Effects of platelet-derived growth factor and transforming growth factor-beta 1 on the synthesis of a large versican-like chondroitin sulfate proteoglycan by arterial smooth muscle cells

Platelet-derived growth factor (PDGF) and transforming growth factor-beta 1 (TGF-beta 1) increase [35S]sulfate incorporation into proteoglycan (PG) by monkey arterial smooth muscle cells but have opposite effects on cell proliferation. The combination of these two growth regulatory peptides has an additive effect on PG synthesis but no effects on cell proliferation. The time course of sulfate incorporation after stimulation indicates that both growth factors cause maximal incorporation of sulfate into glycosaminoglycan chains by 12-18 h. The PG that is most affected is a large CSPG (Mr approximately 1.2 x 10(6)) which can be immunoprecipitated by an antibody against versican, a large CSPG synthesized by human skin fibroblasts. The hydrodynamic size of this molecule increases after PDGF and TGF-beta 1 stimulation, but the size of the core glycoprotein (Mr approximately 450,000) remains the same. Treatment with either growth factor leads to an increase in the amount of core glycoprotein for this PG. This increase correlates with an increase in the steady state level of mRNA identified by hybridization to a cDNA encoding versican. The two growth factors also increase the glycosaminoglycan chain length of this PG accounting for the greater hydrodynamic size of the molecule after stimulation. In contrast, PDGF and not TGF-beta 1 changes the composition of the glycosaminoglycan chains attached to this PG by doubling the ratio of chondroitin 6-sulfate to chondroitin 4-sulfate. These results indicate that although both of these growth factors increase the net synthesis of a large versican like CSPG, they differ in their effects on the structure of the glycosaminoglycan chains. These post-translational modifications may relate to the growth state of the cells.     

Proteoglycan biosynthesis in relation to differentiation of cord blood monocytes in vitro

Human monocytes were obtained from umbilical cord blood and cultured in vitro. By morphological criteria, the neonatal monocytes developed into macrophage-like cells in the course of 3-5 days in culture. The cells were exposed to [35S]sulphate for 24 h, either from day 0-1 or day 9-10 in vitro. The 35S-labelled macromolecules recovered were mainly associated with the medium fraction (approximately 75%) in both day 1 and day 10 cultures. These secretory macromolecules were demonstrated by the use of chondroitinase ABC-digestions to contain predominantly chondroitin sulphate proteoglycan (CSPG). [35S]galactosaminoglycan chains from day 10 cultures were more highly sulphated than the corresponding day 1 species due to the appearance of (glucuronosyl-4,6-diS-N-acetylgalactosamine) disulphated disaccharide units. The galactosaminoglycan chains in neonatal CSPG were found to increase in Mr during cultivation in vitro; from mean Mr of 20,400 to 30,200 (n = 5) in day 1 and day 10 medium proteoglycans, respectively. The corresponding Mr values for adult monocyte [35S]galactosaminoglycan chains were 21,300 and 22,800. On the basis of the concomitant changes in cellular morphology and glycosaminoglycan structure, it is concluded that neonatal monocytes, like monocytes from adults, differentiate into macrophage-like cells in vitro.     

Secreted and cellular proteochondroitin sulfates of a human B lymphoblastoid cell line contain different protein cores.

