Ovarian carcinoma cells synthesize both chondroitin sulfate and heparan sulfate cell surface proteoglycans that mediate cell adhesion to interstitial matrix

Metastatic ovarian carcinoma metastasizes by intra-peritoneal, non-hematogenous dissemination. The adhesion of the ovarian carcinoma cells to extracellular matrix components, such as types I and III collagen and cellular fibronectin, is essential for intra-peritoneal dissemination. The purpose of this study was to determine whether cell surface proteoglycans (a class of matrix receptors) are produced by ovarian carcinoma cells, and whether these proteoglycans have a role in the adhesion of ovarian carcinoma cells to types I and III collagen and fibronectin. Proteoglycans were metabolically labeled for biochemical studies. Both phosphatidylinositol-anchored and integral membrane-type cell surface proteoglycans were found to be present on the SK-OV-3 and NIH:OVCAR-3 cell lines. Three proteoglycan populations of differing hydrodynamic size were detected in both SK-OV-3 and NIH:OVCAR-3 cells. Digestions with heparitinase and chondroitinase ABC showed that cell surface proteoglycans of SK-OV-3 cells had higher proportion of chondroitin sulfate proteoglycans (75:25 of chondroitin sulfate:heparan sulfate ratio), while NIH:OVCAR-3 cells had higher proportion of heparan sulfate proteoglycans (10:90 of chondroitin sulfate:heparan sulfate ratio). RT-PCR indicated the synthesis of a unique assortment of syndecans, glypicans, and CD44 by the two cell lines. In adhesion assays performed on matrix-coated titer plates both cell lines adhered to types I and III collagen and cellular fibronectin, and cell adhesion was inhibited by preincubation of the matrix with heparin, heparan sulfate, chondroitin sulfate, dermatan sulfate, or chondroitin glycosaminoglycans. Treatment of the cells with heparitinase, chondroitinase ABC, or methylumbelliferyl xyloside also interfered with adhesion confirming the role of both heparan sulfate and chondroitin sulfate cell surface proteoglycans as matrix receptors on ovarian carcinoma cells.     

Proteoglycan and glycosaminoglycan synthesis by cultured rat mesangial cells

The synthesis of metabolically labeled proteoglycans and glycosaminoglycans from medium, cell layer and substrate attached material by rat glomerular mesangial cells in culture was characterized. The cellular localization of the labeled proteoglycans and glycosaminoglycans was determined by treating the cells with Flavobacterial heparinase. Of the total sulfated glycosaminoglycans, 33% were heparan sulfate; 55% of the cell layer material was heparan sulfate; 80% of sulfated proteins in the medium were chondroitin sulfate/dermatan sulfate. Putative glycosaminoglycan free chains of heparan sulfate and chondroitin sulfate were found in both the medium and cell layer; 95% of total proteoglycans and most (90%) of the putative heparan sulfate free chains were removed from the cell layer by the heparinase, whereas only 50% of the chondroitin sulfate and 25% of dermatan sulfate were removed. Large amounts of hyaluronic acid labeled with 3H glucosamine were found in the cell layer. In summary, approximately 60% of total sulfated glycoproteins was in the form of putative glycosaminoglycan free chains. Thus rat mesangial cells may synthesize large amounts of putative glycosaminoglycan free chains, which may have biological functions in the glomerulus independent of proteoglycans.     

Major chondroitin sulfate proteoglycans identified in L6J1 myoblast culture

The major proteoglycans from L6J1 rat myoblast culture were identified. The proteoglycans were isolated from different constituents of cell culture: culture medium, extracellular matrix (ECM), and myoblasts. To identify their core proteins, the proteoglycans were treated with enzymes specifically digesting chondroitin/dermatan sulfates or chondroitin sulfates. Subsequent electrophoresis and mass spectrometry revealed versican, collagen XII, and inter-α-trypsin inhibitor classified as chondroitin sulfate proteoglycans and biglycan known to be chondroitin/dermatan sulfate proteoglycan. Versican was identified in ECM and the other proteoglycans in the culture medium. Such difference in localization is likely to be a consequence of different biological functions. Versican, collagen XII, and biglycan are synthesized by myoblasts and inter-α-trypsin inhibitor originates from fetal bovine serum (a culture medium component).     

