Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:3.1.6.12 (chondroitinase)
 2,183 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Heparin, NAcHep, DS, and CS were labeled with deuterium by N-reacetylating, with the deuterated acetic anhydride (CD3CO)2O, GAGs previously N-deacetylated (by hydrazinolysis) to the desired extent. Degrees of deuteration of the present preparations, as determined by 2H- and 1H-NMR were 15%, 51%, 49%, and 79% for heparin, NAcHep, DS, and CS, respectively. The NMR analysis (including the 13C spectra) of the labeled products indicated that deuterium labeling did not involve any substantial modification of the GAG structures. Also NMR signals associated with specific sequences of heparin for antithrombin and of DS for heparin cofactor II were essentially the same in the unlabeled and in the deuterated GAGs. The substantial retention of the original structure was confirmed by data on the degree of sulfation (by conductimetry) and on the electrophoretic mobility in acid buffer. On the other hand, HPLC/SEC data indicated some depolymerization of heparin and DS in the N-deacetylation step of the labeling reactions. HPLC/MS spectrometry permitted a clear identification of disaccharide and tetrasaccharide fragments obtained from deuterated GAGs by enzymic (heparinase, chondroitinase ABC) or chemical depolymerization (deaminative cleavage, Smith degradation), opening new prospects for studies of human pharmacokinetics, with differentiation of exogenous from endogenous GAGs.
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PMID:Preparation and characterization of deuterium-labeled glycosaminoglycans. 799 88

Borrelia burgdorferi adhere to mammalian cells in vitro but neither the ligand(s) nor the receptor(s) has (have) been clearly established. Using an in vitro attachment-inhibition assay, a B. burgdorferi attachment mechanism has been identified. Heparin, heparan sulfate, and dermatan sulfate reduced the attachment of virulent B. burgdorferi strain 297 to HeLa cells by approximately 60%. In addition, virulent, but not avirulent, B. burgdorferi strains B31, N40, and HB19 demonstrated heparin attachment-inhibition. Attachment to Chinese hamster ovary cells deficient in heparan sulfate proteoglycans was reduced by 68% compared to attachment to wild-type cells and was identical to attachment at maximum heparin inhibition to the wild-type cells. Pretreatment of HeLa cell monolayers with heparitinase, heparinase, and chondroitinase ABC, but not with chondroitinase AC, reduced borrelial attachment by approximately 50%. A moderately high affinity, low copy number, promiscuous B. burgdorferi glycosaminoglycan receptor was demonstrated by equilibrium binding studies. A 39-kD polypeptide, purified by heparin affinity chromatography from Triton X-100 extracts derived from virulent borrelia, was a candidate for this receptor. These studies indicate that one mode of B. burgdorferi attachment to eukaryotic cells is mediated by a borrelial glycosaminoglycan receptor attaching to surface-exposed proteoglycans on mammalian cells.
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PMID:Borrelia burgdorferi bind to epithelial cell proteoglycans. 811 83

Binding of urinary protein C inhibitor (PCI) to cultured human epithelial kidney tumor cells (TCL-598) was studied. Binding was dose-dependent, time-dependent, and saturable. Heparin interfered in a dose-dependent way with PCI binding to TCL-598 as did heparan sulfate and to a lesser degree also dermatan sulfate. Pretreatment of TCL-598 with protamine sulfate inhibited subsequent binding of PCI in a dose-dependent manner and > 100 micrograms/ml protamine sulfate reduced binding of PCI to < 10% of the control. Binding of 125I-PCI was specific, and bound 125I-PCI was recovered from the cells by heparin treatment or detached together with intact cells by EDTA treatment, migrated on sodium dodecyl sulfate-polyacrylamide gel electrophoresis with the same mobility (M(r) = 57,000) as unbound 125I-PCI. Furthermore, cell-bound PCI was functionally active as judged from its ability to inhibit the amidolytic activity of urokinase, and its inhibitory activity was stimulated approximately 3-4-fold as compared to fluid-phase PCI. Immunogold electron microscopy revealed that PCI-antigen presented to the cells from the luminal side bound exclusively to that surface in native as well as in prefixed cells. This binding of PCI was abolished in the presence of heparin (50 micrograms/ml) and after pretreatment of the cells either with protamine sulfate (400 micrograms/ml) or with heparinase III (0.5 unit/ml). A slight decrease in PCI binding was seen after pretreatment of the cells with chondroitinase ABC and chondroitinase AC. In contrast, binding of PCI to extracellular matrices of TCL-598 was decreased to approximately 70% after chondroitinase ABC treatment of the extracellular matrices, whereas both heparinase III or chondroitinase AC treatment only reduced matrix-bound PCI to approximately 95%. These data suggest that heparan sulfate-containing proteoglycans are predominantly involved in binding of PCI to the luminal side of TCL-598, while dermatan sulfate-containing proteoglycans, the overall predominant PCI-binding proteoglycans in TCL extracts, are responsible for PCI binding to the extracellular matrix. Heparan sulfate, however, exposed to an environment containing PCI under physiological conditions, might localize PCI and modulate its target enzyme specificity in vivo.
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PMID:Binding of urinary protein C inhibitor to cultured human epithelial kidney tumor cells (TCL-598). The role of glycosaminoglycans present on the luminal cell surface. 818 78