Proteoglycans of the human B lymphoblastoid cell line LICR-LON-HMy2 were metabolically labeled with [35S]sulfate. High-density fractions of 35S-labeled material separated by CsCl gradient ultracentrifugation were further purified by anion exchange chromatography and gel filtration. Two proteoglycans, isolated from cell lysates and culture supernatants, were characterized by gel filtration and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) in combination with enzymatic degradation. Treatment with chondroitinase AC completely degraded the glycosaminoglycan moiety of the proteoglycans. Three to 4 chondroitin sulfate chains (average molecular mass = 26 kDa) were estimated for each of the two proteoglycans. Differences between the proteochondroitin sulfates (CSPG) were observed in the content of N-linked oligosaccharides. After chondroitinase AC treatment the resulting band in SDS-PAGE of the secreted CSPG was sensitive to treatment with endoglycosidase F (Endo F) which further reduced the molecular mass from 30 to 21.5 kDa, whereas the band of the cellular CSPG after chondroitinase AC treatment (molecular mass = 30 kDa) remained resistant to Endo F treatment. The composition of amino acids was different in the protein cores, suggesting differences in the primary structure. Both CSPG contained a high percentage of glycine and serine. For both CSPG a molecular mass of approximately 135 kDa was deduced from the hydrodynamic sizes of the glycosaminoglycan chains obtained after alkaline/borohydride treatment and the migration of the protein/oligosaccharide complexes in SDS-PAGE. 75% of all [35S]sulfate-labeled molecules were found in the culture supernatant and 25% in the cellular fraction. 35S-Labeled material in the culture supernatant consisted exclusively of intact CSPG, whereas 35S-Labeled molecules in the cellular preparation consisted largely of free chondroitin sulfate chains. Only 8.3% of the cellular material, isolated from the microsomal fraction, was intact CSPG. In pulse-chase experiments maximal secretion of CSPG was found after 4 h, comprising approximately 40% of totally synthesized CSPG. From these experiments we tentatively conclude that a small proportion of CSPG synthesized by LICR-LON-HMy2 cells is membrane-associated, a larger portion is secreted, and another portion is intracellularly degraded.     

Modulation of the expression of chondroitin sulfate proteoglycan in stimulated human monocytes

Proteoglycan biosynthesis was studied in human monocytes and monocyte-derived macrophages (MDM) after exposure to typical activators of the monocyte/macrophage system: interferon-gamma (IFN-gamma), lipopolysaccharide (LPS), and phorbol 12-myristate 13-acetate (PMA). By morphological examination, both monocytes and MDM were stimulated by these activators. Treatment with IFN-gamma resulted in a slight decrease in the expression of [35S]chondroitin sulfate proteoglycan (CSPG) in both monocytes and MDM, whereas LPS treatment increased the [35S]CSPG expression 1.8 and 2.2 times, respectively. PMA, in contrast, decreased the CSPG expression 0.4 times in monocytes, whereas MDM were stimulated to increase the biosynthesis 1.9 times. An increase in the sulfate density of the chondroitin sulfate chains was evident following differentiation of monocytes into MDM due to the expression of disulfated disaccharide units of the chondroitin sulfate E type (CS-E). However, monocytes exposed to PMA did also express disaccharides of the chondroitin sulfate E type. Furthermore, the expression of CS-E in MDM was increased 2 times following PMA treatment. An inactive phorbol ester, phorbol 12,13-diacetate, did not affect the expression of CS-E in either monocytes or MDM when compared with control cultures, suggesting that protein kinase C-dependent signal pathways may be involved in the regulation of sulfation of CSPG. Exposure to LPS or IFN-gamma did not lead to any changes in the sulfation of the chondroitin sulfate chains.     

Temporal regulation of hyaluronan and proteoglycan metabolism by human bone cells in vitro

Osteoblasts elaborate a dynamic extracellular matrix that is constructed and mineralized as bone is formed. This matrix is primarily composed of collagen, along with noncollagenous proteins which include glycoproteins and proteoglycans. After various times in culture, human bone cells were labeled with [35S]sulfate, [3H] leucine/proline, or [3H]glucosamine and the metabolism of hyaluronan and four distinct species of proteoglycans (PGs) was assayed in the medium, cell layer, and intracellular pools. These cells produce hyaluronan (Mr approximately 1,400,000; a chondroitin sulfate PG (CSPG), Mr approximately 600,000; a heparan sulfate PG (HSPG), Mr approximately 400,000; and two dermatan sulfate PGs with Mr approximately 270,000 (biglycan, PG I) and Mr approximately 135,000 (decorin, PG II) that distribute between the medium and cell layer. Two days following subculture, 12 h [35S]sulfate steady-state labeling yielded a composition of 24, 27, 31, and 18% for total CSPG, HSPG, biglycan, and decorin, respectively. While HSPG and decorin levels and distribution between medium and cell layer remained relatively constant during steady-state labeling at different times in culture, CSPG and biglycan levels increased dramatically at late stages of growth, and their distribution changed throughout culture. These results were independent of cell density, media depletion, and labeling pool effects. In contrast, hyaluronan synthesis was uncoupled from PG synthesis and apparently density-dependent. Pulse chase labeling at different stages of culture showed that the CSPG and decorin behaved as secretory PGs. Both HSPG and biglycan underwent catabolism, with HSPG possessing a t1/2 of 8 h and biglycan a t1/2 of 4 h. While the rate of HSPG turnover did not appreciably change between early and late culture, that of biglycan decreased. The mRNA for decorin was constant, while that of biglycan changed during culture. These results suggest that each PG possesses a distinct pattern of cellular and temporal distribution that may reflect specific stages in matrix formation and maturation.     