Isolation and characterization of proteoglycans secreted by normal and malignant human mammary epithelial cells

The proteoglycans secreted by a malignant human breast cell line (MDA-MB-231) were compared with the corresponding proteoglycans from a normal human breast cell line (HBL-100). The physicochemical characteristics of these proteoglycans were established by hexosamine analysis, chemical and enzymatic degradations, and dissociative cesium chloride density gradient centrifugation, and by gel filtration before and after alkaline beta-elimination. Both cell lines secreted approximately 70% of the synthesized proteoglycans, which were composed of 20% heparan sulfate and 80% chondroitin sulfate proteoglycans. The MDA cell line secreted large hydrodynamic size (major) and small hydrodynamic size heparan sulfate proteoglycan. In contrast HBL cells secreted only one species having a hydrodynamic size intermediate to the above two. The chondroitin sulfate proteoglycans from MDA medium were slightly larger than the corresponding polymers from HBL medium. All proteoglycans except the small hydrodynamic size heparan sulfate proteoglycan from MDA medium were of high buoyant density. The proteoglycans of both cell lines contained significant proportions of disulfide-linked lower molecular weight components which were more pronounced in the proteoheparan sulfate polymers, particularly those from MDA medium, than in chondroitin sulfate proteoglycans. The glycosaminoglycans of heparan sulfate proteoglycans from MDA medium were more heterogeneous than those from HBL medium. The glycosaminoglycan chains of large hydrodynamic size heparan sulfate proteoglycans from MDA medium were larger in size than those from HBL medium while small hydrodynamic size heparan sulfate proteoglycans contained shorter glycosaminoglycan chains. In contrast to the glycosaminoglycans derived from chondroitin sulfate proteoglycans of both MDA and HBL medium were comparable in size. The heparan sulfate as well as chondroitin sulfate proteoglycans of both cell lines contained both neutral (di- and tetrasaccharides) and sialylated (tri- to hexasaccharides) O-linked oligosaccharides.     

Chondroitin sulfate and heparan sulfate-containing proteoglycans are both partners and targets of basic fibroblast growth factor-mediated proliferation in human metastatic melanoma cell lines

Basic fibroblast growth factor (FGF-2) and its respective tyrosine kinase receptors, form an autocrine loop that affects human melanoma growth and metastasis. The aim of the present study was to examine the possible participation of various glycosaminoglycans, i.e. chondroitin sulfate, dermatan sulfate and heparin on basal and FGF-2-induced growth of WM9 and M5 human metastatic melanoma cells. Exogenous glycosaminoglycans mildly inhibited WM9 cell's proliferation, which was abolished by FGF-2. Treatment with the specific inhibitor of the glycosaminoglycan sulfation, sodium chlorate, demonstrated that endogenous glycosaminoglycan/proteoglycan production is required for both basal and stimulated by FGF-2 proliferation of these cells. Heparin capably restored their growth, and unexpectedly exogenous chondroitin sulfate to WM9 and both chondroitin sulfate and dermatan sulfate to M5 cells allowed FGF-2 mitogenic stimulation. Furthermore, in WM9 cells the degradation of membrane-bound chondroitin/dermatan sulfate stimulates basal growth and even enhances FGF-2 stimulation. The specific tyrosine kinase inhibitor, genistein completely blocked the effects of FGF-2 and glycosaminoglycans on melanoma proliferation whereas the use of the neutralizing antibody for FGF-2 showed that the mitogenic effect of chondroitin sulfate involves the interaction of FGF-2 with its receptors. Both the amounts of chondroitin/dermatan/heparan sulfate and their sulfation levels differed between the cell lines and were distinctly modulated by FGF-2. In this study, we show that chondroitin/dermatan sulfate-containing proteoglycans, likely in cooperation with heparan sulfate, participate in metastatic melanoma cell FGF-2-induced mitogenic response, which represents a novel finding and establishes the central role of sulfated glycosaminoglycans on melanoma growth.     

