Showing papers on "Chondroitin sulfate published in 1994"

The neuronal chondroitin sulfate proteoglycan neurocan binds to the neural cell adhesion molecules Ng-CAM/L1/NILE and N-CAM, and inhibits neuronal adhesion and neurite outgrowth

Abstract: We have previously shown that aggregation of microbeads coated with N-CAM and Ng-CAM is inhibited by incubation with soluble neurocan, a chondroitin sulfate proteoglycan of brain, suggesting that neurocan binds to these cell adhesion molecules (Grumet, M., A. Flaccus, and R. U. Margolis. 1993. J. Cell Biol. 120:815). To investigate these interactions more directly, we have tested binding of soluble 125I-neurocan to microwells coated with different glycoproteins. Neurocan bound at high levels to Ng-CAM and N-CAM, but little or no binding was detected to myelin-associated glycoprotein, EGF receptor, fibronectin, laminin, and collagen IV. The binding to Ng-CAM and N-CAM was saturable and in each case Scatchard plots indicated a high affinity binding site with a dissociation constant of approximately 1 nM. Binding was significantly reduced after treatment of neurocan with chondroitinase, and free chondroitin sulfate inhibited binding of neurocan to Ng-CAM and N-CAM. These results indicate a role for chondroitin sulfate in this process, although the core glycoprotein also has binding activity. The COOH-terminal half of neurocan was shown to have binding properties essentially identical to those of the full-length proteoglycan. To study the potential biological functions of neurocan, its effects on neuronal adhesion and neurite growth were analyzed. When neurons were incubated on dishes coated with different combinations of neurocan and Ng-CAM, neuronal adhesion and neurite extension were inhibited. Experiments using anti-Ng-CAM antibodies as a substrate also indicate that neurocan has a direct inhibitory effect on neuronal adhesion and neurite growth. Immunoperoxidase staining of tissue sections showed that neurocan, Ng-CAM, and N-CAM are all present at highest concentration in the molecular layer and fiber tracts of developing cerebellum. The overlapping localization in vivo, the molecular binding studies, and the striking effects on neuronal adhesion and neurite growth support the view that neurocan may modulate neuronal adhesion and neurite growth during development by binding to neural cell adhesion molecules.

Isolation of a neural chondroitin sulfate proteoglycan with neurite outgrowth promoting properties.

Abstract: Proteoglycans are expressed in various tissues on cell surfaces and in the extracellular matrix and display substantial heterogeneity of both protein and carbohydrate constituents. The functions of individual proteoglycans of the nervous system are not well characterized, partly because specific reagents which would permit their isolation are missing. We report here that the monoclonal antibody 473HD, which binds to the surface of early differentiation stages of murine astrocytes and oligodendrocytes, reacts with the chondroitin sulfate/dermatan sulfate hybrid epitope DSD-1 expressed on a central nervous system chondroitin sulfate proteoglycan designated DSD-1-PG. When purified from detergent-free postnatal days 7 to 14 mouse brain extracts, DSD-1-PG displays an apparent molecular mass between 800-1,000 kD with a prominent core glycoprotein of 350-400 kD. Polyclonal anti-DSD-1-PG antibodies and monoclonal antibody 473HD react with the same molecular species as shown by immunocytochemistry and sequential immunoprecipitation performed on postnatal mouse cerebellar cultures, suggesting that the DSD-1 epitope is restricted to one proteoglycan. DSD-1-PG promotes neurite outgrowth of embryonic day 14 mesencephalic and embryonic day 18 hippocampal neurons from rat, a process which can be blocked by monoclonal antibody 473HD and by enzymatic removal of the DSD-1-epitope. These results show that the hybrid glycosaminoglycan structure DSD-1 supports the morphological differentiation of central nervous system neurons.

Cartilage proteoglycans: structure and potential functions.

Abstract: Hyaline cartilage contains five well-characterized proteoglycans in its extracellular matrix, and it is likely that others exist. The largest in size and most abundant by weight is aggrecan, a proteoglycan that possesses over 100 chondroitin sulfate and keratan sulfate chains. Aggrecan is also characterized by its ability to interact with hyaluronic acid to form large proteoglycan aggregates. Both the high anionic charge on the individual aggrecan molecules endowed by the sulfated glycosaminoglycan chains and the localization within the matrix endowed by aggregate formation are essential for aggrecan function. The molecule provides cartilage with its osmotic properties, which give articular cartilage its ability to resist compressive loads. The other proteoglycans are characterized by their ability to interact with collagen. They are much smaller than aggrecan in size but may be present in similar molar amounts. Decorin, biglycan, and fibromodulin are closely related in protein structure but differ in glycosaminoglycan composition and function. Decorin and biglycan possess one and two dermatan sulfate chains, respectively, whereas fibromodulin bears several keratan sulfate chains. Decorin and fibromodulin both interact with the type II collagen fibrils in the matrix and may play a role in fibrillogenesis and interfibril interactions. Biglycan is preferentially localized in the pericellular matrix, where it may interact with type VI collagen. Finally, type IX collagen can also be considered as a proteoglycan, as its α2(IX) chain may bear a glycosaminoglycan chain. It may serve as a bridge between the collagen fibrils or with the interspersed aggrecan network. © 1994 Wiley-Liss, Inc.