Two immunologically and developmentally distinct chondroitin sulfate proteolglycans in embryonic chick brain.

Two different chondroitin sulfate proteoglycans (CSPG) in embryonic chick brain were distinguished by immunoreactivity either with S103L, a rat monoclonal antibody which reacts specifically with an 11-amino-acid region in the chondroitin sulfate domain of the core protein of chick cartilage CSPG (Krueger, R. C., Jr., Fields, T. A., Mensch, J. R., and Schwartz, N. B. (1990) J. Biol. Chem. 265, 12088-12097), or with HNK-1, a mouse monoclonal antibody which reacts with a 3-sulfoglucuronic acid residue on neural glycolipids and glycoproteins (Chou, D. K. H., Ilyas, A., Evans, J. E. Costello, C., Quarles, R. H., and Jungawala, F. B. (1986) J. Biol. Chem. 261, 11717-11725) but not with both antibodies. This specific immunoreactivity was used to separate the two CSPGs for further characterization. The S103L reactive brain proteoglycan had a core protein of similar size to cartilage CSPG (370 kDa) but exhibited a smaller hydrodynamic size (K(av) of 0.308). It was substituted predominantly with chondroitin sulfate chains and virtually no keratan sulfate chains. The HNK-1 reactive CSPG had a smaller core protein (340 kDa), an even smaller hydrodynamic size (K(av) of 0.564), and was substituted with both chondroitin sulfate and keratan sulfate chains. Glycosidase digestion patterns with endo-beta-galactosidase, N-glycosidase F, neuraminidase, and O-glycosidase, and reactivity with an antibody to the hyaluronate binding region also showed significant differences between the two brain CSPGs. Expression of the S103L reactive brain CSPG was developmentally regulated from embryonic day 7 through 19 with a peak in core protein on day 13, and in mRNA expression at day 10. In contrast the HNK-1 reactive brain CSPG was constitutively present from day 7 through hatching. These data suggest that these two distinct core proteins are immunologically and biochemically unique translation products of two different CSPG genes.     

Multiple forms of heparan sulfate proteoglycans in the Engelbreth-Holm-Swarm mouse tumor. The occurrence of high density forms bearing both heparan sulfate and chondroitin sulfate side chains

Heparan sulfate proteoglycan from the Engelbreth-Holm-Swarm mouse tumor was previously separated into two forms: a high density form (Form HD) and low density form (Form LD). In this study, the two forms were radiolabeled either metabolically with [35S]sulfate or [3H]serine or chemically with 125I. Pulse-chase experiments with [35S]sulfate showed no clear precursor-product relationship between the two forms. Analyses of the labeled proteoglycan samples with heparitinase and chondroitinase ABC indicated that Form LD is a large proteoglycan containing heparan sulfate chains attached to a single core molecule (Mr = 450,000), whereas Form HD is a mixture of small proteoglycans with four different size core molecules (Mr = 34,000, 29,000, 27,000, and 21,000), most, if not all, of which bear both heparan sulfate (Mr = 60,000) and chondroitin sulfate (Mr = 17,000) chains. Glycosaminoglycan-enriched fragments obtained from Form HD by V8 protease digestion were also shown to contain both heparitinase-susceptible chains and chondroitinase ABC-susceptible chains. Tryptic peptide maps of 125I-labeled Form HD and the glycosaminoglycan-enriched fragments derived therefrom were quite different from the corresponding maps for Form LD.     