Isolation and characterization of proteoglycans synthesized by rat myoblasts L6J1 in culture

Proteoglycans synthesized by rat myoblasts L6J1 in culture were isolated using sorbent Q-Sepharose from culture medium, extracellular matrix (ECM), and cells. Elution of the sorbed material in a NaCl gradient separated proteoglycans from the bulk of proteins eluted at low concentration of the salt. Four fractions (fractions I-IV) were obtained for each component of the cell culture, including two proteoglycan fractions for the ECM and culture medium and one fraction for the myoblasts. Proteoglycans of the culture medium were virtually completely represented by proteoglycans of fetal calf serum. With enzymes chondroitinase ABC and heparinase III chondroitin/dermatan sulfate proteoglycans were shown to prevail in all components of the myoblast culture. The core proteins of proteoglycans were characterized by electrophoresis.     

Altered Glycosaminoglycan Chain Structure in a Variant of the C2 Mouse Muscle Cell Line

Abstract: Experiments on the S27 cell line, a variant of the C2 mouse muscle cell line that shows reduced incorporation of ³⁵SO₄ into proteoglycans, suggest that proteoglycans play a role in the clustering of acetylcholine receptors, an early step in synaptogenesis. Thus, unlike the C2 line, S27 myotubes do not form acetylcholine receptor clusters on their surface in aneural cultures and form few clusters in response to agrin. We have examined the proteoglycans synthesized by S27 myotubes to define further the biochemical defect in these cells. Gel filtration analysis of radiolabeled proteoglycans synthesized by C2 and S27 myotubes shows that both cell types express a similarly polydisperse complement of proteoglycans. Both radiolabeled heparan sulfate proteoglycans and chondroitin/dermatan sulfate proteoglycans are reduced in S27 myotubes, with the chondroitin/dermatan sulfate proteoglycans showing a distinct reduction in size. The core protein of perlecan, a major proteoglycan species in muscle, was present in S27 cells and unaltered in electrophoretic mobility. Thus a principal deficiency in S27 cells appears to be a defect in glycosaminoglycan chain elongation.     

Chondroitin sulfate and heparan sulfate proteoglycans of PC12 pheochromocytoma cells

We have isolated and characterized the cell-associated and secreted proteoglycans synthesized by a clonal line of rat adrenal medullary PC12 pheochromocytoma cells, which have been extensively employed for the study of a wide variety of neurobiological processes. Chondroitin sulfate accounts for 70-80% of the [35S] sulfate-labeled proteoglycans present in PC12 cells and secreted into the medium. Two major chondroitin sulfate proteoglycans were detected with molecular sizes of 45,000-100,000 and 120,000-190,000, comprising 14- and 105-kDa core proteins and one or two chondroitin sulfate chains with an average molecular size of 34 kDa. In contrast to the chondroitin sulfate proteoglycans, one major heparan sulfate proteoglycan accounts for most of the remaining 20-30% of the [35S] sulfate-labeled proteoglycans present in the PC12 cells and medium. It has a molecular size of 95,000-170,000, comprising a 65-kDa core protein and two to six 16-kDa heparan sulfate chains. Both the chondroitin sulfate and heparan sulfate proteoglycans also contain O-glycosidically linked oligosaccharides (25-28% of the total oligosaccharides) and predominantly tri- and tetraantennary N-glycosidic oligosaccharides. Proteoglycans produced by the original clone of PC12 cells were compared with those of two other PC12 cell lines (B2 and F3) that differ from the original clone in morphology, adhesive properties, and response to nerve growth factor. Although the F3 cells (a mutant line derived from B2 and reported to lack a cell surface heparan sulfate proteoglycan) do not contain a large molecular size heparan sulfate proteoglycan species, there was no significant difference between the B2 and F3 cells in the percentage of total heparan sulfate released by mild trypsinization, and both the B2 and F3 cells synthesized cell-associated and secreted chondroitin sulfate and heparan sulfate proteoglycans having properties very similar to those of the original PC12 cell line but with a reversed ratio (35:65) of chondroitin sulfate to heparan sulfate.     

