing guinea pigs and epiphyseal cartilage from Beagle puppie were separated into three fractions, representing non-mineralized, low mineralized, and high mineralized, tissue, by centrifuging finely ground material in acetone/bromoform density gradients. Following extraction under dissociative conditions, the proteoglycans were fractionated by density gradient ultracentrifugation under associative and dissociative conditions.With the onset of mineralization, the cartilage lost approximately half its content of proteoglycans. The proteoglycans remaining in the calcified cartilage differed in composition and in size from those of nonmineralized tissue. With the increased mineral content of the tissues the ratios of protein to polysaccharide, of chondroitin sulfate to keratan sulfate, and of 4-sulfated to 6-sulfated chondroitin sulfate increased in the proteoglycan fraction. Furthermore, gel chromatograms indicated decreased proportions of very high molecular weight proteoglycans, in mineralized tissue.     

Isolation of proteoglycans from human articular cartilage.

Proteoglycans were extracted from normal human articular cartilage of various ages with 4M-guanidinium chloride and were purified and characterized by using preformed linear CsCl density gradients. With advancing age, there was a decrease in high-density proteoglycans of low protein/uronic acid weight ratio and an increase in the proportion of lower-density proteoglycans, richer in keratan sulphate and protein. Proteoglycans of each age were also shown to disaggregate in 4M-guanidinium chloride and at low pH and to reaggregate in the presence of hyaluronic acid and/or low-density fractions. Osteoarthrotic-cartilage extracts had an increased content of higher-density proteoglycans compared with normal cartilage of the same age, and results also suggested that these were not mechanical or enzymic degradation products, but were possibly proteoglycans of an immature nature.     

Human colon adenocarcinoma is associated with specific post-translational modifications of versican and decorin

In this study, the amounts and the fine structural characteristics of versican and decorin present in human colon adenocarcinomas (HCC) were investigated and compared with those in human normal colon (HNC). HCC is characterized by significant increase in the amounts of versican and decorin (13- and 8-fold in terms of protein, respectively). These two proteoglycans (PGs) were the predominant in HCC (86% of total uronic acid). In HNC, versican and decorin contained both chondroitin sulfate/dermatan sulfate chains (CS/DS), with DS to be the predominant one (90-93%). The molecular sizes (M(r)s) estimated for DS and CS chains were 25-28 and 21-28 kDa, respectively. In CS/DS chains isolated from both versican and decorin, 4-sulfated disaccharides accounted for 79-86% of total disaccharide units, respectively, whereas lower amounts of 6- and non-sulfated units were also recorded. In contrast, the tumor-associated versican and decorin were of smaller hydrodynamic size with lower glycosaminoglycan (GAG) content per PG molecule as compared with those found in HNC. In HCC, both PGs contained mainly CS chains (up to 86%) and the M(r)s of CS and DS chains were also found to be of smaller size (12 and 16 kDa, respectively). The sulfation patterns of CS/DS chains from both PGs were also significantly different. They were composed mainly of 6-sulfated disaccharides (63-70%), whereas 4-sulfated units accounted for 23-31%. A significant increase in the proportion of non-sulfated disaccharides was also recorded. These findings indicate that the colon adenocarcinoma is characterized by a remarkable increase in the concentration of versican and decorin. Furthermore, these PGs are significantly modified at the post-translational level, i.e. the type, length and the sulfation pattern of their GAG chains. These specific structural alterations of versican and decorin may influence the biology of cancer cells in HCC.     

Structural characterization of the bovine tracheal chondroitin sulfate chains and binding of Plasmodium falciparum-infected erythrocytes

