Activation of the autophagy, c-FOS and ubiquitin expression, and nucleolar alterations in Schwann cells precede demyelination in tellurium-induced neuropathy

The method of affinity coelectrophoresis was used to study the binding of nine representative glycosaminoglycan (GAG)-binding proteins, all thought to play roles in nervous system development, to GAGs and proteoglycans isolated from developing rat brain. Binding to heparin and non-neural heparan and chondroitin sulfates was also measured. All nine proteins-laminin-1, fibronectin, thrombospondin-1, NCAM, L1, protease nexin-1, urokinase plasminogen activator, thrombin, and fibroblast growth factor-2-bound brain heparan sulfate less strongly than heparin, but the degree of difference in affinity varied considerably. Protease nexin-1 bound brain heparan sulfate only 1.8-fold less tightly than heparin (Kdvalues of 35 vs. 20 nM, respectively), whereas NCAM and L1 bound heparin well (Kd approximately 140 nM) but failed to bind detectably to brain heparan sulfate (Kd>3 microM). Four proteins bound brain chondroitin sulfate, with affinities equal to or a few fold stronger than the same proteins displayed toward cartilage chondroitin sulfate. Overall, the highest affinities were observed with intact heparan sulfate proteoglycans: laminin-1's affinities for the proteoglycans cerebroglycan (glypican-2), glypican-1 and syndecan-3 were 300- to 1800-fold stronger than its affinity for brain heparan sulfate. In contrast, the affinities of fibroblast growth factor-2 for cerebroglycan and for brain heparan sulfate were similar. Interestingly, partial proteolysis of cerebroglycan resulted in a >400-fold loss of laminin affinity. These data support the views that (1) GAG-binding proteins can be differentially sensitive to variations in GAG structure, and (2) core proteins can have dramatic, ligand-specific influences on protein-proteoglycan interactions.     

Syndecans, heparan sulfate proteoglycans, maintain the proteolytic balance of acute wound fluids

An imbalance between proteases and antiproteases is thought to play a role in the inflammatory injury that regulates wound healing. The activities of some proteases and antiproteases found in inflammatory fluids can be modified in vitro by heparin, a mast cell-derived glycosaminoglycan. Because syndecans, a family of cell surface heparan sulfate proteoglycans, are the major cellular source of heparin-like glycosaminoglycan, we asked whether syndecans modify protease activities in vivo. Syndecan-1 and syndecan-4 ectodomains are shed into acute human dermal wound fluids (Subramanian, S. V., Fitzgerald, M. L., and Bernfield, M. (1997) J. Biol. Chem. 272, 14713-14720). Moreover, purified syndecan-1 ectodomain binds cathepsin G (Kd = 56 nM) and elastase (Kd = 35 nM) tightly and reduces the affinity of these proteases for their physiological inhibitors. Purified syndecan-1 ectodomain protects cathepsin G from inhibition by alpha1-antichymotrypsin and squamous cell carcinoma antigen 2 and elastase from inhibition by alpha1-proteinase inhibitor by decreasing second order rate constants for protease-antiprotease associations (kass) by 3700-, 32-, and 60-fold, respectively. Both enzymatic degradation of heparan sulfate and immunodepletion of the syndecan-1 and -4 in wound fluid reduce these proteolytic activities in the fluid, indicating that the proteases in the wound environment are regulated by interactions with syndecan ectodomains. Thus, syndecans are shed into acute wound fluids, where they can modify the proteolytic balance of the fluid. This suggests a novel physiological role for these soluble heparan sulfate proteoglycans.     

Hypereosinophilic syndrome in a child mosaic for a congenital triplication of the short arm of chromosome 8

Fibroblast growth factors are important heparin binding, mitogenic proteins. The binding site in heparin and heparan sulfate for fibroblast growth factor-2 (basic fibroblast growth factor) has been described as rich in glucosamine-2-sulfate 1-->4 linked to iduronic acid-2-sulfate. The glucosamine residue in the heparin binding site is also 6-sulfated. A new glycosaminoglycan, acharan sulfate, has been chemically modified to prepare a polysaccharide, N-sulfoacharan sulfate, consisting of glucosamine-2-sulfate 1-->4 linked to iduronic acid-2-sulfate. Acharan sulfate binds very weakly to fibroblast growth factor-2 while N-sulfoacharan sulfate binds with nearly the same affinity as heparin. Mitogenicity studies were performed using heparan sulfate-free cells stably transfected with fibroblast growth factor receptor-1. Acharan sulfate inhibits heparin's enhancement of fibroblast growth factor-2 mitogenic activity, without affecting cell viability, while N-sulfoacharan sulfate shows heparin-like activity but at a greatly reduced level. These results suggest additional mechanisms not requiring high affinity glycosaminoglycan binding to fibroblast growth factor-2 may be important in its mitogenic activity.     