A large chondroitin sulfate proteoglycan (PG-M) synthesized before chondrogenesis in the limb bud of chick embryo

Extraction of stage 22-23 chick embryo limb buds that had been metabolically labeled with [35S]sulfate yielded heparan sulfate proteoglycan, small chondroitin sulfate proteoglycan, and large chondroitin sulfate proteoglycan (designated PG-M). PG-M constituted over 60% of the total macromolecular [35S]sulfates. It was larger in hydrodynamic size, richer in protein, and contained fewer chondroitin sulfate chains as compared to the predominant proteoglycan (PG-H, Mr congruent to 1.5 X 10(6)) of chick embryo cartilage. The chondroitin sulfate chains were notable for their large size (Mr greater than or equal to 60,000) and high content of nonsulfated chondroitin units (about 20% of the total hexosamine). Hexosamine-containing chains corresponding in size to N-linked and O-linked oligosaccharides were also present. The core protein was rich in serine, glutamic acid (glutamine), and glycine which together comprised about 38% of the total amino acids. Following chondroitinase AC II (or ABC) digestion, core molecules were obtained which migrated on sodium dodecyl sulfate gel electrophoresis as a doublet of bands with approximately Mr = 550,000 (major) and 500,000, respectively. The Mr = 550,000 core glycoprotein was structurally different from the core glycoprotein (Mr congruent to 400,000) of PG-H, as ascertained by tryptic peptide mapping and immunochemical criteria. Immunofluorescent localization of PG-M showed that the intensity of PG-M staining progressively became higher in the core mesenchyme region than in the peripheral loose mesenchyme, closely following the condensation of mesenchymal cells. Since the cell condensation process has been shown to begin with the increase of fibronectin and type I collagen concentration, the similar change in PG-M distribution suggests that PG-M plays an important role in the cell condensation process by means of its interaction with fibronectin and type I collagen.     

Immunological characterization of a basement membrane-specific chondroitin sulfate proteoglycan

Reichert's membrane, an extraembryonic membrane present in developing rodents, has been proposed as an in vivo model for the study of basement membranes. We have used this membrane as a source for isolation of basement membrane proteoglycans. Reichert's membranes were extracted in a guanidine/3-[(3-cholamidopropyl)dimethylammonio]-1- propanesulfonate buffer followed by cesium chloride density-gradient ultracentrifugation under dissociative conditions. The proteoglycans were subsequently purified from the two most dense fractions (greater than 1.3 g/ml) by ion-exchange chromatography. Mice were immunized with the proteoglycan preparation and four mAbs recognizing the core protein of a high-density, buoyant chondroitin sulfate proteoglycan were raised. Confirmation of antibody specificity was carried out by the preparation of affinity columns made from each of the mAbs. Chondroitin sulfate proteoglycans (CSPGs) were purified from both supernatant and tissue fractions of Reichert's membranes incubated in short-term organ culture in the presence of radiolabel. The resultant affinity-purified proteoglycan samples were examined by gel filtration, SDS-PAGE, and immunoblotting. This proteoglycan is of high molecular weight (Mr = 5-6 x 10(5)), with a core protein of Mr = approximately 1.5-1.6 x 10(5) and composed exclusively of chondroitin sulfate chains with an average Mr = 1.6-1.8 x 10(4). In addition, a CSPG was purified from adult rat kidney, whose core protein was also Mr = 1.6 x 10(5). The proteoglycan and its core protein were also recognized by all four mAbs. Indirect immunofluorescence of rat tissue sections stained with these antibodies reveal a widespread distribution of this proteoglycan, localized specifically to Reichert's membrane and nearly all basement membranes of rat tissues. In addition to heparan sulfate proteoglycans, it therefore appears that at least one CSPG is a widespread basement membrane component.     