Sequestration of Plasmodium falciparum-infected red blood cells (IRBCs) in the human placenta is mediated by chondroitin 4-sulfate (C4S). A cytoadherence assay using chondroitin sulfate proteoglycans (CSPGs) is widely used for studying C4S-IRBC interactions. Bovine tracheal chondroitin sulfate A (CSA) preparation lacking a major portion of core protein has been frequently used for the assay. Here the CSPG purified from bovine trachea and CSA were assessed for IRBC binding and the CS chains studied in detail for structure-activity relationship. The IRBCs bound at significantly higher density to the CSPG than CSA. The CS chains of CSPG/CSA are heterogeneous with varying levels of 4- and 6-sulfates, which are distributed such that approximately 80% of the 4-sulfated disaccharides are present as single and blocks of two or three separated by one to three 6-sulfated disaccharides. The remainder of the 4-sulfated disaccharides is present in blocks composed of 4-12 units, separated by 6-sulfated disaccharides. In the IRBC adherence inhibition analysis, CSA fragments with 88%-92% 4-sulfate were significantly less inhibitory than the intact CSA, indicating that the regions consisting of shorter 4-sulfated blocks efficiently bind IRBCs despite the presence of relatively high levels of 6-sulfate. This is because the 6-sulfated disaccharides have unsubstituted C-4 hydroxyls that are crucial for IRBC binding. The presence of high levels of 6-sulfate, however, significantly interfere with the IRBC binding activity of CSA, which otherwise would more efficiently bind IRBCs. Thus our study revealed the distribution pattern of 4- and 6-sulfate in bovine tracheal CSA and structural basis for IRBC binding.     

Isolation and characterization of matrix proteoglycans from human nasal cartilage. Compositional and structural comparison between normal and scoliotic tissues

The content, types and the fine structures of proteoglycans (PGs) present in human normal nasal cartilage (HNNC) were investigated and compared with those in human scoliotic nasal cartilage (HSNC). Three PG types were identified in both HNNC and HSNC; the large-sized high buoyant density aggrecan, which is the predominant PG population, and the small-sized low buoyant density biglycan and decorin. HSNC contained a significantly higher amount of keratan sulfate (KS)-rich aggrecan (30%) of smaller hydrodynamic size as compared to HNNC. The average molecular sizes (M(r)s) of aggecan-derived chondroitin sulfate (CS) chains in both HNNC and HSNC were identical (18 kDa), but they significantly differ in disaccharide composition, since CS isolated from HSNC contained higher proportions of 6-sulfated disaccharides as compared to those from HNNC. Scoliotic tissue contained also higher amounts (67%) of the small PGs, biglycan and decorin as compared to HNNC. It is worth noticing that both normal and scoliotic human nasal cartilage contain also non-glycanated forms of decorin and biglycan. Dermatan sulfate (DS) was the predominant glycosaminoglycan (GAG) present on biglycan and decorin in both tissues. The small PGs-derived CS chains in both normal and scoliotic cartilage had the same M(r) (20 kDa), whereas DS chains from scoliotic cartilage were of greater M(r) (32 kDa) than those from normal cartilage (24 kDa). Furthermore, scoliotic tissue-derived DS chains contained higher amounts of iduronate (20%) as compared to those of normal cartilage (12%). Disaccharide analysis of small PGs showed that both HNNC and HSNC were rich in 4-sulfated disaccharides and in each case, the small size PGs contained a considerably higher proportion of 4-sulfated disaccharides than the aggrecan of the same tissue. The higher amounts of matrix PGs identified in scoliotic tissue as well as the differences in fine chemical composition of their GAG chains may reflect the modified architecture and functional failure of scoliotic tissue.     

The sulfation pattern of chondroitin sulfate from articular cartilage explants in response to mechanical loading

Chondrocytes within articular cartilage experience complete unloading between loading cycles thereby utilizing mechanical signals to regulate their own anabolic and catabolic activities. Structural alterations of proteoglycans (PGs) during aging and the development of osteoarthritis (OA) have been reported; whether these can be attributed to altered load or compression is largely unknown. We report here on experiments in which the effect of intermittent loading on the fine structure of newly synthesized chondroitin sulfate (CS) in bovine articular cartilage explants was examined. Tissues were subjected for 6 days to cyclic compressive pressure using a sinusoidal waveform of 0.1, 0.5 or 1.0 Hz frequency with a peak stress of 0.5 MPa for a period of 5, 10 or 20 s, followed by an unloading period lasting 10, 100 or 1000 s. During the final 18 h of the culture, cartilage explants were radiolabeled with 50 microCi/ml D-6-[3H]glucosamine, and newly synthesized as well as endogenous CS chains were isolated after proteinase solubilization of the tissue. CS chains were depolymerized with chondroitinase ABC and ACII, and the 3H-digestion products were quantified after fractionation by high-performance anion-exchange chromatography using a CarboPac PA1 column. Intermittently applied cyclic mechanical loading did not affect the proportion of 4- and 6-sulfated disaccharide repeats, but caused a significant decrease in the abundance of the 4,6-disulfated nonreducing terminal galNAc residues. In addition, loading induced elongation of CS chains. Taken together, these data provide evidence for the first time that long-term in vitro loading results in marked and reproducible changes in the fine structure of newly synthesized CS, and that accumulation of such chains may in turn modify the physicochemical and biological response of articular cartilage. Moreover, data presented here suggest that in vitro dynamic compression of cartilage tissue can induce some of the same alterations in CS sulfation that have previously been shown to occur during the development of degenerative joint diseases such as OA.     

