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.     

Apoptosis induced by granzyme B-glycosaminoglycan complexes: implications for granule-mediated apoptosis in vivo

Lymphocyte granule-mediated apoptosis occurs by perforin-mediated intracellular delivery of granule-associated serine proteases (granzymes). A granule-associated proteoglycan, namely serglycin, that contains chondroitin 4-sulfate (CS) glycosaminoglycans is present in the granules of cytotoxic cells. Serglycin acts as scaffold for packaging the positively charged granzymes and probably chaperones the proteases secreted extracellularly. To learn how the interaction of granzyme B (GrB) with serglycin might influence the apoptotic potential of this proteases, we have evaluated a model system where desalted CS is combined with isolated human granzyme. CS-GrB complexes were very stable, remaining undissociated in salt concentrations upwards to 500 mM (pH 7.4). On the basis of a capture enzyme immunoassay that accurately detects GrB, equivalent amounts of active free and CS-GrB, delivered by perforin or adenovirus, efficiently induced apoptosis in Jurkat cells and produced a similar time-dependent increase in caspase-3-like activity. CS-GrB processed isolated caspases-3 and -7 less efficiently than free granzyme. However, when added to cytosolic extracts, rates of processing were nearly equivalent for the two forms, suggesting cationic GrB may nonspecifically bind cytosolic proteins, leading to reduce proteolytic activity. Finally, GrB was found to be exocytosed from lymphocyte-activated killer cells as a neutral, high macromolecular weight complex, which possessed apoptotic activity. Collectively, the results indicate that neutral, high m.w. GrB has the capacity to induce cell death and will be useful to study the mechanism of cytotoxic cell-mediated apoptosis in vitro.     

Serglycin-independent Release of Active Mast Cell Proteases in Response to Toxoplasma gondii Infection

Earlier studies identified serglycin proteoglycan and its heparin chains to be important for storage and activity of mast cell proteases. However, the importance of serglycin for secretion and activity of mast cell proteases in response to parasite infection has been poorly investigated. To address this issue, we studied the effects on mast cell proteases in serglycin-deficient and wild type mice after peritoneal infection with the obligate intracellular parasite Toxoplasma gondii. In line with previous results, we found severely reduced levels of cell-bound mast cell proteases in both noninfected and infected serglycin-deficient mice. However, serglycin-deficient mice secreted mast cell proteases at wild type levels at the site of infection, and enzymatic activities associated with mast cell proteases were equally up-regulated in wild type and serglycin-deficient mice 48 h after infection. In both wild type and serglycin-deficient mice, parasite infection resulted in highly increased extracellular levels of glycosaminoglycans, including hyaluronan and chondroitin sulfate A, suggesting a role of these substances in the general defense mechanism. In contrast, heparan sulfate/heparin was almost undetectable in serglycin-deficient mice, and in wild type mice, it was mainly confined to the cellular fraction and was not increased upon infection. Furthermore, the heparan sulfate/heparin population was less sulfated in serglycin-deficient than in wild type mice indicative for the absence of heparin, which supports that heparin production is dependent on the serglycin core protein. Together, our results suggest that serglycin proteoglycan is dispensable for normal secretion and activity of mast cell proteases in response to peritoneal infection with T. gondii.     

Glycan and glycosaminoglycan binding properties of stromal cell-derived factor (SDF)-1alpha

We show here that cell surface glycosaminoglycans (GAGs) are involved in the binding of stromal cell-derived factor (SDF)-1alpha to CD4(+)lymphoid CEM or monocytic U937 cells, inasmuch as pretreating the cells with heparitinase or chondroitinase inhibits SDF-1alpha binding by 40-41% and 31-35%, respectively. Soluble heparin or chondroitin sulfate partially but significantly inhibits SDF-1alpha binding to the cells by 45-52% and 42-56%, respectively, while dextran has no significant effect. Taken together, these results indicate the role of GAGs in SDF-1alpha attachment to the cells. However, the effects of heparitinase and chondroitinase as well as those of heparin and chondroitin sulfate are not additive, which suggests that SDF-1alpha may attach to the cells through different GAGs, and also through other ligands. Soluble mannan also inhibits SDF-1alpha binding to the cells by 30-33%. Additivity between this effect and that of heparin or chondroitin sulfate is observed. Therefore, beside GAGs, mannose-containing species may also be involved in SDF-1alpha attachment to the cells. Accordingly, SDF-1alpha specifically binds to heparin-agarose and mannose-divinylsulfone agarose affinity matrices, and these interactions are inhibited respectively by soluble heparin, chondroitin sulfate, and mannan. We have previously shown that gp120 of X4 strain HIV-1LAI presents specific carbohydrate-binding properties for mannosylated derivatives, including mannan, and for GAGs including heparin. The present data therefore indicate that, in the same manner as HIV-1 Env, SDF-1alpha can interact with GAGs and glycans at the cell surface.     