Heparin was extracted and purified from beef intestinal mucosa. The two components, fast moving heparin and slow moving heparin were purified by selective precipitation as barium salts. Heparan sulfate was extracted and purified from beef spleen. Dermatan sulfate was purified from beef intestinal mucosa and chondroitin sulfate from bovine trachea. The purity of the purified glycosaminoglycans was evaluated by agarose-gel and cellulose polyacetate electrophoresis and by specific optical rotation. The relative molecular masses of glycosaminoglycans were estimated by high performance-size exclusion chromatography and the sulfate to carboxyl ratio by titrimetric analysis. The disaccharide pattern of heparin, fast moving and slow moving heparins and heparan sulfate were determined by specific enzymatic cleavage using heparinase I, II and III; the disaccharide composition of dermatan sulfate and chondroitin sulfate was evaluated by cleavage by chondroitinase ABC. The disaccharides obtained by enzymatic cleavage were qualitatively and quantitatively analysed by strong anion exchange-high performance liquid chromatography. The sulfate to carboxyl ratios of glycosaminoglycans were also determined by this technique and compared with the values obtained by titrimetric analysis.
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PMID:Extraction, purification and evaluation of structures and physico-chemical properties of glycosaminoglycans. 835 79

Cell adhesion to extracellular matrix molecules such as fibronectin involves complex transmembrane signaling processes. Attachment and spreading of primary fibroblasts can be promoted by interactions of cell surface integrins with RGD-containing fragments of fibronectin, but the further process of focal adhesion and stress fiber formation requires additional interactions. Heparin-binding fragments of fibronectin can provide this signal. The COOH-terminal heparin-binding domain of fibronectin contains five separate heparin-binding amino acid sequences. We show here that all five sequences, as synthetic peptides coupled to ovalbumin, can support cell attachment. Only three of these sequences can promote focal adhesion formation when presented as multicopy complexes, and only one of these (WQPPRARI) retains this activity as free peptide. The major activity of this peptide resides in the sequence PRARI. The biological response to this peptide and to the COOH-terminal fragment may be mediated through cell surface heparan sulfate proteoglycans because treatment of cells with heparinase II and III, or competition with heparin, reduces the response. Treatment with chondroitinase ABC or competition with chondroitin sulfate does not.
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PMID:A synthetic peptide from the COOH-terminal heparin-binding domain of fibronectin promotes focal adhesion formation. 837 70

Heparin significantly increased the amount of newly synthesized apolipoprotein E (apoE) released by HepG2 cells. Culturing cells in the presence of 10 micrograms/ml of heparin for 2-6 days caused a 1.3-fold (day 2) to 3-fold (day 6) increase of extracellular apoE without affecting the total amount of apoE synthesized by the cells. The amounts of apoA-I and apoB produced by HepG2 cells were unaffected by heparin. Surprisingly, short-term treatment with heparin (15-30 min) also increased extracellular apoE by 2- to 3-fold. In this situation, heparin exerted its effect on apoE post-translationally. Among glycosaminoglycans (GAGs), only heparan sulfate mimicked heparin at a concentration of 10 micrograms/ml; hyaluronic acid and the chondroitin sulfates were effective only at a higher concentration (100 micrograms/ml). Extracellular apoE was not increased by treating cells with anti-apoE antiserum or a heparin-binding peptide of apoE (amino acids 130-169). Removal of cell surface-associated GAGs by culturing cells in 4-methylumbelliferyl-beta-D-xyloside ablated the effect of heparin on apoE. ApoE was released from cells by treatment with heparinases I and III, but not by chondroitinase ABC. The results provide evidence that a heparin-releasable pool of newly synthesized apoE is associated with cell surface GAGs that resemble heparin and/or heparan sulfate.
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PMID:Heparin releases newly synthesized cell surface-associated apolipoprotein E from HepG2 cells. 838 53