Sulfation of chondroitin sulfate in human articular cartilage. The effect of age, topographical position, and zone of cartilage on tissue composition.

The chondroitin ABC lyase digestion products of normal human femoral condyle articular cartilage and of purified aggrecan were analyzed for their mono- and nonsulfated disaccharide composition. Changes in the total tissue chemistry were most pronounced during the period from birth to 20 years of age, when the -[GlcAbeta,3GalNAc6]- disaccharide content increased from approximately 50% to 85% of the total disaccharide content and there was a concomitant decrease in the content of the 4-sulfated disaccharide. In general, the disaccharide content of the deeper layers of immature cartilage were richer in the 4-sulfated residue than the upper regions of the tissue. As the tissue aged and decreased in thickness, the disaccharide composition became more evenly 6-sulfated. The newly synthesized chondroitin sulfate chains had a similar composition to the endogenous chains and also underwent the same age and zonal changes. The monoclonal antisera 3B3(+) and 2B6(+) were used to immunolocalize the unsaturated 6- and 4-sulfated residues generated at the reducing termini of the chondroitin sulfate chains by digestion with chondroitin ABC lyase, and these analyses indicated that the sulfation pattern at this position did not necessarily reflect the internal disaccharide composition of the chains. In summary, the sulfation pattern of chondroitin sulfate disaccharides from human normal articular cartilage varies with the age of the specimen, the position (topography) on the joint surface, and the zone of cartilage analyzed. Furthermore, these changes in composition are a consequence of both extracellular, post-translational processing of the core protein of aggrecan and changes in the sulfotransferase activity of the chondrocyte.     

Structure of chondroitin sulfates analyses of the products formed from chondroitin sulfates A and C by the action of the chondroitinases C and AC from Flavobacterium heparinum

The structures of chondroitin sulfate A from whale cartilage and chondroitin sulfate C from shark cartilage have been examined with the aid of the chondroitinases AC and C from Flavobacterium heparinum. The analyses of the products formed from the chondroitin sulfates by the action of the chondroitinases have shown that three types of oligosaccharides compose the structure of chondroitin sulfate A, namely, a dodeca-, hexa- and a tetra-saccharide, containing five, two and one 4-sulfated disaccharides per 6-sulfated disaccharide residue, respectively. The polymer contains an average of 3 mol of each oligosaccharide per mol of chondroitin sulfate A. Each mol of chondroitin sulfate C contains an average of 5 mol of 4-sulfated disaccharide units. A tetra-saccharide containing one 4-sulfated disaccharide and one 6-sulfated disaccharide was isolated from this mucopolysaccharide by the action of the chondroitinase C, indicating that the 4-sulfated disaccharides are not linked together in one specific region but spaced in the molecule.     

The effect of retinoic acid on proteoglycan turnover in bovine articular cartilage cultures