Binding and internalization of exogenous glycosaminoglycans in weakly and highly metastatic rhabdomyosarcoma cells

The fate of exogenous glycosaminoglycans in cultures of strongly (RMS 0) and weakly (RMS 8) metastatic rat rhabdomyosarcoma cells was studied. The time course and concentration dependence of binding and internalization of the radiolabeled sulfated glycosaminoglycans were determined. Weakly metastatic cells took up heparin, heparan and dermatan sulfates into their pericellular compartment at a higher rate than the strongly metastatic RMS 0 cells. The RMS 8 cells exhibited about two times more binding sites for these iduronic acid containing glycosaminoglycans, and internalized higher amounts of them than the RMS 0 cells. The uptake of the chondroitin sulfate into the peri- and intracellular compartments of both cell types was about 5-15% of that of the other glycosaminoglycans studied. The specificity of displacement of the pericellular heparin and dermatan sulfate by the unlabeled glycosaminoglycans indicates the involvement of specific structural features of the polysaccharide chains in the interactions of glycosaminoglycans with the surface of rhabdomyosarcoma cells, beside ionic forces due to the polyanionic character of the glycosaminoglycans. Heparin and heparan sulfate degradation products, mainly large oligosaccharides, were recovered from the surface of RMS 0 cells but were absent on the surface of the RMS 8 cells. About 30% of the internalized heparin and heparan sulfate was present in the partially degraded form in both cell types. Oligosaccharides derived from glycosaminoglycans were not released into the medium. The decrease in the amount of iduronic acid containing glycosaminoglycans internalized by the highly invasive cells seems to be correlated with an increased cell-associated degradation and with an apparent loss of glycosaminoglycan binding sites on the cell surface.     

Isolation and characterization of proteoglycans synthesized by adult human gingival fibroblasts in vitro

The proteoglycans synthesized by fibroblasts derived from healthy human gingivae were isolated and characterized. The largest medium proteoglycan was excluded from Sepharose CL-4B but not from Sepharose CL-2B; it was recovered in the most-dense density gradient fraction and identified as a chondroitin sulfate proteoglycan. The medium contained two smaller proteoglycans; one contained predominantly chondroitin sulfate proteoglycan, while the other was comprised predominantly of dermatan sulfate proteoglycan and was quantitatively the major species. The largest proteoglycan in the cell layer fraction, excluded from both Sepharose CL-2B and Sepharose CL-4B, was found in the least-dense density gradient fraction and contained heparan sulfate and chondroitin sulfate proteoglycan. It could be further dissociated by treatment with detergent, suggesting an intimate association with cell membranes. Two other proteoglycan populations of intermediate size were identified in the cell layer extracts which contained variable proportions of heparan sulfate, dermatan sulfate, or chondroitin sulfate proteoglycan. Some small molecular weight material indicative of free glycosaminoglycan chains was also associated with the cell layer fraction. Carbohydrate analysis of the proteoglycans demonstrated the glycosaminoglycan chains to have approximate average molecular weights of 25,000. In addition, N- and O-linked oligosaccharides which were associated with the proteoglycans appeared to be sulfated in varying degrees.     

Stimulation of human arterial smooth muscle cell chondroitin sulfate proteoglycan synthesis by transforming growth factor-beta

Summary Human platelet-derived transforming growth factor-beta (TGF-beta) is a cell-type specific promotor of proteoglycan synthesis in human adult arterial cells. Cultured human adult arterial smooth muscle cells synthesized chondroitin sulfate, dermatan sulfate, and heparan sulfate proteoglycans, and the percent composition of these three proteoglycan subclasses varied to some extent from cell strain to cell strain. However, TGF-beta consistently stimulated the synthesis of chondroitin sulfate proteoglycan. Both chondroitin 4- and chondroitin 6-sulfate were stimulated by TGF-beta to the same extent. TGF-beta had no stimulatory effect on either class of [³⁵S]sulfate-labeled proteoglycans which appeared in an approximately 1:1 and 2:1 ratio of heparan sulfate to dermatan sulfate of the medium and cell layers, respectively, of arterial endothelial cells. Human adult arterial endothelial cells synthesized little or no chondroitin sulfate proteoglycan. Pulse-chase labeling revealed that the appearance of smooth muscle cell proteoglycans into the medium over a 36-h period equaled the disappearance of labeled proteoglycans from the cell layer, independent of TGF-beta. Inhibitors of RNA synthesis blocked TGF-beta-stimulated proteoglycan synthesis in the smooth muscle cells. The incorporation of [³⁵S]methionine into chondroitin sulfate proteoglycan core proteins was stimulated by TGF-beta. Taken together, the results presented indicate that TGF-beta stimulates chondroitin sulfate proteoglycan synthesis in human adult arterial smooth muscle cells by promoting the core protein synthesis. Supported in part by grants from the Public Health Service, U.S. Department of Health and Human Services, Washington, DC (CA 37589 and HL 33842), RJR Nabisco, Inc., and Chang Gung Biomedical Research Foundation (CMRP 291).     

