Showing papers on "Chondroitin sulfate published in 1986"

Binding of hyaluronate and chondroitin sulphate to liver endothelial cells

Abstract: Hyaluronate is taken up and metabolized in liver endothelial cells by means of a receptor. To characterize the interaction with the receptor, two preparations of 3H-labelled hyaluronate, of Mr 4 X 10(5) and 6.4 X 10(6), and a series of hyaluronate oligosaccharides were bound to cultured liver endothelial cells at 7 degrees C. The dissociation constant varied between 4.6 X 10(-6) M for an octasaccharide and 9 X 10(-12) M for the largest polymer. The Mr-dependence for the series of oligosaccharides was explained by the increased probability of binding due to the repetitive sequence along the chain. The high affinity of high-Mr hyaluronate for the receptor could also be mainly ascribed to this effect, which rules out any major contribution of co-operative multiple-site attachment to the cell surface. Each liver endothelial cell can bind 10(5) oligosaccharides, about 10(4) molecules with Mr 4 X 10(5) and about 10(3) molecules with Mr 6.4 X 10(6). This is explained by mutual exclusion of large molecules from the cell surface. Chondroitin sulphate is also bound to liver endothelial cells. Inhibition studies showed that it binds to the same receptor as hyaluronate and with an affinity that is about 3-fold higher than that of hyaluronate of the same degree of polymerization.

Intestinal mucosal mast cells from rats infected with Nippostrongylus brasiliensis contain protease-resistant chondroitin sulfate di-B proteoglycans.

Abstract: Rats infected with the helminth Nippostrongylus brasiliensis were injected i.p. with 2 mCi of [35S] sulfate on days 13, 15, 17, and 19 after infection. The intestines were removed from animals on day 20 or 21 after infection, the intestinal cells were obtained by collagenase treatment and mechanical dispersion of the tissue, and the 35S-labeled mucosal mast cells (MMC) were enriched to 60 to 65% purity by Percoll centrifugation. The cell-associated 35S-labeled proteoglycans were extracted from the MMC-enriched cell preparation by the addition of detergent and 4 M guanidine HCl and were partially purified by density gradient centrifugation. The isolated proteoglycans were of approximately 150,000 m.w., were resistant to pronase degradation, and contained highly sulfated chondroitin sulfate side chains. Analysis by high-performance liquid chromatography of chondroitinase ABC-treated 35S-labeled proteoglycans from these rat MMC revealed that the chondroitin sulfate chains consisted predominantly of disaccharides with the disulfated di-B structure (IdUA-2SO4----GalNAc-4SO4) and disaccharides with the monosulfated A structure (G1cUA----GalNAc-4SO4). The ratio of disaccharides of the di-B to A structure ranged from 0.4 to 1.6 in three experiments. Small amounts of chondroitin sulfate E disaccharides (GlcUA----GalNAc-4,6-diSO4) were also detected in the chondroitinase ABC digests of the purified rat MMC proteoglycans, but no nitrous acid-susceptible heparin/heparan sulfate glycosaminoglycans were detected. The presence in normal mammalian cells of chondroitin sulfate proteoglycans that contain such a high percentage of the unusual disulfated di-B disaccharide has not been previously reported. The rat intestinal MMC proteoglycans are the first chondroitin sulfate proteoglycans that have been isolated from an enriched population of normal mast cells. They are homologous to the chondroitin sulfate-rich proteoglycans of the transformed rat basophilic leukemia-1 cell and the cultured interleukin 3-dependent mouse bone marrow-derived mast cell, in that these chondroitin sulfate proteoglycans as well as rat serosal mast cell heparin proteoglycans are all highly sulfated, protease-resistant proteoglycans.

A single gene in mast cells encodes the core peptides of heparin and chondroitin sulfate proteoglycans.