This paper describes proteoglycan catabolism by adult bovine articular cartilage treated with retinoic acid as a means of stimulating the loss of this macromolecule from the extracellular matrix of cartilage. Addition of retinoic acid (10(-12)-10(-6) M) to adult bovine articular cartilage which had been labeled with [35S]sulfate for 6 h after 5 days in culture, resulted in a dose-dependent increase in the rate of loss of 35S-labeled proteoglycans from the matrix of the tissue. Concomitant with this loss was a decrease in the proteoglycan content of the tissue. Incubation of cultures treated with 1 microM retinoic acid, at 4 degrees C, or with 0.5 mM cycloheximide, resulted in a significant decrease in the rate of retinoic acid-induced loss of proteoglycans and demonstrated cellular involvement in this process. Analysis of the 35S-labeled proteoglycans remaining in the matrix showed that the percentage of radioactivity associated with the small proteoglycan species extracted from the matrix of articular cartilage explants labeled with [35S]sulfate after 5 days in culture was 15% and this increased to 22% in tissue maintained in medium alone. In tissue treated with 1 microM retinoic acid for 6 days, the percentage of radioactivity associated with the small proteoglycan was 58%. Approximately 93% of the 35S-labeled proteoglycans released into the medium of control and retinoic acid-treated cultures was recovered in high density fractions after CsCl gradient centrifugation and eluted on Sepharose CL-2B as a broad peak with a Kav of 0.30-0.37. Less than 17% of these proteoglycans was capable of aggregating with hyaluronate. These results indicate that in both control and retinoic acid-treated cultures the larger proteoglycan species is lost to the medium at a greater rate than the small proteoglycan species. The effect of retinoic acid on proteoglycan turnover was shown to be reversible. Cartilage cultures maintained with retinoic acid for 1 day then switched to medium with 20% (v/v) fetal calf serum for the remainder of the culture period exhibited decreased rates of loss of 35S-labeled proteoglycans from the matrix and increased tissue hexuronate contents to levels near those observed in tissue maintained in medium with 20% (v/v) fetal calf serum throughout. Furthermore, following switching to 20% (v/v) fetal calf serum, the relative proportions of the 35S-labeled proteoglycan species remaining in the matrix of these cultures were similar to those of control cultures.     

Buffer induced modification of 6-sulfated disaccharide in chondroitin lyase digestions

High-performance liquid chromatographic analyses of chondroitin lyase AC or ABC hydrolysates revealed unexpected high content of material coeluting with the nonsulfated disaccharide 2-acetamido-2-deoxy-3-O-(β-d-gluco-4-enepyranosyl uronic acid)-d-galactose. Incubation of a commercial preparation of the 6-sulfated disaccharide, 2-acetamido-2-deoxy-3-O-(β-d-gluco-4-enepyranosyl uronic acid)-6-O-sulfo-d-galactose with “enriched Tris buffer” generated material coeluting with nonsulfated disaccharide. The amount of material exhibiting this anomalous chromatographic behavior was proportional to the amount of 6-sulfated disaccharide added to the incubation mixture. This suggested a precursor/product relationship between the 6-sulfated disaccharide and the anomalous peak. The result was specific for the 6-sulfated disaccharide: incubation of the 4-sulfated disaccharide, 2-acetamido-2-deoxy-3-O-(β-d-gluco-4-enepyranosyl uronic acid)-4-O-sulfo-d-galactose, with enriched Tris buffer did not generate material with anomalous chromatographic properties. When [³⁵S]sulfate labeled cartilage glycosaminoglycans were hydrolyzed with chondroitin lyases, some of the radioactivity coeluted with the nonsulfated disaccharide. Thus, buffer-induced modification of 6-sulfated disaccharide was not caused by hydrolysis of ester sulfate. Although the proportion of the 6-sulfated disaccharide which was recovered in the anomalous peak was constant for incubations done simultaneously, incubations done at different times gave variable results. Thus, control incubations of 6-sulfated disaccharide with chondroitinase buffer must be included with each reaction series to allow correction for the proportion of the material eluting with nonsulfated disaccharide which is actually 6-sulfated.     

Characterization of proteoglycans synthesized by rat thyroid cells in culture and their response to thyroid-stimulating hormone

A Fisher rat thyroid cell line was maintained in culture and the cells were labeled with [3H]glucosamine, [35S]sulfate, and [35S]cysteine to examine the synthesis of proteoglycans. 3H and 35S radioactivity from these precursors were incorporated into both chondroitin sulfate (CS) and heparan sulfate (HS) proteoglycans. CS proteoglycans were almost exclusively secreted into the medium while HS proteoglycans remained mainly associated with the cell layer. Single chain glycosaminoglycans released by papain digestion or alkaline borohydride treatment of either the CS or HS proteoglycans had average molecular weights of approximately 30,000 on Sepharose CL-6B chromatography. Both CS and HS proteoglycans were relatively small and contained only one or two glycosaminoglycans chains. 3H and 35S incorporation into both CS and HS proteoglycans were increased by thyroid-stimulating hormone (TSH) in a dose-dependent manner, which is in part explained by an adenylate cyclase-dependent mechanism as indicated by a similar effect in response to dibutyryl cAMP. TSH enhanced the incorporation of 35S into CS from [35S]cysteine about 1.5-fold and that from [35S]sulfate about 2-fold. This result demonstrated that the increased 35S incorporation from the [35S]sulfate precursor reflects an actual increase in sulfate incorporation and is not simply a result from an apparent increase in specific activity of the phosphoadenosine phosphosulfate donor. Analysis of disaccharides from chondroitinase digests revealed that the proportion of non-sulfated, 4-sulfated, and 6-sulfated disaccharides was not altered appreciably by TSH. These results, together with the disproportionate increase in 3H incorporation into CS from [3H]glucosamine, indicated that TSH increased the specific activity of the 3H label as well. Chase experiments revealed that CS proteoglycans were rapidly (t1/2 = 15 min) secreted into the medium and that the degradation of cell-associated proteoglycans was enhanced by TSH.     