Glycosaminoglycans differentially bind HARP and modulate its biological activity

Heparin affin regulatory peptide (HARP) is a polypeptide belonging to a family of heparin binding growth/differentiation factors. The high affinity of HARP for heparin suggests that this secreted polypeptide should also bind to heparan sulfate proteoglycans derived from cell surface and extracellular matrix defined as extracellular compartments. Using Western blot analysis, we detected HARP bound to heparan sulfate proteoglycans in the extracellular compartments of MDA-MB 231 and MC 3T3-E1 as well as NIH3T3 cells overexpressing HARP protein. Heparitinase treatment of BEL cells inhibited HARP-induced cell proliferation, and the biological activity of HARP in this system was restored by the addition of heparin. We report that heparan sulfate, dermatan sulfate, and to a lesser extent, chondroitin sulfate A, displaced HARP bound to the extracellular compartment. Binding analyses with a biosensor showed that HARP bound heparin with fast association and dissociation kinetics (kass = 1.6 x 10(6) M-1 s-1; kdiss = 0.02 s-1), yielding a Kd value of 13 nM; the interaction between HARP and dermatan sulfate was characterized by slower association kinetics (kass = 0.68 x 10(6) M-1 s-1) and a lower affinity (Kd = 51 nM). Exogenous heparin, heparan sulfate, and dermatan sulfate potentiated the growth-stimulatory activity of HARP, suggesting that corresponding proteoglycans could be involved in the regulation of the mitogenic activity of HARP.     

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

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

Proteoglycan synthesis by normal and neoplastic human transitional epithelial cells

Metabolically 35S-labeled proteoglycans were isolated from cell-associated matrices and media of confluent cultures of human normal transitional epithelial cells and HCV-29T transitional carcinoma cells. On Sepharose CL-4B columns, the cell-associated proteoglycans synthesized from both cell types separated into three identical size classes, termed CI, CII, and CIII. Normal epithelial cell C-fractions eluted in a 22:34:45 proportion and contained 64%, 64%, and 72% heparan sulfate, whereas corresponding HCV-29T fractions eluted in a 29:11:60 proportion, and contained 91%, 77%, and 70% heparan sulfate, respectively. Medium proteoglycans from normal cells separated into two size classes in a proportion of 6:94 and were composed of 35% and 50% heparan sulfate. HCV-29T medium contained only one size class of proteoglycans consisting of 23% heparan sulfate. The remaining percentages were accounted for by chondroitin/dermatan sulfate. On isopycnic CsCl gradients, proteoglycan fractions from normal cells had buoyant densities that were higher than the corresponding fractions from HCV-29T cells. DEAE-Sephacel chromatography showed that cell and medium associated heparan sulfate from HCV-29T cells was consistently of lower charge density (undersulfated) than that from normal epithelial cells. In contrast, the chondroitin/dermatan sulfate of HCV-29T was of a charge density similar to that of normal cells. These as well as other structural and compositional differences in the proteoglycan may account, at least in part, for the altered behavioral traits of highly invasive carcinoma cells.     

Structure and properties of an under-sulfated heparan sulfate proteoglycan synthesized by a rat hepatoma cell line