Abstract: The diversity of the genes encoding mammalian proteoglycan peptide cores was explored using a cDNA clone that encodes the partial sequence of a cell surface/pericellular matrix-localized chondroitin sulfate proteoglycan. Thus we were able to detect the expression of the gene(s) encoding the intracellular chondroitin sulfate proteoglycan produced by a variety of rat and mouse mucosal-like mast cells and the intracellular heparin proteoglycan synthesized by rat serosal mast cells. The cDNA from the proteoglycan cDNA clone pPG-1 was fractionated into two discrete fragments, one of which contained the nucleotides encoding the serine-glycine repeat sequence (pPG-B) and the other of which contained sequences on the 3' side of the repeat (pPG-M). As assessed by Southern blot analysis, pPG-B identified a large gene family, whereas pPG-M identified a single DNA fragment in the rat genome. When the pPG-1 insert and the two subcloned probes pPG-B and pPG-M were used to analyze RNA extracted from the rat and mouse mucosal-like mast cells and the rat serosal mast cells, the same major RNA species was detected at 1.3 kilobases with both probes. These data suggest that the gene responsible for the peptide core of the extracellular chondroitin sulfate proteoglycan synthesized by the rat yolk sac cell line is also the gene that encodes the core peptides of the secretory granule-localized chondroitin sulfate and heparin proteoglycans.

Immunocytochemical investigation on the distribution of small chondroitin sulfate-dermatan sulfate proteoglycan in the human.

Abstract: Polyclonal antibodies against the core protein of the small chondroitin sulfate-dermatan sulfate proteoglycan from human skin fibroblast secretions were used, after affinity-purification, as a probe to study localization of crossreactive material in several human tissues by indirect immunocytochemistry. In contrast to skin, kidney, and the adventitial layer of aorta, positive staining of brain, liver, cartilage, and intimal and medial layers of aorta required pre-treatment of tissue sections with chondroitin ABC lyase. In all tissues investigated, antigenic material was present in the interstitial space. Filamentous structures were perpendicularly oriented towards basement membranes. In liver, specific staining was seen along the sinusoidal walls. Reticular fibers with or without focal condensations were seen in cerebral cortex and cerebellum. The results suggest a role of small chondroitin sulfate-dermatan sulfate proteoglycan in cell-matrix interactions.

Conditioning of native substrates by chondroitin sulfate proteoglycans during cardiac mesenchymal cell migration.

Abstract: It is generally proposed that embryonic mesenchymal cells use sulfated macromolecules during in situ migration Attempts to resolve the molecular mechanisms for this hypothesis using planar substrates have been met with limited success In the present study, we provide evidence that the functional significance of certain sulfated macromolecules during mesenchyme migration required the presence of the endogenous migratory template; ie, native collagen fibrils Using three-dimensional collagen gel lattices and whole embryo culture procedures to produce metabolically labeled sulfated macromolecules in embryonic chick cardiac tissue, we show that these molecules were primarily proteoglycan (PG) in nature and that their distribution was class specific; ie, heparan sulfate PG, the minor labeled component (15%), remained pericellular while chondroitin sulfate (CS) PG, the predominately labeled PG (85%), was associated with collagen fibrils as "trails" of 50-60-nm particles when viewed by scanning electron microscopy Progressive "conditioning" of collagen with CS-PG inhibited the capacity of the template to support subsequent cell migration Lastly, metabolically labeled, PG-derived CS chains were compared with respect to degree of sulfation in either the C-6 or C-4 position by chromatographic separation of chondroitinase AC digestion products Results from temporal and regional comparisons of in situ-labeled PGs indicated a positive correlation between the presence of mesenchyme and an enrichment of disaccharide-4S relative to that from regions lacking mesenchyme (ie, principally myocardial tissue) The suggestion of a mesenchyme-specific CS-PG was substantiated by similarly examining the PGs synthesized solely by cardiac mesenchymal cells migrating within hydrated collagen lattice in culture These data were incorporated into a model of "substratum conditioning" which provides a molecular mechanism by which secretion of mesenchyme-specific CS-PGs not only provides for directed and sustained cell movement, but ultimately inhibits migration of the cell population as a whole

Proteoglycan-collagen relationships in developing chick and bovine tendons. Influence of the physiological environment.