In vitro studies in our laboratory have indicated that heparan sulfate proteoglycans (HSPGs) play an important role in murine embryo implantation. In order to investigate the potential function of HSPGs in human implantation, two human cell lines (RL95 and JAR) were used to model uterine epithelium and embryonal trophectoderm, respectively. A heterologous cell-cell adhesion assay was developed to determine if binding of JAR cells to RL95 cells was heparan sulfate-dependent. Labeled, single cell suspensions of JAR cells attached to confluent monolayers of RL95 cells in a dose- and time-dependent manner. Heparin-like glycosaminoglycans and JAR cell proteoglycans competitively inhibited JAR cell adhesion to RL95 cells by 50% or more. A panel of chemically modified heparins were used to demonstrate that O-sulfation and amino group substitution were critical for inhibition of cell-cell adhesion. Treatment with chlorate, an inhibitor of ATP-sulfurylase, resulted in a 56% reduction in cell-cell binding compared to untreated controls. Heparinase and chondroitinase ABC markedly inhibited JAR-RL95 binding, while chondroitinase AC had no significant effect. These observations indicated that HSPGs as well as dermatan sulfate-containing proteoglycans participated in cell-cell binding. Collectively, these results indicate that initial binding interactions between JAR and RL95 cells is mediated by cell surface glycosaminoglycans (GAGs) with heparin-like properties (i.e., heparan sulfate and dermatan sulfate). These observations are consistent with an important role for HS and heparin-like GAGs as well as their corresponding binding sites in early stages of human trophoblast-uterine epithelial cell binding.
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PMID:Heparin-like glycosaminoglycans participate in binding of a human trophoblastic cell line (JAR) to a human uterine epithelial cell line (RL95). 846 65

The signals that trigger the cytodifferentiation of oligodendrocytes (OLGs) are largely unknown. Using as a model system cultures of pure OLGs, we have shown that adhesion to a substratum initiates myelinogenesis (Yim SH, Szuchet S, Polak PE, J Biol Chem 261:11808-11815, 1986). It was of interest to investigate whether components such as proteoglycans (PGs) play any role in the biology of OLGs as it pertains to myelinogenesis. We set out to determine first, whether OLGs carry PGs; second, the nature of the association of these components with OLG plasma membrane; and third, if and how these PGs are modulated by OLG-substratum interaction. We compared the expression and characteristics of PGs extracted with different solvents from nonattached (B3.f) and attached (B3.fA) OLGs. B3.f and B3.fA OLG cultures were labeled with carrier-free 35SO4(2-) in serum-free medium. After removing excess label, OLGs were treated with heparin to extract susceptible components. Pellets were then exposed to 1% Triton X-100 plus 0.1 M NaCl and subsequently to 4 M guanidine-HCl plus 0.5 M NaCl. Solutions containing extracted material were characterized by size-exclusion chromatography, SDS-PAGE, and enzymatic degradation. Herein we report that (1) OLGs display [35S]PGs on their surface within 24 hr of substratum adhesion, and (2) these PGs can be operationally classified as peripheral and integral. We further show that the peripheral PGs are of high and intermediate size as assessed by size-exclusion chromatography and are segregated within the plasma membrane in such a way that the species with intermediate mass are extracted while OLGs remain adhered, whereas the high-molecular-weight species are only extracted after OLGs have been detached. Heparin also dislodges a number of sulfated proteins/Gps. Only a single class--high molecular weight--of integral PGs was identified; this PG requires guanidine-HCl for extraction. All PGs belong to the heparan sulfate class as evidenced by their degradation with heparitinase and their lack of susceptibility to chondroitinase ABC. The common theme of our findings is that these macromolecules have basal levels of expression in the nonadhered OLGs but undergo an adhesion-induced enhancement in their syntheses. We postulate that these PGs (1) play a role in OLG-substratum adhesion and hence myelinogenesis, and (2) may be determinants in establishing OLG polarity. Such polarization is the first overt sign of OLG functional differentiation and occurs prior to any morphological differentiation, e.g., extension of processes does not occur until 48 hr later when the plasma membrane is already polarized.
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PMID:Oligodendrocyte proteoglycans: modulation by cell-substratum adhesion. 847 42