Heparin affects the interaction of kininogen on endothelial cells

In the plasma kallikrein-kinin system, it has been shown that when plasma prekallikrein (PK) and high molecular weight kininogen (HK) assemble on endothelial cells, plasma kallikrein (huPK) becomes available to cleave HK, releasing bradykinin, a potent mediator of the inflammatory response. Because the formation of soluble glycosaminoglycans occurs concomitantly during the inflammatory processes, the effect of these polysaccharides on the interaction of HK on the cell surface or extracellular matrix (ECM) of two endothelial cell lines (ECV304 and RAEC) was investigated. In the presence of Zn⁺², HK binding to the surface or ECM of RAEC was abolished by heparin; reduced by heparan sulfate, keratan sulfate, chondroitin 4-sulfate or dermatan sulfate; and not affected by chondroitin 6-sulfate. By contrast, only heparin reduced HK binding to the ECV304 cell surface or ECM. Using heparin-correlated molecules such as low molecular weight dextran sulfate, low molecular weight heparin and N-desulfated heparin, we suggest that these effects were mainly dependent on the charge density and on the N-sulfated glucosamine present in heparin. Surprisingly, PK binding to cell- or ECM-bound-HK and PK activation was not modified by heparin. However, the hydrolysis of HK by huPK, releasing BK in the fluid phase, was augmented by this glycosaminoglycan in the presence of Zn²⁺. Thus, a functional dichotomy exists in which soluble glycosaminoglycans may possibly either increase or decrease the formation of BK. In conclusion, glycosaminoglycans that accumulated in inflammatory fluids or used as a therapeutic drug (e.g., heparin) could act as pro- or anti-inflammatory mediators depending on different factors within the cell environment.     

Expression of externally-disposed heparin/heparan sulfate binding sites by uterine epithelial cells

A class of high-affinity binding sites that preferentially bind heparin/heparan sulfate have been identified on the external surfaces of mouse uterine epithelial cells cultured in vitro. [3H]Heparin binding to these surfaces was time-dependent, saturable, and was blocked specifically by the inclusion of unlabeled heparin or endogenous heparan sulfate in the incubation medium. A variety of other glycosaminoglycans did not compete for these binding sites. The presence of sulfate on heparin influenced, but was not essential for, recognition of the polysaccharide by the cell surface binding sites. [3H]-Heparin bound to the cell surface was displaceable by unlabeled heparin, but not chondroitin sulfate. Treatment of intact cells on ice with trypsin markedly reduced [3H]heparin binding, indicating that a large fraction of the surface binding sites were associated with proteins. Scatchard analyses revealed a class of externally disposed binding sites for heparin/heparan sulfate exhibiting an apparent Kd of approximately 50 nM and present at a level of 1.3 x 10(6) sites per cell. Approximately 9-14% of the binding sites were detectable at the apical surface of cells cultured under polarized conditions in vitro. Detachment of cells from the substratum with EDTA stimulated [3H]heparin binding to cell surfaces. These observations suggested that most of the binding sites were basally distributed and were not primarily associated with the extracellular matrix. Collectively, these observations indicate that specific interactions with heparin/heparan sulfate containing molecules can take place at both the apical and basal cell surfaces of uterine epithelial cells. This may have important consequences with regard to embryo-uterine and epithelial-basal lamina interactions.     

Conformational Transitions in 3D Model of Bovine Testicular Hyaluronidase during Molecular Docking with Glycosaminoglycan Ligands

In silico molecular docking of the trimer repeating unit of chondroitin sulfate (sulfated hexasaccharide) and tetramer repeating unit of heparin (sulfated octasaccharide) to the 3D model of bovine testicular hyaluronidase by the methods of computational chemistry demonstrated the presence of eight significant binding sites for these ligands (cs1–cs8). The interaction of the active site of the enzyme with the heparin ligand, which inactivates the enzyme, and the protective effect of the chondroitin sulfate ligands bound to the surface sites of the biocatalyst molecule were theoretically studied using calculation approaches. We sequentially determined binding sites for the chondroitin sulfate ligands (in positions cs2, cs4, cs7, cs8 or cs1, cs2, cs4, cs7, cs8) critical for the protein structure stabilization, whose occupancy is theoretically sufficient to prevent irreversible deformations of the enzyme molecule when the heparin ligand is introduced into the cavity of its active site. Theoretical detection of these ‘sensibility points’ on the hyaluronidase globule indicates the possibility of regulating its functioning under the binding of the glycosaminoglycan ligands that initiate the fine formation of an effective type of the surface electrostatic potential. The interaction of the glycosaminoglycan ligands with hyaluronidase is mainly determined by electrostatic forces.     