Abstract: Developing and mature flexor digitorum tendons of chick and bovine origin were analyzed for nucleic acid, hydroxyproline, hyaluronate, chondroitin sulfate and sulfated glycosaminoglycan hexuronate Collagen fibril diameters were determined by electron microscopy Results are compared with similar data from rat tail tendon Tissue hydroxyproline (mg/g) increased rapidly in early embryogenesis, while collagen fibril diameters remained relatively constant and small Hyaluronate and chondroitin sulfate were present in considerable quantities, (phase I) A rapid increase in fibril diameters in midterm ‘pregnancy’ coincided with a decrease in hyaluronate and chondroitin sulfate contents (phase II) The transition from phase Ito phase II was associated with the first loading by active muscles of the tendon, independently of whether the animal was pre- or post partum The probable existence of a developmental program intrinsic to tendon itself, is discussed The data are compared with predictions from three diffe

The melanoma proteoglycan: restricted expression on microspikes, a specific microdomain of the cell surface.

Abstract: A cell surface chondroitin sulfate proteoglycan associated with human melanomas and defined by mAb's F24.47 and 48.7 has been characterized biochemically and localized by indirect immunogold electron microscopy. These antibodies recognize distinct epitopes on the intact proteoglycan. In addition, mAb 48.7 also recognizes an epitope on a 250,000-D glycoprotein and is therefore similar to antibody 9.2.27 (described by Bumol, T.F., and R.A. Reisfeld, 1982, Proc. Natl. Acad. Sci. USA., 79:1245-1249). Furthermore, it was shown that the glycosaminoglycan chains released by alkaline borohydride treatment of the proteoglycan recognized by mAb 48.7 had a size of approximately 60,000 D. Since the intact proteoglycan was estimated to be 420,000 D, there are probably three chondroitin sulfate chains attached to the 250,000-D core glycoprotein. Furthermore, an oligosaccharide fraction containing 42% of the 3H activity (glucosamine as precursor) was isolated. Immunolocalization studies using whole-mount electron microscopy revealed that the chondroitin sulfate proteoglycan was present almost exclusively on microspikes, a microdomain of the melanoma cell surface. These processes were present as 1-2-micron structures on the upper cell surface and as longer (up to 20 micron) structures at the cell periphery. Peripheral microspikes were involved in the initial interactions between adjacent cells and formed complex footpads that made contact with the substratum. Immunogold-labeled cells were also thin sectioned and the specific localization of the chondroitin sulfate proteoglycan antigen was quantitated. The data confirmed the results of whole-mount microscopy and demonstrated a statistically significant association of the antigen with the microspike processes as compared with other areas of the cell surface. By using two different mAb's (48.7 and F24.47) that recognize epitopes on either the core glycoprotein or the intact proteoglycan, respectively, we have demonstrated that both molecules have the same restricted distribution at the cell surface. The specific localization of the antigen to microspikes at the cell surface suggests it may play a role in cell-cell contact and cell-substratum adhesion, which could be important in the metastatic process.

The effect of chronic inflammation on gingival connective tissue proteoglycans and hyaluronic acid

Abstract: Proteoglycans have been isolated and analysed from extracts of normal and chronically inflamed human gingiva in order to determine the effects of chronic inflammation on these important soft connective tissue extracellular macromolecules. The uronic acid content of glycosaminoglycans isolated by papain digestion of normal and inflamed gingiva did not differ significantly. Likewise, electrophoretic analysis revealed that the content of hyaluronic acid, heparan sulfate, dermatan sulfate and chondroitin sulfate was similar. The sulfated glycosaminoglycans from both sources eluted from a Sepharose C1-6B column with a Kav of 0.45 (approximate Mr 25,000). However, hyaluronic acid from normal gingiva was predominantly of a large size eluting in the void volume of a Sepharose. CL-6B column, while that isolated form inflamed tissue was mostly a small molecular weight species which eluted in the included volume of a Sepharose CL-6B column. Using dissociative conditions, intact proteoglycans could be more readily extracted from inflamed tissues (90% of the total tissue uronic acid) than from normal tissues where only 80% of the total tissue uronic acid was extractable. Even though DEAE-Sephacel ion-exchange chromatography revealed no differences in charge between normal and inflamed gingival proteoglycans, Sepharose CL-4B chromatography revealed more molecular size polydispersity in samples from inflamed tissue than from normal tissue. Taken together, these results indicate that while hyaluronic acid is depolymerized in inflamed tissue, no evidence of sulfated glycosaminoglycan degradation was found. Therefore, the most likely cause for disruption to the molecular integrity of the proteoglycans is via proteolytic alteration to the proteoglycan core protein.