Plasmodium falciparum: Assessment of parasite-infected red blood cell binding to placental chondroitin proteoglycan and bovine tracheal chondroitin sulfate A

In pregnant women infected with Plasmodium falciparum, the infected red blood cells (IRBCs) sequester in placenta by binding to the chondroitin 4-sulfate (C4S) chains of low sulfated chondroitin sulfate proteoglycan (CSPG). Placental CSPG, the natural receptor for IRBC adherence in the placenta, is the ideal molecule for studying structural interactions in IRBC adhesion to C4S, adhesion inhibitory antibody responses, and identification of parasite adhesive protein(s). However, because of difficulty involved in purifying placental CSPG, the commercially available bovine tracheal chondroitin sulfate A (bCSA), a copolymer having structural features of both C4S and C6S, has been widely used. To determine the validity of bCSA for C4S-IRBC interaction studies, we comparatively evaluated the characteristics of IRBC binding to placental CSPG and bCSA using three commonly used parasite strains. The results indicate that, in all three parasites studied, the characteristics of IRBC binding to placental CSPG and bCSA are qualitatively similar, but the binding capacity with respect to both the number of IRBCs bound per unit area of coated surface and binding strength is significantly higher for CSPG than bCSA regardless of whether parasites were selected on CSPG or bCSA. These results demonstrate that placental CSPG is best suited for studying interactions between parasite adhesive protein(s) and C4S, and have implications in understanding C4S-IRBC structural interactions.     

Structure and interactions of proteoglycans in the extracellular matrix produced by cultured human fibroblasts.

Subconfluent cultures of human embryonic skin fibroblasts were labelled with [35S]sulphate for 3 days, after which cell-free extracellular matrix was isolated. A chondroitin sulphate proteoglycan (CSPG) and a heparan sulphate proteoglycan (HSPG) were purified from the matrix. Chromatography on Sepharose CL-2B gave peak Kav. values of 0.35 and 0.38 respectively for the CSPG and the HSPG. The polysaccharide chains released from the two PGs were of similar size (Kav. 0.50 on Sepharose CL-4B). Approx. 50% of the CSPG showed affinity for hyaluronic acid (HA). However, it differed immunologically from the HA-aggregating CSPG of human articular cartilage, and had a larger core protein (apparent molecular mass 290 kDa) than had the cartilage PG. Neither metabolically [35S]sulphate-labelled PGs, isolated from the medium of fibroblast cultures, nor chemically 3H-labelled polysaccharides (HA, CS, HS and heparin) were incorporated into the extracellular matrix when added to unlabelled cell cultures. These results indicate that the matrix PGs are not derived from the PGs present in the medium and that an interation between polysaccharide chains and matrix components is not sufficient for incorporation of PGs into the matrix. Incubation of cell-free 35S-labelled matrix with unlabelled polysaccharides did not lead to the release of any 35S-labelled material, supporting this conclusion. Furthermore, so-called 'link proteins' were not present in the fibroblast cultures, indicating that the CSPGs were anchored in the matrix in a manner different from the link-stabilized association of CSPG with HA in chondrocyte matrix. The identification of a proteinase, secreted by fibroblasts in culture, that after activation with heparin has the ability to release 35S-labelled PGs from the matrix may also indicate that the core proteins are important for the association of the PGs to the matrix.     

Nanomelic chondrocytes synthesize a glycoprotein related to chondroitin sulfate proteoglycan core protein

Chicken embryos homozygous for the autosomal recessive gene nanomelia exhibit cartilage defects, synthesize low levels of cartilage chondroitin sulfate proteoglycan (CSPG), and are missing the CSPG core protein (Argraves, W. S., McKeown-Longo, P. J., and Goetinck, P. F. (1981) FEBS Lett. 131, 265). In our studies of nanomelic chondrocytes in culture, we detected neither sulfate-labeled CSPG nor its Mr 370,000 core protein. However, in immunoprecipitation reactions using both polyclonal and monoclonal antibodies directed against the cartilage CSPG core protein, we identified a protein of Mr 300,000 that contains an epitope found in the hyaluronic acid-binding region of the normal core protein. This protein was also detected among products synthesized by chondrocytes obtained from phenotypically normal embryos resulting from matings between parents heterozygous for nanomelia. Sensitivity to endoglycosidase H indicated that the product is a glycoprotein with attached mannose-rich oligosaccharides. Pulse-chase studies revealed the disappearance of the glycoprotein after 6 h of chase, but no detectable formation of proteoglycan. Our results suggest that although nanomelic chondrocytes are deficient in the production of normal CSPG and its core protein, they do synthesize a smaller, immunologically related glycoprotein that does not undergo the post-translational processing characteristic of the normal cartilage core protein.     