Cell-associated proteoheparan sulfate mediates binding and uptake of thrombospondin in cultured porcine vascular endothelial cells.

[125I]Thrombospondin (TSP) binds to porcine endothelial cells in a specific, saturable and time-dependent fashion and is endocytosed by a receptor-mediated process. The N-terminal heparin-binding domain is necessary for the interaction with the cell surface. Binding and uptake is inhibited by heparin and to a much smaller extent by other vascular glycosaminoglycans. Chemical modification of lysine and arginine residues of TSP, but not treatment of the molecule with neuraminidase, resulted in a pronounced loss of binding at the cell surface. Treatment of cells with heparitinase but not with chondroitin ABC lyase caused inhibition of binding and uptake of TSP. Inhibition of sulfation of proteoglycans on the cell surface by chlorate leads to a dose and time-dependent inhibition of binding and degradation of TSP. In the presence of chlorate, newly synthesized TSP is not incorporated into the cell matrix but mainly released into the culture medium, whereas localization and incorporation of newly synthesized fibronectin is not altered. A cell surface proteoheparan sulfate was identified as TSP binding macromolecule by affinity chromatography. The data emphasize the role of heparan sulfate proteoglycan as a receptor-like molecule for the specific interaction with thrombospondin.     

Characterization of sugar binding by osteoclast inhibitory lectin

Osteoclast inhibitory lectin (OCIL) is a membrane-bound C-type lectin that blocks osteoclast differentiation and, via binding to its cognate receptor NKRP1D, inhibits natural killer cell-mediated cytotoxicity. OCIL is a member of the natural killer cell receptor C-type lectin group that includes CD69 and NKRP1D. We investigated carbohydrate binding of soluble recombinant human and mouse OCIL in enzyme-linked immunosorbent assay-based assays. OCIL bound immobilized high molecular weight sulfated glycosaminoglycans, including fucoidan, lambda-carrageenan, and dextran sulfate, but not unsulfated dextran or sialated hyaluronic acid. Carbohydrate binding was Ca(2+)-independent. Binding of immobilized low molecular weight glycosaminoglycans, including chondroitin sulfate (A, B, and C forms) and heparin, was not observed. However, the soluble forms of these low molecular weight glycosaminoglycans competed for OCIL binding of immobilized fucoidan (as did soluble fucoidan, dextran sulfate, and lambda-carrageenan), indicating that OCIL does recognize these carbohydrates. Inhibition constants for chondroitin sulfate A and heparin binding were 380 and 5 nm, respectively. Immobilized and soluble monosaccharides did not bind OCIL. The presence of saturating levels of fucoidan, dextran sulfate, and lambda-carrageenan did not affect OCIL inhibition of osteoclast formation. The fucoidan-binding lectins Ulex europaeus agglutinin I and Anguilla anguilla agglutinin did not block osteoclast formation or affect the inhibitory action of OCIL. Although the osteoclast inhibitory action of OCIL is independent of sugar recognition, we have found that OCIL, a lectin widely distributed, but notably localized in bone, skin, and other connective tissues, binds a range of physiologically important glycosaminoglycans, and this property may modulate OCIL actions upon other cells.     

Glycosaminoglycans interact selectively with chemokines and modulate receptor binding and cellular responses.

Chemokines selectively recruit and activate a variety of cells during inflammation. Interactions between cell surface glycosaminoglycans (GAGs) and chemokines drive the formation of haptotactic or immobilized gradients of chemokines at the site of inflammation, directing this recruitment. Chemokines bind to glycosaminoglycans on human umbilical vein endothelial cells (HUVECs) with affinities in the micromolar range: RANTES > MCP-1 > IL-8 > MIP-1alpha. This binding can be competed with by soluble glycosaminoglycans: heparin, heparin sulfate, chondroitin sulfate, and dermatan sulfate. RANTES binding showed the widest discrimination between glycosaminoglycans (700-fold), whereas MIP-1alpha was the least selective. Almost identical results were obtained in an assay using heparin sulfate beads as the source of immobilized glycosaminoglycan. The binding of chemokines to glycosaminoglycan fragments has a strong length dependence, and optimally requires both N- and O-sulfation. Isothermal titration calorimetry data confirm these results; IL-8 binds heparin fragments with a K(d) of 0.39-2.63 microM, and requires five saccharide units to bind each monomer of chemokine. In membranes from cells expressing the G-protein-coupled chemokine receptors CXCR1, CXCR2, and CCR1, soluble GAGs inhibit the binding of chemokine ligands to their receptors. Consistent with this, heparin and heparin sulfate could inhibit IL-8-induced neutrophil calcium flux. Chemokines can therefore form complexes with both cell surface and soluble GAGs; these interactions have different functions. Soluble GAG chemokines complexes are unable to bind the receptor, resulting in a block of the biological activity. Previously, we have shown that cell surface GAGs present chemokines to the G-protein-coupled receptors, by increasing the local concentration of protein. A model is presented which brings together all of these data. The selectivity in the chemokine-GAG interaction suggests selective disruption of the haptotactic gradient may be an achievable therapeutic approach in inflammatory disease.     