Immunocytochemical localization of chondroitin sulfates in the interphotoreceptor matrix of the normal and dystrophic rat retina.

Abstract: The interphotoreceptor matrix (IPM) is a mixture of proteoglycans and glycoproteins through which metabolites must pass in transit between the retinal pigment epithelium (RPE) and the photoreceptor cells. In order to localize various species of chondroitin sulfates in the IPM of the normal and dystrophic rat retina, we have used monoclonal antibodies directed against 6-sulfated chondroitin sulfate (6S), 4-sulfated chondroitin sulfate (4S) and unsulfated chondroitin (OS). Immunofluorescence and immunoperoxidase methods were carried out on frozen and wax-embedded sections of rat eyes. In the normal rat retina, strong labeling with the 6S antibody was observed in the basal IS/OS zone and, to a lesser extent, in the photoreceptor interstices extending to the apical RPE surface. In the RCS retina, the IPM in the basal IS/OS zone and much of the outer segment debris zone were labeled intensely with 6S antibody, but no strong labeling was present at the apical RPE surface. The labeling pattern with OS antibody w...

A proposed structure of chondroitin 6-sulfate proteoglycan of human normal and adjacent atherosclerotic plaque.

Abstract: Chondroitin sulfate proteoglycan monomers were prepared from intima media minces of grossly normal human aorta and adjacent fatty fibrous atherosclerotic plaques. Glycosaminoglycan chains prepared from monomer from normal aorta displayed a normal distribution profile on Ultrogel ACA 54 with a Kav of 0.48, whereas those of atherosclerotic aorta displayed a bimodal distribution (major peak, Kav 0.35; minor peak, Kav 0.70). The Mr of glycosaminoglycans from normal aorta was estimated to be 1.5 X 10(4). For atherosclerotic aorta, the majority of chains were 2.0 X 10(4) while the smaller population was 1.2 X 10(4). All glycosaminoglycans were identified as chondroitin sulfate sulfated at the C-6 position. The amino acid compositions of both core proteins were similar with Mr of about 1.6 X 10(5). After beta-elimination in the presence of sodium borohydride prior to acid hydrolysis, chondroitin sulfate proteoglycan from normal aorta had reductions in serine from 109 to 68 and in threonine from 117 to 55. For the monomer from atherosclerotic plaque, reductions in serine and threonine, respectively, were from 103 to 81 and from 107 to 77 residues per 1000. The results suggested fewer chondroitin sulfate chains and oligosaccharides on the core protein in the proteoglycan of atherosclerotic plaque. Compared to normal aorta, substituted serines and threonines in the proteoglycan of atherosclerotic plaque were about half, respectively, 38% vs 21% for serine, 53% vs 28% for threonine. It is estimated that in atherosclerotic plaque there are fewer, but longer, chondroitin sulfate chains per core protein, translating into a smaller overall monomer size in atherosclerotic plaque.

Effects of heparan sulfate removal on attachment and reattachment of fibroblasts and endothelial cells

Abstract: Human skin fibroblasts and calf aorta endothelial cells were grown as tissue culture monolayers in the presence of [35S]sulfate in order to label the glycosaminoglycan portions of proteoglycans for investigation of their role in cell attachment. The [35S]glycosaminoglycans were then selectively removed from the cell monolayers by the addition of various glycosaminoglycan-degrading enzymes. As previously described, in contrast to trypsin treatment none of these enzymes removed any cells from the culture plates. Incubation with a preparation from Flavobacterium heparinum left only small stubs of [35S]glycosaminoglycans on the cell monolayers, indicating that all the cell-surface proteoheparan [35S]sulfate and proteochondroitin [35S]sulfate was accessible to this enzyme preparation. The treatment did not change the amount or time of incubation with trypsin necessary for release of the cells from the monolayers. Thus, cell attachment was not weakened by removal of heparan sulfate or chondroitin sulfate. In contrast, neither fibroblasts nor endothelial cells in suspension would reattach in the presence of the F. heparinum preparation while reattachment occurred readily in the presence of chondroitin ABC lyase. This provides evidence that heparan sulfate, but not chondroitin sulfate, is involved in the process of cell attachment even though neither is necessary for maintaining attachment.