Differential release of proteoglycans during human B lymphocyte maturation

Proteoglycans interact with soluble proteins such as growth factors and thereby regulate extracellular signals. During B lymphocyte maturation, secretion of proteoglycans may be functionally related to the different requirements of the respective maturation stage. In order to address this question we compared structures of proteoglycans released by three B lymphocyte lines which correspond to different maturation stages. Plasma-cell type U266 cells secreted the largest proteoglycans (150 kDa), followed by mature B cells JOK-1 (130 kDa) and pre-B cells Nalm 6 (90 kDa). On average, secreted proteoglycans carried four glycosaminoglycan chains with molecular masses ranging each from 32 kDa (U266) to 23 kDa (Nalm 6). All three cell lines secreted more than 90% of their proteoglycans possessing chondroitin sulfate chains having chondroitin-4-sulfate (delta Di-4S) as the prevalent disaccharide unit. In these proteochondroitin sulfates, unsulfated chondroitin (delta Di-0S) was present in smaller quantities and chondroitin-6-sulfate (delta Di-6S)-containing proteoglycan was released only by Nalm 6 and U266 cells. Cell line Nalm 6 exclusively produced proteochondroitin sulfate, whereas in culture medium of JOK-1 and U266 a small amount of proteoheparan sulfate was found also. In all three cell lines, treatment with chondroitinase ABC released a protein of 30 kDa and chemical deglycosylation resulted in a core protein of 21 kDa. In addition to pure proteochondroitin sulfate, a small portion of proteoheparan sulfate with a protein moiety of 30 kDa was detected after heparitinase treatment in supernatants of JOK-1 and U266. Thus, our results indicate that released proteoglycans may undergo modulations in their glycosaminoglycan moieties during B-cell differentiation. This may have functional consequences at the level of growth factor regulation.     

Formation of promutagenic methylation damage in tissue-DNA of mice treated with antischistosomal agents

BACKGROUND: Cultured bovine corneal endothelial cells (CEC) synthesize heparan sulfate and dermatan sulfate containing proteoglycans and distribute them between different compartments. METHODS AND RESULTS: [35S]sulfate labelled proteoglycans are found associated with the cell layer, secreted into the culture medium and deposited into the underlaying extracellular matrix. In the presence of basic fibroblast growth factor (bFGF)-a strong mitogen for CEC-subconfluent cells incorporate [35S]sulfate into the sulfated proteoglycans at a rate three times higher as compared with the proteoglycans of CEC in the absence of bFGF. The enhanced proteoglycan synthesis is accompanied with a shift in the proteoglycan distribution pattern. While in control cells the cell-associated heparan sulfate accounts for about 30% of the total glycosaminoglycans under the influence of bFGF the HS percentage increases to approximately 60%. CONCLUSIONS: CEC synthesize and deposit endogenous bFGF into the extracellular matrix. Heparitinase treatment of the extracellular matrix releases bFGF activity which is able to stimulate the 35S incorporation into proteoglycans in a comparable manner as exogenous bFGF but does not influence the proteoglycan distribution pattern. Pretreatment of the matrix-bound bFGF activity with polyclonal antibodies against bFGF abolishes its stimulating activity.     

Prospective study of the significance of contact sensitization caused by metal implants

Cell nuclei of mouse hepatoma contain various proteoglycans (PG) which include heparan sulfate proteoglycan (HS-PG), dermatan sulfate proteoglycan (DS-PG), and chondroitin sulfate proteoglycan (CS AC-PG). The latter is not found in cell nuclei of normal mouse liver. Heparan sulfate (HS) and dermatan sulfate (DS) are the main constituents of carbohydrate chains of nuclear proteoglycans of tumor and normal cells, respectively. Changes in the composition of nuclear PG during malignant transformation are discussed considering the concept of their possible involvement in the regulation of cell mitotic activity.     