The relevance of glycosaminoglycan sulfates to Apo E induced lipid uptake by hepatocyte monolayers

The binding of Apolipoprotein E supplemented triglyceride emulsions to sulfated glycosaminoglycans demonstrated specificity for the carbohydrate polymers. Glucosamine containing glycosaminoglycans with relatively less sulfate had little affinity for the Apo E emulsion whereas those with more sulfate (i.e. heparin and sulfated heparans) effectively bound the emulsion. Galactosamine containing glycosaminoglycans (chondroitin 4 sulfate and dermatan sulfate) demonstrated no binding. The Apo E induced uptake of triglyceride emulsions by hepatocytes was inhibited by highly sulfated polysaccharides (i.e. heparin, dextran sulfate) but other glycosaminoglycans which did not bind the emulsion were ineffective in this inhibition. The same sulfated compounds which inhibited the hepatocyte Apo E emulsion interaction effectively released hepatic lipase from isolated heptic perfusions. Glycosaminoglycan sulfates which did not bind the Apo E supplemented emulsions and did not inhibit hepatocyte association were ineffective in releasing lipase. A heparan mixture isolated from human liver was much less effective in inhibiting Apo E induced association of emulsions with hepatocytes, than heparin. A highly sulfated octasaccharide fraction isolated from bovine liver heparin inhibited more effectively than the human heparans but less than the heparin. Inhibition of Apo E mediated hepatocyte emulsion association was produced by a one hour exposure of the cells to either heparinase or heparanase. The heparanase was more active than the heparinase and both were effective in the presence of protease inhibitors. Enzymes hydrolyzing chondroitin sulfates and hyaluronic acid were ineffective in inhibiting the Apo E induced association. The specific binding of human low density lipoprotein to the hepatocyte was much less effected by the heparanase exposure than the Apo E mediated binding.     

A novel mechanism for protein delivery: granzyme B undergoes electrostatic exchange from serglycin to target cells

The molecular interaction of secreted granzyme B-serglycin complexes with target cells remains undefined. Targets exposed to double-labeled granzyme B-serglycin complexes show solely the uptake of granzyme B. An in vitro model demonstrates the exchange of the granzyme from serglycin to immobilized, sulfated glycosaminoglycans. Using a combination of cell binding and internalization assays, granzyme B was found to exchange to sulfated glycosaminoglycans and, depending on the cell type, to higher affinity sites. Apoptosis induced by purified granzyme B and cytotoxic T-cells was diminished in targets with reduced cell surface glycosaminoglycan content. A mechanism of delivery is proposed entailing electrostatic transfer of granzyme B from serglycin to cell surface proteins.     

Bioengineered human heparin with anticoagulant activity

Heparin is a carbohydrate anticoagulant used clinically to prevent thrombosis, however impurities can limit its efficacy. Here we report the biosynthesis of heparin-like heparan sulfate via the recombinant expression of human serglycin in human cells. The expressed serglycin was also decorated with chondroitin/dermatan sulfate chains and the relative abundance of these glycosaminoglycan chains changed under different concentrations of glucose in the culture medium. The recombinantly expressed serglycin produced with 25 mM glucose present in the culture medium was found to possess anticoagulant activity one-seventh of that of porcine unfractionated heparin, demonstrating that bioengineered human heparin-like heparan sulfate may be a safe next-generation pharmaceutical heparin.     