Chondroitin sulfate proteoglycans of bovine cornea: structural characterization and assessment for the adherence of Plasmodium falciparum-infected erythrocytes

The structures of the bovine corneal chondroitin sulfate (CS) chains and the nature of core proteins to which these chains are attached have not been studied in detail. In this study, we show that structurally diverse CS chains are present in bovine cornea and that they are mainly linked to decorin core protein. DEAE-Sephacel chromatography fractionated the corneal chondroitin sulfate proteoglycans (CSPGs) into three distinct fractions, CSPG-I, CSPG-II, and CSPG-III. These CSPGs markedly differ in their CS and dermatan sulfate (DS) contents, and in particular the CS structure-the overall sulfate content and 4- to 6-sulfate ratio. In general, the CS chains of the corneal CSPGs have low to moderate levels (15-64%) of sulfated disaccharides and 0-30% DS content. Structural analysis indicated that the DS disaccharide units in the CS chains are segregated as large blocks. We have also assessed the suitability of the corneal CSPGs as an alternative to placental CSPG or the widely used bovine tracheal chondroitin sulfate A (CSA) for studying the structural interactions involved in the adherence of Plasmodium falciparum-infected red blood cells (IRBCs) to chondroitin 4-sulfate. The data demonstrate that the corneal CSPGs efficiently bind IRBCs, and that the binding strength is either comparable or significantly higher than the placental CSPG. In contrast, the IRBC binding strength of bovine tracheal CSA is markedly lower than the human placental and bovine corneal CSPGs. Thus, our data demonstrate that the bovine corneal CSPG but not tracheal CSA is suitable for studying structural interactions involved in IRBC-C4S binding.     

Chondroitin sulfate proteoglycan expression during neuronal development.

Proteoglycans of developing chick brain were distinguished on the basis of reactivity with four well characterized antibody reagents (S103L, to the CS-rich domain; HNK-1, to 6-sulfated glucuronic acid; 1-C-3, to the HABr region and 5-D-4, to KS chains). One chondroitin sulfate proteoglycan reacted exclusively with S103L and 1-C-3 and not with the other two antibodies, hence is designated the S103L reactive brain CSPG. The other proteoglycan reacted exclusively with HNK-1 and 5-D-4 and not with S103L and 1-C-3, hence it is designated the HNK-1 reactive brain CSPG. In addition to these immunological distinctions, the S103L and HNK-1 CSPGs exhibited significant biochemical differences at both the protein and carbohydrate levels. Most interestingly, both CSPGs were found in all regions of the brain, and were expressed in a developmentally regulated pattern. The S103L CSPG was not detectable prior to embryonic day 7, increased to a maximum at day 13-15 and declined by day 20 in most brain regions examined. In contrast, the HNK-1 CSPG was present as early as embryonic day 4 and remained constant through hatching. Neuronal cultures established from embryonic day 6 (E6) cerebral hemispheres represent an in vitro paradigm that mimics in vivo neuronal development and differentiation. In this culture system we found that the expression of the S103L and HNK-1 CSPG followed a pattern similar to that observed in developing brain and further, that neurons are probably the sole source of S103L CSPG in cerebral cortex during neuroembryogenesis.     

Effect of beta-D-xyloside on the glomerular proteoglycans. I. Biochemical studies