Extracellular matrix and the maintenance of the differentiated state: proteoglycans synthesized by replated chondrocytes and nonchondrocytes

It has been previously shown that undifferentiated stage 23 to 24 chick limb bud mesenchymal cells can be maintained in culture under conditions which promote chondrogenesis. As the chondrocytes mature in vitro, their proteoglycan synthesis progresses through a specific and reproducible biosynthetic program. By the eighth day of culture, the chondrocytes are making proteoglycans that are similar to proteoglycans isolated from adult animal tissues. Relative to the Day 8 proteoglycans, the proteoglycans synthesized by chick limb bud chondrocytes earlier in culture have a smaller monomer size, longer chondroitin sulfate chains, shorter keratan sulfate chains, a higher ratio of chondroitin-6-sulfate to chondroitin-4-sulfate, and a decreased ability to interact with hyaluronic acid. We have reported a procedure to remove the cells from Day 8 cultures and strip away most, if not all, of the extracellular matrix. In addition, the chondrocytes can be separated from the 40-50% nonchondrocytic cells normally found in Day 8 cultures, and the two cell populations replated separately. This report describes the analysis of the proteoglycans synthesized by replated cells; this analysis demonstrates quantitative and qualitative differences between chondrocyte and nonchondrocyte proteoglycans. The overall rate of proteoglycan synthesis is fourfold higher and the rate of synthesis of high buoyant density proteoglycans 30-fold higher for replated chondrocytes relative to nonchondrocytes. Qualitatively, more newly synthesized nonchondrocyte proteoglycans partition at lower buoyant density on CsCl equilibrium density gradients than do chondrocyte proteoglycans. Nonchondrocyte proteoglycans are of two major classes: One has a monomer size slightly smaller than that of Day 8 chondrocyte proteoglycan, but has much longer glycosaminoglycan chains. The other is considerably smaller than Day 8 chondrocyte proteoglycans, but has glycosaminoglycans of slightly larger size. In contrast, replated chondrocytes synthesize, even as soon as 4.5 hr after replating, proteoglycans that are identical to Day 8 chondrocyte proteoglycan in monomer size, in glycosaminoglycan chain size, in aggregability, and in the ratio of 6-sulfated to 4-sulfated chondroitin. Since denuding mature Day 8 chondrocytes of their extracellular matrix does not cause them to recapitulate their developmentally regulated program for the biosynthesis of proteoglycans, it is concluded that the quality of mature chondrocyte proteoglycan is not altered by the absence of extracellular matrix.     

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.     

Isolation and characterization of matrix proteoglycans from human nasal cartilage: Compositional and structural comparison between normal and scoliotic tissues