Glycosaminoglycan specificity of a heparin-binding peptide

Glycosaminoglycans are complex sulfated polysaccharides with a diverse range of biological functions. Three glycosaminoglycan standards--chondroitin sulfate, dermatan sulfate and heparin--were characterized during this study. The interaction of the heparin binding site of protein C inhibitor, represented by the peptide sequence 264-283, in solution with the above glycosaminoglycan standards was studied. Circular dichroism spectroscopy was used to determine the dominant secondary structure induced in the peptide upon binding the relevant glycosaminoglycans. The various glycosaminoglycans induced different secondary structures. The level of induced secondary structure by dermatan sulfate and heparin was approximately twice that induced by chondroitin sulfate. For chondroitin sulfate and heparin, alpha-helix was the dominant ordered secondary structure, whereas for dermatan sulfate the beta-strand conformation dominated. The order of secondary structure induction of the protein C inhibitor peptide by the glycosaminoglycans paralleled the reported biological activities of these glycosaminoglycans for mediation of the biological activity in the intact protein. The strength of the interaction of dermatan sulfate and heparin with the protein C inhibitor peptide was measured by determining the concentration of salt required to inhibit 50% of the interaction. The values determined were 0.1 and 0.3 M salt for dermatan sulfate and heparin, respectively. These results show that different glycosaminoglycans can support different secondary structures in the protein C inhibitor peptide.     

The core protein of the chondroitin sulfate proteoglycan phosphacan is a high-affinity ligand of fibroblast growth factor-2 and potentiates its mitogenic activity

Using a radioligand binding assay we have demonstrated that phosphacan, a chondroitin sulfate proteoglycan of nervous tissue that also represents the extracellular domain of a receptor-type protein tyrosine phosphatase, shows saturable, reversible, high-affinity binding (Kd approximately 6 nM) to fibroblast growth factor-2 (FGF-2). Binding was reduced by only approximately 35% following chondroitinase treatment of the proteoglycan, indicating that the interaction is mediated primarily through the core protein rather than the glycosaminoglycan chains. Immunocytochemical studies also showed an overlapping localization of FGF-2 and phosphacan in the developing central nervous system. At concentrations of 10 microg protein/ml, both native phosphacan and the core protein obtained by chondroitinase treatment potentiated the mitogenic effect of FGF-2 (5 ng/ml) on NIH/3T3 cells by 75-90%, which is nearly the same potentiation as that produced by heparin at an equivalent concentration. Although studies on the role of proteoglycans in mediating the binding and mitogenic effects of FGF-2 have previously focused on cell surface heparan sulfate, our results indicate that the core protein of a chondroitin sulfate proteoglycan may also regulate the access of FGF-2 to cell surface signaling receptors in nervous tissue.     

The cytoplasmic domain of syndecan-1 is required for cytoskeleton association but not detergent insolubility. Identification of essential cytoplasmic domain residues

Syndecan-1 is a member of a gene family of multifunctional transmembrane heparan sulfate proteoglycans that bind a variety of extracellular ligands and possess highly conserved non-catalytic cytoplasmic domains. It has been shown that antibody-mediated clustering of syndecan-1 causes the proteoglycan to become associated with microfilaments and insoluble in non-ionic detergent. A series of truncation and point mutations of the syndecan-1 core protein was constructed to identify specific structural features that were required for these characteristics. The transmembrane domain but not the cytoplasmic domain was required for cell surface expression of syndecan-1. Deletion of the COOH-terminal 11 amino acids of the cytoplasmic domain had no effect, while deletion of an additional 12 amino acids abolished microfilament association. Mutation of a conserved tyrosine residue within the latter region also abolished microfilament association. In contrast, mutation of 2 tyrosine residues outside this region had no effect. Deletion of the entire cytoplasmic domain (except for a short stop-transfer sequence) did not affect insolubility of the proteoglycan in detergent. Analysis of a form of syndecan-1 that lacked glycosaminoglycan acceptor sites revealed that covalently attached glycosaminoglycans were not required for cell surface expression, microfilament association, or detergent insolubility. These results demonstrate that microfilament association is a function of a subregion within the cytoplasmic domain and suggest that insolubility in detergent is a function of the transmembrane domain.     

Multiple heparan sulfate chains are required for optimal syndecan-1 function

Syndecans have three highly conserved sites available for heparan sulfate attachment. To determine if all three sites are required for normal function, a series of mutated syndecans having two, one, or no heparan sulfate chains were expressed in ARH-77 cells. Previously, we demonstrated that expression of wild-type syndecan-1 on these myeloma cells mediates cell-matrix and cell-cell adhesion and inhibits cell invasion into collagen gels. Here we show that to optimally mediate each of these activities, all three sites of heparan sulfate attachment are required. Generally, an increasing loss of syndecan-1 function occurs as the number of heparan sulfate attachment sites decreases. This loss of function is not the result of a decrease in either the total amount of cell surface heparan sulfate or syndecan-1 core protein. In regard to cell invasion, cells expressing syndecan-1 bearing a single heparan sulfate attachment site exhibit a hierarchy of function based upon the position of the site within the core protein; the presence of an available attachment site at serine 47 confers the greatest level of activity, while serine 37 contributes little to syndecan-1 function. However, when all three heparan sulfate chains are present, significantly greater biological activity is observed than is predicted by the sum of the activities occurring when the chains act individually. This synergy provides a functional basis for the evolutionary conservation of the three heparan sulfate attachment sites on syndecans and supports the idea that molecular heterogeneity, which is characteristic of proteoglycans, contributes to their functional diversity.     