The effect of p-nitrophenyl-beta-D-xylopyranoside on glomerular extracellular matrices (glomerular basement membrane and mesangial matrix) proteoglycans was studied. The proteoglycans of rat kidneys were labeled with [35S]sulfate in the presence or absence of beta- xyloside (2.5 mM) by using an isolated organ perfusion system. The proteoglycans from the glomeruli and perfusion medium were isolated and characterized by Sepharose CL-6B chromatography and by their behavior in CsCl density gradients. With xyloside treatment there was a twofold decrease in 35S-labeled macromolecules in the tissues but a twofold increase in those recovered in the medium as compared with the control. The labeled proteoglycans extracted from control kidneys eluted as a single peak with Kav = 0.25 (Mr = approximately 130,000), and approximately 95% of the radioactivity was associated with heparan sulfate proteoglycan (HS-PG), the remainder with chondroitin (or dermatan) sulfate proteoglycan (CS-PG). In the xyloside-treated kidneys, the proteoglycans extracted from the tissue eluted as two peaks, Kav = 0.25 (Mr = approximately 130,000) and 0.41 (Mr = approximately 46,000), which contained approximately 40 and approximately 60% of the total radioactivity, respectively. The first peak contained mostly the HS-PG (approximately 90%) while the second peak had a mixture of HS-PG (approximately 70%) and CS-PG (approximately 30%). In controls, approximately 90% of the radioactivity, mostly HS-PG, was confined to high density fractions of a CsCl density gradient. In contrast, in xyloside experiments, both HS- PG and CS-PG were distributed in variable proportions throughout the gradient. The incorporated 35S activity in the medium of xyloside- treated kidneys was twice that of the controls and had three to four times the amount of free chondroitin (or dermatan) sulfate glycosaminoglycan chains. The data suggest that beta-xyloside inhibits the addition of de novo synthesized glycosaminoglycan chains onto the core protein of proteoglycans and at the same time stimulates the synthesis of chondroitin or dermatan sulfate chains which are mainly discharged into the perfusion medium.     

Cell-associated proteoheparan sulfate from bovine arterial smooth muscle cells

Cell-associated proteoheparan sulfate has been isolated from bovine arterial smooth muscle cells preincubated with [35S]sulfate or a combination of [3H]glucosamine and [35S]methionine. The purified proteoheparan sulfate had an apparent Mr of 200,000 on calibrated Sepharose CL-2B columns. The glycosaminoglycan component (Mr approximately 30,000) was identified as heparan sulfate by its susceptibility to specific enzymatic and chemical degradation. After degradation of the proteoheparan sulfate by microbial heparitinase the resulting protein core had an apparent Mr of 92,000 on SDS-polyacrylamide gels. Its mobility was similar in the absence and presence of reducing agents indicating that the protein core consists of a single polypeptide chain. Pulse-chase experiments revealed that about 40% of the cell layer-associated proteoheparan sulfate was released into the medium, while the remainder was internalized and converted to smaller species through a series of degradation steps. Initially there was a proteolytical cleavage of the protein core generating glycosaminoglycan peptide intermediates with polysaccharides chains similar in size to the original. The half-life of the native proteoheparan sulfate was found to be about 4 h.     

Proteoglycans synthesized by cultured mouse osteoblasts

Proteoglycan synthesis in nonmineralizing osteoblast cultures was investigated. Cultures were labeled with [35S]sulfate or [3H]serine, and proteoglycans were extracted from medium and cell layer with 4 M guanidine HCl. Labeled material was subjected to Sepharose CL-4B and DEAE-Sephacel chromatography and polyacrylamide gel electrophoresis. The size and composition of the glycosaminoglycan chains and the protein core size were determined. Two proteoglycan populations were isolated by Sepharose CL-4B chromatography: a minor excluded species with chondroitin sulfate chains of apparent Mr 25,000 and a smaller population (Kav = 0.43) accounting for 80% of the total labeled material. This small population resolved into two species by polyacrylamide gel electrophoresis. Both species contain dermatan sulfate chains of apparent Mr 40,000 and a core protein with Mr 45,000 on sodium dodecyl sulfate gels. With the exception of their glycosaminoglycan composition these species appear similar to those extracted from bone. In addition, high molecular weight hyaluronic acid and glycosaminoglycan peptides were found in cell extracts.     

Cell density-dependent expression of chondroitin sulfate proteoglycan in cultured human monocytes