The content, types and the fine structures of proteoglycans (PGs) present in human normal nasal cartilage (HNNC) were investigated and compared with those in human scoliotic nasal cartilage (HSNC). Three PG types were identified in both HNNC and HSNC; the large-sized high buoyant density aggrecan, which is the predominant PG population, and the small-sized low buoyant density biglycan and decorin. HSNC contained a significantly higher amount of keratan sulfate (KS)-rich aggrecan (30%) of smaller hydrodynamic size as compared to HNNC. The average molecular sizes (M_rs) of aggecan-derived chondroitin sulfate (CS) chains in both HNNC and HSNC were identical (18 kDa), but they significantly differ in disaccharide composition, since CS isolated from HSNC contained higher proportions of 6-sulfated disaccharides as compared to those from HNNC. Scoliotic tissue contained also higher amounts (67%) of the small PGs, biglycan and decorin as compared to HNNC. It is worth noticing that both normal and scoliotic human nasal cartilage contain also non-glycanated forms of decorin and biglycan. Dermatan sulfate (DS) was the predominant glycosaminoglycan (GAG) present on biglycan and decorin in both tissues. The small PGs-derived CS chains in both normal and scoliotic cartilage had the same M_r (20 kDa), whereas DS chains from scoliotic cartilage were of greater M_r (32 kDa) than those from normal cartilage (24 kDa). Furthermore, scoliotic tissue-derived DS chains contained higher amounts of iduronate (20%) as compared to those of normal cartilage (12%). Disaccharide analysis of small PGs showed that both HNNC and HSNC were rich in 4-sulfated disaccharides and in each case, the small size PGs contained a considerably higher proportion of 4-sulfated disaccharides than the aggrecan of the same tissue. The higher amounts of matrix PGs identified in scoliotic tissue as well as the differences in fine chemical composition of their GAG chains may reflect the modified architecture and functional failure of scoliotic tissue.     

Characterization of the dermatan sulfate proteoglycans, DS-PGI and DS-PGII, from bovine articular cartilage and skin isolated by octyl-sepharose chromatography

Two forms of dermatan sulfate proteoglycans, called DS-PGI and DS-PGII, have been isolated from both bovine fetal skin and calf articular cartilage and characterized. The proteoglycans were isolated using either (a) molecular sieve chromatography under conditions where DS-PGI selectively self-associates or (b) chromatography on octyl-Sepharose, which separates DS-PGI from DS-PGII based on differences in the hydrophobic properties of their core proteins. The NH2-terminal amino acid sequence of DS-PGI from skin and cartilage is identical. The NH2-terminal amino acid sequence of DS-PGII from skin and cartilage is identical. However, the amino acid sequence data and tryptic peptide maps demonstrate that the core proteins of DS-PGI and DS-PGII differ in primary structure. In DS-PGI from bovine fetal skin, 81-84% of the glycosaminoglycan was composed of IdoA-GalNAc(SO4) disaccharide repeating units. In DS-PGI from calf articular cartilage, only 25-29% of the glycosaminoglycan was composed of IdoA-GalNAc(SO4). In DS-PGII from bovine fetal skin, 85-93% of the glycosaminoglycan was IdoA-GalNAc(SO4), whereas in DS-PGII from calf articular cartilage, only 40-44% of the glycosaminoglycan was IdoA-GalNAc(SO4). Thus, analogous proteoglycans from two different tissues, such as DS-PGI from skin and cartilage, possess a core protein with the same primary structure, yet contain glycosaminoglycan chains which differ greatly in iduronic acid content. These differences in the composition of the glycosaminoglycan chains must be determined by tissue-specific mechanisms which regulate the degree of epimerization of GlcA-GalNAc(SO4) into IdoA-GalNAc(SO4) and not by the primary structure of the core protein.     

Preparation of chondroitin sulfate libraries containing disulfated disaccharide units and inhibition of thrombin by these chondroitin sulfates

Chondroitin sulfate (CS) containing GlcA-GalNAc(4,6-SO₄) (E unit) and CS containing GlcA(2SO₄)-GalNAc(6SO₄) (D unit) have been implicated in various physiological functions. However, it has been poorly understood how the structure and contents of disulfated disaccharide units in CS contribute to these functions. We prepared CS libraries containing E unit or D unit in various proportions by in vitro enzymatic reactions using recombinant GalNAc 4-sulfate 6-O-sulfotransferase and uronosyl 2-O-sulfotransferase, and examined their inhibitory activity toward thrombin. The in vitro sulfated CSs containing disulfated disaccharide units showed concentration-dependent direct inhibition of thrombin when the proportion of E unit or D unit in the CSs was above 15–17%. The CSs containing both E unit and D unit exhibited higher inhibitory activity toward thrombin than the CSs containing either E unit or D unit alone, if the proportion of the total disulfated disaccharide units of these CSs was comparable. The thrombin-catalyzed degradation of fibrinogen, a physiological substrate for thrombin, was also inhibited by the CS containing both E unit and D unit. These observations indicate that the enzymatically prepared CS libraries containing various amounts of disulfated disaccharide units appear to be useful for elucidating the physiological function of disulfated disaccharide units in CS.