Age-related changes in populations of aortic glycosaminoglycans: species with low affinity for plasma low-density lipoproteins, and not species with high affinity, are preferentially affected

Glycosaminoglycans were extracted from the intima and media layers of normal human thoracic aortas from donors of different ages. The arterial segments were devoid of macroscopically visible lesions obtained from patients who had no clinically evident cardiovascular disease. Total glycosaminoglycan content increases during the first 40 years of life. Changes in the content of hyaluronic acid and heparan sulfate are less noticeable. The content of chondroitin sulfate (mainly the 6-isomer) increases, whereas dermatan sulfate remains constant. Plasma LDL-affinity chromatography of dermatan sulfate+chondroitin 4/6-sulfate fractions allowed the separation of LDL high- and low-affinity glycosaminoglycan species. Remarkably, only glycosaminoglycan species with low affinity for plasma LDL increase with age in the disease-free areas of human thoracic aortas studied. These results suggest that age-related changes in glycosaminoglycan composition of the arterial wall do not contribute to increased deposition of plasma LDL. However, the alternative explanation that individuals with arterial glycosaminoglycans that avidly bind LDL would develop early and severe cardiovascular disease and would thus be excluded from our analysis cannot be ruled out.     

Isolation and characterization of chondroitin sulfate proteoglycans from embryonic quail that influence neural crest cell behavior

The movement of neural crest cells is controlled in part by extracellular matrix. Aggrecan, the chondroitin sulfate proteoglycan from adult cartilage, curtails the ability of neural crest cells to adhere, spread, and move across otherwise favorable matrix substrates in vitro. Our aim was to isolate, characterize, and compare the structure and effect on neural crest cells of aggrecan and proteoglycans purified from the tissues through which neural crest cells migrate. We metabolically radiolabeled proteoglycans in E2.5 quail embryos and isolated and characterized proteoglycans from E3.3 quail trunk and limb bud. The major labeled proteoglycan was highly negatively charged, similar in hydrodynamic size to chick limb bud versican/PG-M, smaller than adult cartilage aggrecan but larger than reported for embryonic sternal cartilage aggrecan. The molecular weight of the iodinated core protein was about 400 kDa, which is more than reported for aggrecan but less than that of chick versican/PG-M. The proteoglycan bore chondroitin sulfate glycosaminoglycan chains of 45 kDa, which is larger than those of aggrecan. It lacked dermatan sulfate, heparan sulfate, or keratan sulfate chains. It bound to collagen type I, like aggrecan, but not to fibronectin (unlike versican/PG-M), collagen type IV, or laminin-1 in solid-phase assays and it bound to hyaluronate in gel-shift assays. When added at concentrations between 10 and 30 microg/ml to substrates of fibronectin, trunk proteoglycan inhibited neural crest cell spreading and migration. Attenuation of cell spreading was shown to be the most sensitive and titratable measure of the effect on neural crest cells. This effect was sensitive to digestion with chondroitinase ABC. Similar cell behavior was also produced by aggrecan and the small dermatan sulfate proteoglycan decorin; however, 30-fold more aggrecan was required to produce an effect of similar magnitude. When added in solution to neural crest cells which were already spread and migrating on fibronectin, the embryonic proteoglycan rapidly and reversibly caused complete rounding of the cells, being at least 30-fold more potent than aggrecan in this activity.     

Paget''s disease of bone

The protein encoded by ras and src protooncogenes are frequently activated in a constitutive state in human colorectal cancer. In this study, we investigated the effect of oncogenic p21ras and Py-MT/pp60c-src on the synthesis of syndecan-1, a membrane anchored proteoglycan playing a role in cell-matrix interaction and neoplastic growth control. To this end, we used Caco-2 cells transfected with an activated (Val-12) human Ha-ras gene or the polyoma middle T (Py-MT) oncogene, a constitutive activator of pp60c-src tyrosine kinase activity. As compared to control vector-transfected Caco-2 cells, both oncogene-transfected cells exhibited: (1) a decrease in syndecan-1 specific activity; (2) a decrease in size and sulfation of syndecan-1 ectodomain glycosaminoglycan side chains; and (3) an active heparanase specifically degrading the heparan sulfate chains. In conclusion, the tumorigenic progression induced by oncogenic p21ras or Py-MT/pp60c-src is associated with marked alterations of syndecan-1 at the post-translational level.     