A rat hepatoma cell line was shown to synthesize heparan sulfate and chondroitin sulfate proteoglycans. Unlike cultured hepatocytes, the hepatoma cells did not deposit these proteoglycans into an extracellular matrix, and most of the newly synthesized heparan sulfate proteoglycans were secreted into the culture medium. Heparan sulfate proteoglycans were also found associated with the cell surface. These proteoglycans could be solubilized by mild trypsin or detergent treatment of the cells but could not be displaced from the cells by incubation with heparin. The detergent-solubilized heparan sulfate proteoglycan had a hydrophobic segment that enabled it to bind to octyl- Sepharose. This segment could conceivably anchor the molecule in the lipid interior of the plasma membrane. The size of the hepatoma heparan sulfate proteoglycans was similar to that of proteoglycans isolated from rat liver microsomes or from primary cultures of rat hepatocytes. Ion-exchange chromatography on DEAE-Sephacel indicated that the hepatoma heparan sulfate proteoglycans had a lower average charge density than the rat liver heparan sulfate proteoglycans. The lower charge density of the hepatoma heparan sulfate can be largely attributed to a reduced number of N-sulfated glucosamine units in the polysaccharide chain compared with that of rat liver heparan sulfate. Hepatoma heparan sulfate proteoglycans purified from the culture medium had a considerably lower affinity for fibronectin-Sepharose compared with that of rat liver heparan sulfate proteoglycans. Furthermore, the hepatoma proteoglycan did not bind to the neoplastic cells, whereas heparan sulfate from normal rat liver bound to the hepatoma cells in a time-dependent reaction. The possible consequences of the reduced sulfation of the heparan sulfate proteoglycan produced by the hepatoma cells are discussed in terms of the postulated roles of heparan sulfate in the regulation of cell growth and extracellular matrix formation.     

Cathepsin X binds to cell surface heparan sulfate proteoglycans

Glycosaminoglycans have been shown to be important regulators of activity of several papain-like cathepsins. Binding of glycosaminoglycans to cathepsins thus directly affects catalytic activity, stability or the rate of autocatalytic activation of cathepsins. The interaction between cathepsin X and heparin has been revealed by affinity chromatography using heparin-Sepharose. Conformational changes were observed to accompany heparin-cathepsin X interaction by far UV-circular dichroism at both acidic (4.5) and neutral (7.4) pH. These conformational changes promoted a 4-fold increase in the dissociation constant of the enzyme-substrate interaction and increased 2.6-fold the kcat value also. The interaction between cathepsin X and heparin or heparan sulfate is specific since dermatan sulfate, chondroitin sulfate, and hyaluronic acid had no effect on the cathepsin X activity. Using flow cytometry cathepsin X was shown to bind cell surface heparan sulfate proteoglycans in wild-type CHO cells but not in CHO-745 cells, which are deficient in glycosaminoglycan synthesis. Moreover, fluorescently labeled cathepsin X was shown by confocal microscopy to be endocytosed by wild-type CHO cells, but not by CHO-745 cells. These results demonstrate the existence of an endocytosis mechanism of cathepsin X by the CHO cells dependent on heparan sulfate proteoglycans present at the cell surface, thus strongly suggesting that heparan sulfate proteoglycans can regulate the cellular trafficking and the enzymatic activity of cathepsin X.     

Proteoglycans synthesized and secreted by pancreatic islet beta-cells bind amylin

Pancreatic islet amyloid deposits in type 2 diabetes are associated with decreased islet beta-cell function. They contain both amylin (islet amyloid polypeptide), the beta-cell-derived unique fibrillogenic component, and heparan sulfate proteoglycans (HSPGs). We hypothesized that beta-cell HSPGs contribute to islet amyloidogenesis. [35S]Sulfate-labeled proteoglycans from islet-derived beta-TC3 cell cultures eluted from diethylaminoethyl Sephacel at 0.35M NaCl. Chromatography on Sepharose CL-4B and SDS-PAGE analysis revealed distinct populations of proteoglycans. Medium HSPGs eluted at K(av) approximately 0.18 and 0.50 with glycosaminoglycan chains of approximately 28 and 19 kDa, respectively. A third population containing chondroitin/dermatan sulfate eluted at K(av) approximately 0.70 with glycosaminoglycan chains of approximately 10 kDa. A single size class of heparan and chondroitin/dermatan sulfate proteoglycans in the cell layer eluted at K(av) approximately 0.40 with glycosaminoglycan chains of approximately 19 kDa. Medium and cell layer proteoglycans bound exclusively to fibrillogenic amylin, as determined by gel mobility shift assays, indicating a possible role for beta-cell-derived proteoglycans in islet amyloid formation.     