Interactions of the chondroitin sulfate proteoglycan phosphacan, the extracellular domain of a receptor-type protein tyrosine phosphatase, with neurons, glia, and neural cell adhesion molecules.

Abstract: Phosphacan is a chondroitin sulfate proteoglycan produced by glial cells in the central nervous system, and represents the extracellular domain of a receptor-type protein tyrosine phosphatase (RPTP zeta/beta). We previously demonstrated that soluble phosphacan inhibited the aggregation of microbeads coated with N-CAM or Ng-CAM, and have now found that soluble 125I-phosphacan bound reversibly to these neural cell adhesion molecules, but not to a number of other cell surface and extracellular matrix proteins. The binding was saturable, and Scatchard plots indicated a single high affinity binding site with a Kd of approximately 0.1 nM. Binding was reduced by approximately 15% after chondroitinase treatment, and free chondroitin sulfate was only moderately inhibitory, indicating that the phosphacan core glycoprotein accounts for most of the binding activity. Immunocytochemical studies of embryonic rat spinal phosphacan, Ng-CAM, and N-CAM have overlapping distributions. When dissociated neurons were incubated on dishes coated with combinations of phosphacan and Ng-CAM, neuronal adhesion and neurite growth were inhibited. 125I-phosphacan bound to neurons, and the binding was inhibited by antibodies against Ng-CAM and N-CAM, suggesting that these CAMs are major receptors for phosphacan on neurons. C6 glioma cells, which express phosphacan, adhered to dishes coated with Ng-CAM, and low concentrations of phosphacan inhibited adhesion to Ng-CAM but not to laminin and fibronectin. Our studies suggest that by binding to neural cell adhesion molecules, and possibly also by competing for ligands of the transmembrane phosphatase, phosphacan may play a major role in modulating neuronal and glial adhesion, neurite growth, and signal transduction during the development of the central nervous system.

Binding of the protease-sensitive form of PrP (prion protein) to sulfated glycosaminoglycan and congo red [corrected]

Abstract: Congo red and certain sulfated glycans are potent inhibitors of protease-resistant PrP accumulation in scrapie-infected cells. One hypothesis is that these inhibitors act by blocking the association between protease-resistant PrP and sulfated glycosaminoglycans or proteoglycans (e.g., heparan sulfate proteoglycan) that is observed in amyloid plaques of scrapie-infected brain tissue. Accordingly, we have investigated whether the apparent precursor of protease-resistant PrP, protease-sensitive PrP, binds to Congo red and heparin, a highly sulfated glycosaminoglycan with an inhibitory potency like that of heparan sulfate. Protease-sensitive PrP released from the surface of mouse neuroblastoma cells bound to heparin-agarose and Congo red-glass beads. Sucrose density gradient fractionation provided evidence that at least some of the PrP capable of binding heparin-agarose was monomeric. Free Congo red blocked PrP binding to heparin and vice versa, suggesting that these ligands share a common binding site. The relative efficacies of pentosan polysulfate, Congo red, heparin, and chondroitin sulfate in blocking PrP binding to heparin-agarose corresponded with their previously demonstrated potencies in inhibiting protease-resistant PrP accumulation. These results are consistent with the idea that sulfated glycans and Congo red inhibit protease-resistant PrP accumulation by interfering with the interaction of PrP with an endogenous glycosaminoglycan or proteoglycan.

Changes in cartilage metabolism in arthritis are reflected by altered serum and synovial fluid levels of the cartilage proteoglycan aggrecan. Implications for pathogenesis.

Abstract: The metabolism of the cartilage proteoglycan aggrecan was studied in patients with osteoarthritis (OA, n = 83), rheumatoid arthritis (RA, n = 127), and in controls (n = 117) using monoclonal antibody-based radioimmunoassays for glycosaminoglycans in the serum and synovial fluid (SF) to detect epitope 846 on chondroitin sulfate (probably only on recently synthesized molecules) and a keratan sulfate (KS) epitope AN9PI, present on intact and degraded molecules. Epitope 846 levels were always elevated in SF over serum (mean 38-fold in OA and 8.6-fold in RA) being highest in OA patients with the longest disease duration and greatest loss of cartilage, and lowest in RA joints with high leucocyte counts. Serum levels were more often elevated in RA (56%) than in OA (19%) and probably reflect increased aggrecan synthesis in diseased joints. KS levels were higher in SF than in serum in 69% of patients (up to 2.3-fold); levels were inversely (OA) and directly (RA) related to SF leucocyte counts. Serum KS was reduced in both diseases and in RA was inversely related to both systemic and joint inflammation markers. SF 846 levels were inversely related to SF KS in both diseases. These epitopes may provide a measure of the balance between cartilage synthesis and degradation in these diseases.