Glycosaminoglycans can modulate extracellular localization of the wingless protein and promote signal transduction

Wingless, the Drosophila homologue of the proto-oncogene Wnt-1, encodes a secreted glycoprotein that regulates differentiation and proliferation of nearby cells. Here we report on the biochemical mechanism(s) by which the wingless signal is transmitted from cell to cell. When expressed in S2 cells, the majority (approximately 83%) of secreted wingless protein (WG) is bound to the cell surface and extracellular matrix through specific, noncovalent interactions. The tethered WG can be released by addition of exogenous heparan sulfate and chondroitin sulfate glycosaminoglycans. WG also binds directly to heparin agarose beads with high affinity. These data suggest that WG can bind to the cell surface via naturally occurring sulfated proteoglycans. Two lines of evidence indicate that extracellular glycosaminoglycans on the receiving cells also play a functional role in WG signaling. First, treatment of WG-responsive cells with glycosaminoglycan lyases reduced WG activity by 50%. Second, when WG-responsive cells were preincubated with 1 mM chlorate, which blocks sulfation, WG activity was inhibited to near-basal levels. Addition of exogenous heparin to the chlorate-treated cells was able to restore WG activity. Based on these results, we propose that WG belongs to the group of growth factor ligands whose actions are mediated by extracellular proteoglycan molecules.     

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

The proteoglycans synthesized by fibroblasts derived from healthy human adult dental pulps have been isolated and characterized on the basis of their glycosaminoglycan content, molecular size and charge. The proteoglycans were identified by their labelling with [35S] sulphate and susceptibility to digestion by papain. The sulphated glycosaminoglycans associated with the proteoglycans were identified following specific enzymatic and chemical degradations as chondroitin sulphate, dermatan sulphate and heparan sulphate. Dermatan sulphate and chondroitin sulphate and heparan sulphate were the principal glycosaminoglycans associated with the cell layers. The proteoglycans could be fractionated on the basis of their charge and size into a number of heterogeneous pools. The principal proteoglycans isolated were small and contained either chondroitin sulphate or dermatan sulphate and most likely correspond to decorin and biglycan. Other molecules with features similar to versican and syndecan were also identified.     

Binding of human phospholipase A2 type II to proteoglycans. Differential effect of glycosaminoglycans on enzyme activity

Phospholipase A2 acting on low density lipoproteins in the extracellular arterial intima may form proinflammatory lipid mediators. Human nonpancreatic secretory phospholipase A2 has three regions that may associate with sulfated glycosaminoglycans. The apoB-100 molecule in low density lipoproteins also has glycosaminoglycan binding regions that could mediate its retention in the arterial intima. Here we report that human nonpancreatic phospholipase A2 isolated from a transfected cell line binds to glycosaminoglycans secreted by cultured human arterial smooth muscle cells. A gel mobility shift assay showed that the affinity of phospholipase A2 for glycosaminoglycans from a heparan sulfate/chondroitin sulfate proteoglycan was higher than for chondroitin sulfate glycosaminoglycans from a larger versican-like proteoglycan. Affinity chromatography confirmed these results. All glycosaminoglycans tested, at concentrations up to 100 microM, increased the activity of phospholipase A2 toward phosphatidylcholine liposomes. Above this concentration, heparan sulfate and heparin inhibited the enzyme. Heparin and chondroitin 6-sulfate increased phospholipase A2 activity on low density lipoproteins up to 4-fold at 100 microM, whereas heparan sulfate had no effect. The results indicate that human nonpancreatic secretory phospholipase A2 interacts with proteoglycans via their glycosaminoglycan moiety and that the enzyme activity may be modulated by the association of the enzyme and its substrate to the sulfated polysaccharides.     

Interferon-gamma inhibits 35S incorporation in heparan sulfate synthesized by human skin fibroblasts

Glycosaminoglycans synthesized by human skin fibroblasts were simultaneously radiolabelled with D-[1-(3H)]glucosamine and Na2(35)SO4. Considering 3H incorporation, we found that IFNgamma increased the production of glycosaminoglycan synthesis, including hyaluronic acid, heparan and chondroitin/dermatan sulfate. In contrast, the production of heparan and chondroitin/dermatan sulfate was slightly decreased on the basis of the 35S signal. Furthermore, when heparan sulfate was treated with nitrous acid, the release of free 35S was greater in control than in treated cells, although the 3H patterns of depolymerization with this agent were similar. These data demonstrate that IFNgamma inhibits the incorporation of sulfate from extracellular medium into heparan sulfate.