NCAM-Mediated Adhesion of Transfected Cells to Agrin

The vertebrate neural cell adhesion molecule NCAM mediates heterophilic adhesion to heparan sulfate proteoglycans in embryonic chick brain membranes. In this study, mouse L cells transfected with chicken NCAM were used to identify two of these ligands as agrin and the target of the 6C4 monoclonal antibody. A third heparan sulfate proteoglycan, perlecan, appeared not to support NCAM-mediated adhesion. Enzymatic degradation of chon-droitin sulfates decreased adhesion in agrin-containing membrane fractions but increased adhesion if the agrin had previously been removed by immunoprecipitation, suggesting that interactions between heparan sulfate and chondroitin sulfate proteoglycans have important influences on adhesion. Our experiments support the view that NCAM can interact with multiple, but not with all, heparan sulfate and chondroitin sulfate proteoglycans in chick brain membranes in both positive and negative ways to influence cell adhesion.     

Biosynthesis of glycosaminoglycans and proteoglycans by the lymph node

Previous studies of hyaluronan uptake and catabolism by lymph nodes indicated that the nodes might also add some HA of low molecular weight to the unabsorbed fraction that passes through from afferent to efferent lymph vessels.The ability of lymph nodes to synthesise HA and proteoglycans was therefore examined (i) by perfusion of [³H] acetate through an afferent lymph vessel in vivo, and recovery of labeled products from the efferent lymph vessel and from the node after perfusion; and (ii) by tissue culture of lymph nodes with [³H] acetate.Perfusion of lymph nodes with [³H] acetate in situ yielded: (a), in outflowing lymph, small amounts of chondroitin/dermatan sulfate within the first hour which continued to be produced for up to 24[emsp4 ]h; heparin in the second hour and HA in the third. In the nodes removed 17 to 19[emsp4 ]h later, equal amounts of hyaluronan and chondroitin/dermatan sulfate and heparan sulfate proteoglycans were detected. In the tissue culture of lymph nodes: (1) HA, heparin and proteoglycans of heparan sulfate and chondroitin/dermatan sulfate were released into the medium but in the cell extract only heparan sulfate proteoglycan was detected; and (ii) molecular weight of the released hyaluronan ranged widely but was mostly less than 4–5×10⁵[emsp4 ]D; heparan sulfate proteoglycan was 2.8×10⁴ to 9.4×10⁵[emsp4 ]D; heparin 7.9×10⁴[emsp4 ]D and chondroitin sulfate 1.3×10⁴[emsp4 ]D, suggesting that the chondrotin sulfate were released from their proteoglycans core by enzymic degradation.It is concluded that lymph nodes can release HA, heparin, heparan sulfate and chondroitin/dermatan sulfate proteoglycans into efferent lymph but the amount of hyaluronan is likely to be small without immune or other stimulation and its molecular weight is lower than in other tissues.     

Reduced Sulfation of Chondroitin Sulfate but Not Heparan Sulfate in Kidneys of Diabetic db/db Mice

Heparan sulfate proteoglycans are hypothesized to contribute to the filtration barrier in kidney glomeruli and the glycocalyx of endothelial cells. To investigate potential changes in proteoglycans in diabetic kidney, we isolated glycosaminoglycans from kidney cortex from healthy db/+ and diabetic db/db mice. Disaccharide analysis of chondroitin sulfate revealed a significant decrease in the 4-O-sulfated disaccharides (D0a4) from 65% to 40%, whereas 6-O-sulfated disaccharides (D0a6) were reduced from 11% to 6%, with a corresponding increase in unsulfated disaccharides. In contrast, no structural differences were observed in heparan sulfate. Furthermore, no difference was found in the molar amount of glycosaminoglycans, or in the ratio of hyaluronan/heparan sulfate/chondroitin sulfate. Immunohistochemical staining for the heparan sulfate proteoglycan perlecan was similar in both types of material but reduced staining of 4-O-sulfated chondroitin and dermatan was observed in kidney sections from diabetic mice. In support of this, using qRT-PCR, a 53.5% decrease in the expression level of Chst-11 (chondroitin 4-O sulfotransferase) was demonstrated in diabetic kidney. These results suggest that changes in the sulfation of chondroitin need to be addressed in future studies on proteoglycans and kidney function in diabetes.