Histamine and chondroitin sulfate E proteoglycan released by cultured human colonic mucosa: indication for possible presence of E mast cells.

Abstract: An association between the release of histamine and chondroitin sulfate E proteoglycan (PG) was demonstrated in human colonic mucosa (HCM). Colonic biopsy samples incorporated [35S]sulfate (2.7 X 10(6) +/- 188 X 10(3) cpm/mg of wet tissue; mean +/- SEM, n = 5) into PG, which was partially released into the culture medium during the incubation period. Ascending thin-layer chromatography of the released 35S-labeled PG after its digestion by chondroitin ABC lyase (chondroitinase, EC 4.2.2.4) followed by autoradiography yielded three products that migrated in the position of monosulfated disaccharides of N-acetylgalactosamine 4-sulfate and N-acetylgalactosamine 6-sulfate and of an oversulfated disaccharide possessing N-acetylgalactosamine 4,6-disulfate. Cultured colonic mucosa released 23.6 +/- 3.7 ng of histamine per mg of wet tissue (mean +/- SEM, n = 16) without any specific trigger. Comparison by linear regression analysis of the release of histamine and chondroitin [35S]sulfate E PG revealed a correlation coefficient (r) of 0.7 (n = 16; P less than 0.005). Histological examination of the colonic biopsies revealed the presence of many mast cells in various degrees of degranulation in the mucosa and submucosa, most of which were found in the submucosa. Incubation of the HCM biopsies in the presence of anti-human IgE revealed 58% +/- 12% (mean +/- SEM, n = 3) enhancement in the release of chondroitin [35S]sulfate E PG and 64% +/- 10% (mean +/- SEM, n = 4) of histamine release. The above correlation, the observation that most of the mast cells showed various degrees of degranulation, and the lack of heparin synthesis as opposed to the synthesis and immunological release of chondroitin sulfate E strongly suggest that the E mast cell exists in the human colon.

Distribution of hyaluronic acid and sulfated glycosaminoglycans during blood-vessel development in the chick chorioallantoic membrane.

Abstract: This study uses histochemical methods to determine the ultrastructural distribution of specific glycosaminoglycans (GAGs) during the development of blood vessels in the chick chorioallantoic membrane (CAM) and to correlate changes in GAG composition with the significant structural events in the development of these vessels. Tissues were stained with tannic acid, ruthenium red, and high iron diamine and digested in various GAG-degrading enzymes to identify specific GAGs. The results are consistent with a role for hyaluronic acid in the formation, alignment, or migration of the capillary plexus of the CAM and a role for sulfated GAGs (heparan sulfate, chondroitin sulfate, dermatan sulfate) in the differentiation and development of arterial and venous vessels of the chorioallantoic membrane.

Macular corneal dystrophy. Lack of keratan sulfate in serum and cornea.

Abstract: An ELISA assay using a monoclonal antibody (ET-4-A-4) that recognizes a sulfated carbohydrate epitope in both keratan sulfate type I (corneal) and type II (skeletal) was employed to quantify keratan sulfate in serum and corneal tissue from patients with macular corneal dystrophy (MCD). This assay disclosed significant quantities of keratan sulfate in the serum in 45 healthy individuals (251 +/- 78 ng/ml), and in 66 patients with various corneal diseases (273 +/- 101 ng/ml). In contrast keratan sulfate was not detected (less than 2 ng/ml) in the serum of 16 patients with histopathologically confirmed MCD. Keratan sulfate was also detected in extracts of normal corneas and corneal tissue with a variety of pathologic conditions, but was virtually absent in corneal tissue from five patients with MCD. In corneas with MCD the chondroitin sulfate/keratan sulfate ratio was considerably higher than that of all normal and pathologic corneas studied. Since keratan sulfate in the serum appears to be derived predominantly from the normal turnover of cartilage these studies strongly suggest that the defect in keratan sulfate synthesis in MCD is not restricted to corneal cells and that MCD is one manifestation of a systemic disorder of keratan sulfate. The cartilage changes, however, do not have clinical significance. Moreover, since keratan sulfate can be detected in the blood of newborns it should be possible to diagnose MCD prior to corneal opacification.