Monocytes were isolated and established in vitro at different cell densities. The incorporation of [35S]sulfate into macromolecules in monocytes (day 1 in culture) and monocyte-derived macrophages (day 5 in culture) was found to increase with decreasing cell density in approximately the same way in both day 1 and day 5 cell cultures. [35S]Sulfate was found to be incorporated almost exclusively into chondroitin sulfate proteoglycan (CSPG) in both high and low density monocyte and monocyte-derived macrophage cultures. The molecular size of the [35S]CSPGs produced by the high and low cell density cultures were not found to differ as judged by gel chromatography elution patterns. The molecular size and the structure of the glycosaminoglycan chains were found to be almost similar in high and low density day 1 and day 5 cultures. Only a small degree of proteoglycan degradation could be observed in both high and low density cultures. Furthermore, cell density-dependent differences in CSPG biosynthesis could be observed already 2 h after the establishment of the cultures, indicating that a process of "down-regulation" in high density cultures was already in operation. The glycosaminoglycan synthesis in high cell density day 1 cultures could be increased slightly following exposure to 0.5 mM benzyl-beta-D-xyloside, but not to the same level as that observed in untreated low cell density cultures. By contrast, the expression of 35S-macromolecules by cells cultured at high cell density for 5 days could be increased by xyloside treatment almost to the same level as that observed in the low density cultures.     

Chondroitin 6-sulfate oligosaccharides as immunological determinants of chick proteoglycans

Monoclonal antibodies to hyaluronidase-treated chondroitin sulfate proteoglycan (CSPG) were used to study the immunological determinants of chick cartilage proteoglycan. The determinants recognized by the antibodies were studied by a radioimmune inhibition assay utilizing hyaluronidase-treated [35S]CSPG. Hyaluronidase-treated CSPG inhibits the reaction of four clonal antibodies, S54C, S103L, S11D, and P100D, with [35S]CSPG, but to varying degrees. Only the reaction of S103L is inhibited to a considerable extent by undigested CSPG, indicating that hyaluronidase treatment exposes determinants specific for the other three antibodies. These findings are consistent with the earlier conclusion that S103L is specific for a protein determinant (Dorfman et al., 1980). Only the reaction of S54C is not significantly inhibited by chondroitinase ABC-digested CSPG. This result indicates that chondroitinase ABC digestion can also expose determinants recognized by S11D and P100D but that such digestion removes the determinant recognized by S54C. Of the four antibodies tested, only the reaction of S54C with hyaluronidase-treated [35S]CSPG is significantly inhibited by chondroitin-6-SO4 tetra- and hexasaccharide (59 and 43% inhibition, respectively, at a concentration of 1333 microM). The reaction of S54C is inhibited to a lesser extent by chondroitin tetra- and hexasaccharide (28 and 26% inhibition, respectively, at a concentration of 1333 microM). In contrast, chondroitin-4-SO4 oligosaccharides do not inhibit the reactions of any of the clonal antibodies. These result suggest that S54C recognizes a determinant that contains chondroitin-6-SO4 oligosaccharide, attached via the linkage oligosaccharide to core protein.     

Sulfated proteoglycans synthesized by Neuro 2a neuroblastoma cells: Comparison between cells with and without ganglioside-induced neurites

Mouse neuroblastoma Neuro 2a cells are known to extend neurite-like processes in response to gangliosides added to the culture medium. We compared the structural features of proteoglycans (PG) synthesized by conventional Neuro 2a cells with those of neurite-bearing cells. Two different proteoglycans labeled with [³⁵S]sulfate, namely, chondroitin sulfate proteoglycan (CS-PG) and heparan sulfate proteoglycan (HS-PG), were found both in the cell layer and in the culture medium of the conventional cells. CS-PG isolated from the cell layer had a K_av value of 0.38 on Sepharose CL-6B, and had CS side chains with Mr of 27,000. HS-PG in the cell layer was slightly larger (K_av of 0.33) in terms of hydrodynamic size than CS-PG, and the apparent Mr of the heparan sulfate side chains was 10,000. The structural parameters of CS-PG and HS-PG isolated from the medium were almost identical to those of the PGs in the cell layer. In addition to these PGs, single-chain HS, with an average Mr of 2,500, was observed only in the cell layer and this component was the major sulfated component in the cell layers of both control and ganglioside treated cells. The neurite-bearing cells also synthesized both CS-PG and HS-PG which were very similar in hydrodynamic size to those synthesized by the conventional cells, but the size of HS side chains was greater. Radioactivity, as³⁵S, of each sulfated component from the gangliosideteated culture seemed to be slightly less than that of the corresponding component from the control culture. These findings indicate that the marked morphological change in Neuro 2a cells, induced by gangliosides is not accompanied by major changes in the synthesis of PGs.