TSG-6, an arthritis-associated hyaluronan binding protein, forms a stable complex with the serum protein inter-alpha-inhibitor

Abstract: TSG-6 is a secreted 35-kDa glycoprotein, inducible by TNF and IL-1. The N-terminal portion of TSG-6 shows sequence homology to members of the cartilage link protein family of hyaluronan binding proteins. The C-terminal half of TSG-6 contains a so-called CUB domain, characteristic for developmentally regulated proteins. High levels of TSG-6 protein are found in the synovial fluid of patients with rheumatoid arthritis and some other arthritic diseases. Here we show that TSG-6 readily formed a complex with a protein present in human, bovine, rabbit, and mouse serum. This complex was stable during SDS-PAGE under reducing conditions, and in the presence of 8 M urea. The protein that binds TSG-6 was purified from human serum and identified as inter-alpha-inhibitor (I alpha I) by N-terminal microsequencing. Microsequencing of the complex itself revealed the presence of TSG-6 and two of the three polypeptide chains of I alpha I (bikunin and HC2). Experiments with recombinant TSG-6 and I alpha I purified from human serum showed that the TSG-6/I alpha I complex is rapidly formed even in the apparent absence of other proteins at 37 degrees C, but not at 4 degrees C. The TSG-6/I alpha I complex was cleaved by chondroitin sulfate ABC lyase, suggesting that cross-linking by chondroitin sulfate is required for the stability of the complex.(ABSTRACT TRUNCATED AT 250 WORDS)

Aminosugar and glycosaminoglycan composition for the treatment and repair of connective tissue

Abstract: A therapeutic composition for the protection, treatment and repair of connective tissue in humans and animals and a method for the treatment of connective tissue in humans and animals by the administration of the composition. The composition includes aminosugars and glycosaminoglycans. The aminosugar is selected from a group consisting of glucosamine, glucosamine salts and mixtures thereof. The glycosaminoglycan is selected from a group consisting of chondroitin, chondroitin sulfate and mixtures thereof. The therapeutic composition may also include a soluble manganese salt for humans and animals having a deficiency of manganese.

The structure, function and turnover of aggrecan, the large aggregating proteoglycan from cartilage.

Abstract: Aggrecan, the large aggregating proteoglycan from cartilage contains chondroitin sulphate and keratan sulphate attached to a multidomain protein core. It aggregates by binding to hyaluronan and this is further stabilised by a separate globular link protein. There are two structurally related N-terminal globular domains, G1 and G2, of which only G1 and not G2 is involved in aggregation. The interglobular domain joining G1 and G2 contains proteinase sensitive sequences which appear to be the key site for cleavage during aggrecan turnover. Much of the keratan sulphate and all of the chondroitin sulphate is attached to the long extended glycosaminoglycan attachment region. The function of the C-terminal G3 domain is unknown. It contains a mammalian type C lectin and complement regulatory protein motifs. These may have interactive properties that contribute to matrix organisation. There is also an alternatively spliced form with an epidermal growth factor-like motif. The carbohydrate composition of aggrecan varies with cartilage source, development and age and is heterogeneous in each sample. There is evidence of a close control of chondroitin sulphate synthesis that determines chain length and disaccharide composition and which change during development and in pathology. Monoclonal antibodies that recognise specific sequences within chondroitin sulphate chains enable some of these changes in fine structure to be detected. Progressive digestion of chains with chondroitinase AC II has provided evidence of a pattern of sulphation, with 6-sulphated disaccharides more abundant towards the protein core, although the disaccharide next to the linkage region is predominantly non-sulphated.

Microtiter plate adherence assay and receptor analogs for Mycoplasma hyopneumoniae.

Abstract: A microtiter plate adherence assay for Mycoplasma hyopneumoniae was established by use of purified swine tracheal cilia which contained receptors for the mycoplasmas. M. hyopneumoniae bound specifically to plates coated with solubilized cilia. The binding was dependent on both the concentration of cilia and the number of mycoplasmas. Dextran sulfate, heparin, chondroitin sulfate, laminin, mucin, and fucoidan significantly inhibited the binding of the mycoplasmas. The six inhibitors also disrupted the adherence of the mycoplasmas to intact ciliated cells. Preincubation with either mycoplasmas or cilia indicated that heparin, mucin, fucoidan, and chondroitin sulfate interacted with the adhesive molecules on the surface of the mycoplasmas, while laminin blocked the receptors in cilia. The basis for the inhibition induced by dextran sulfate was unknown. Treatment of cilia with neuraminidase appeared to promote adherence of the mycoplasmas, whereas treatment of cilia with sodium metaperiodate decreased binding. These results indicate that receptors for M. hyopneumoniae in the ciliated epithelium of the respiratory tract of pigs are glycoconjugate in nature.