We have studied the binding, uptake, and degradation of a recombinant form of apolipoprotein[a] (r-apo[a]) using a cultured cell model. In HepG2 cells and in human fibroblasts, r-apo[a] complexed with low density lipoprotein(LDL) is bound and internalized via high affinity (Kd = 10 nM) receptors; in both cell types, low affinity (Kd = 200-300 nM) sites also mediate free apo[a] uptake. Using competition studies, we found that the high affinity binding component corresponds to the LDL receptor. Involvement of the LDL receptor in r-apo[a] uptake by fibroblasts was confirmed using fibroblasts derived from an individual homozygous for familial hypercholesterolemia; in contrast to normal fibroblasts, these cells lacked the high affinity r-apo[a] binding component. Cell association of 125I-labeled r-apo[a] was increased and decreased concomitantly with the up- and down-regulation of the LDL receptor in response to a number of compounds. The addition of alpha 2-macroglobulin as well as treatment with heparinase, chondroitinase ABC, and sodium chlorate did not decrease total specific binding of r-apo[a], suggesting that neither the low density lipoprotein receptor-related protein nor cell surface proteoglycans are involved in r-apo[a] clearance. The low affinity binding component present in both fibroblasts and HepG2 cells likely corresponds to the plasminogen receptor, as binding of r-apo[a] to these sites was specifically decreased by the addition of plasminogen or the lysine analogue epsilon-aminocaproic acid, but not by the addition of tissue-type plasminogen activator. Heparin abolished uptake of r-apo[a] by the LDL receptor component only; this indicates that apo[a] must be associated with LDL to be cleared by this receptor. In contrast, free apo[a] can be effectively cleared by the plasminogen receptor which may represent a significant route of clearance for free apo[a] in vivo.
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PMID:Interaction of a recombinant form of apolipoprotein[a] with human fibroblasts and with the human hepatoma cell line HepG2. 872 15

The mitogenic action of insulin-like growth factors (IGFs) on target cells is determined by interaction with signaling IGF-I receptors and modulated by interactions with IGF-binding proteins (IGFBPs). IGFBP-3, an abundant IGFBP that binds IGF-I and IGF-II with high affinity, can form soluble inhibitory complexes with the IGFs that prevent them from binding to IGF-I receptors. Alternatively, IGFBP-3 can bind to the cell surface and possibly potentiate IGF action or act independently of the IGFs. Previous studies showed that heparin inhibited IGFBP-3 binding to the cell surface and increased its accumulation in the medium, suggesting that it might act as a competitive inhibitor of IGFBP-3 binding to structurally similar heparan sulfate proteoglycans on the cell surface. We evaluated this hypothesis by binding 125I-labeled recombinant glycosylated human IGFBP-3 to human fetal skin fibroblasts (GM-10) and to C6 rat glioma cells at 12 C. Heparin inhibited [125I]IGFBP-3 binding more effectively than chondroitin sulfate and dextran sulfate. Complete digestion of cell surface heparan sulfate and chondroitin sulfate glycosaminoglycans using heparitinase and chondroitinase ABC, however, did not significantly decrease IGFBP-3 binding. Quantitative removal was demonstrated by analysis of parallel cultures of cells whose glycosaminoglycans had been biosynthetically labeled using Na2 35SO4. These results suggested that IGFBP-3 did not bind to heparan sulfate glycosaminoglycans on the cell surface, and that the inhibition of IGFBP-3 binding by heparin most likely resulted from its direct interaction with the heparin-binding domains of IGFBP-3. When [125I]IGFBP-3 was incubated with GM-10 fibroblasts or C6 glioma cells at 37 C for 4 h, only 10% of the bound ligand remained associated with the cell surface; approximately 90% of the cell-associated radio-activity was internalized and could be recovered in lysates of acid-washed cells. Incubation with IGF-I or heparin decreased the total cell-associated radioactivity, but did not affect internalization. These results suggest that direct interaction of heparin or IGF-I with IGFBP-3 inhibits its ability to bind to the surface of GM-10 fibroblasts and C6 glioma cells.
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PMID:Heparin inhibition of insulin-like growth factor-binding protein-3 binding to human fibroblasts and rat glioma cells: role of heparan sulfate proteoglycans. 882 97


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