Glycans are involved in RANTES binding to CCR5 positive as well as to CCR5 negative cells

We show that cell surface glycans, sialic acid and mannose-containing species, are involved beside glycosaminoglycans (GAGs), heparan sulfate and chondroitin sulfate in the binding of full length (1--68) RANTES not only to CCR5 positive human primary lymphocytes or macrophages but also to CCR5 negative monocytic U937 cells. Pretreating the cells with neuraminidase, heparitinase, chondroitinase or adding soluble glycans such as mannan or GAGs (heparin or chondroitin sulfate), significantly inhibited RANTES binding. Such effects were not observed with truncated (10--68) RANTES. Heat-denaturation of (1--68) RANTES strongly decreased its binding to the cells, demonstrating involvement of the three-dimensional structure. Accordingly, full length, but not truncated (10--68) RANTES, specifically bound to soluble mannan as well as to mannose-divinylsulfone-agarose affinity matrix and to soluble heparin or chondroitin sulfate as well as to heparin-agarose. Soluble heparin exerts, depending on its concentration, inhibitory or enhancing effects on RANTES binding to mannose-divinylsulfone-agarose, which indicates that RANTES interaction with glycans is modulated by GAGs. These data demonstrate that full length RANTES, but not its (10--68) truncated counterpart, interacts with glycans and GAGs, in soluble forms or presented either by affinity matrices or CCR5 positive as well as CCR5 negative cells.     

Role of glycosaminoglycans for binding and infection of hepatitis B virus

Many parts of the life cycle of hepatitis B virus (HBV) infection of hepatocytes have been unravelled, but the attachment and entry process leading to infection is largely unknown. Using primary Tupaia hepatocyte cultures as an in vitro infection system, we determined that HBV uses cell-surface heparan sulfate proteoglycans as low-affinity receptor, because HBV infection was inhibited by heparin (IC50: 5 μg ml⁻¹) or other higher-sulfated polymers, but not by lower-sulfated glycosaminoglycans, such as chondroitin sulfate. Pretreatment of primary hepatocytes with heparinase decreased viral binding and inhibited HBV infection completely. Interestingly, after preS1-dependent viral binding at 16°C to the cell surface, subsequent infection could still be inhibited by HBV preS1-lipopeptides, but not by heparin any more, suggesting a shift of the virus to a high-affinity receptor. In summary, we suggest following multistep attachment process: in vivo , HBV is initially trapped within the liver in the space of Dissé by heparan sulfate proteoglycans. Thereafter, HBV binds via its preS1 attachment site and the N-terminal myristic acid to a yet unknown, high-affinity receptor that confers uptake in a yet unknown compartment.     

Complexing of fibronectin glycosaminoglycans and collagen

Collagen-fibronectin complexes, formed by binding of fibronectin to gelatin or collagen insolubilized on Sepharose, were found to bind 20–40% of radioactivity in [³⁵S]heparin. Fibronectin attached directly to Sepharose also bound [³⁵S]heparin, while gelatin-Sepharose without fibronectin did not. Unlabeled heparin and highly sulfated heparan sulfate efficiently inhibited the binding of [³⁵S]heparin, hyaluronic acid and dermatan sulfate were slightly inhibitory, while chondroitin sulfates and heparan sulfate with a low sulfate content did not inhibit.The interaction of heparin with fibronectin bound to gelatin resulted in complexes which required higher concentrations of urea to dissociate than complexes of fibronectin and gelatin alone. Heparin as well as highly sulfated heparan sulfate and hyaluronic acid brought about agglutination of plastic beads coated with gelatin when fibronectin was present. Neither fibronectin nor glycosaminoglycans alone agglutinated the beads.It is proposed that the multiple interactions of fibronectin, collagen and glycosaminoglycans revealed in these assays could play a role in the deposition of these substances as an insoluble extracellular matrix. Alterations of the quality or quantity of any one of these components could have important effects on cell surface interactions, including the lack of cell surface fibronectin in malignant cells.     

Binding and endocytosis of thrombospondin and thrombospondin fragments in endothelial cell cultures analyzed by cuprolinic blue staining, colloidal gold labeling, and silver enhancement techniques

We investigated the distribution of thrombospondin-specific binding sites and the uptake of thrombospondin-gold conjugates in cultured porcine endothelial cells by light and electron microscopy. Colloidal gold marker and silver enhancement techniques were applied for cytochemical detection of monomeric thrombospondin and fragments of thrombospondin. Thrombospondin binds to granular and fibrillar structures and to sites of cell-cell contact on the cell surface, as indicated by many proteoglycan-cuprolinic blue precipitates. Cell migration tracks on the culture dish bottom are most heavily stained. Labeling of intact thrombospondin and of proteolytic fragments of thrombospondin with colloidal gold followed by silver intensification enables one to detect its binding and uptake in endothelial cells. Binding to the cell surface and uptake of thrombospondin-gold particles was inhibited by heparin but not by hyaluronic acid or chondroitin sulfate. The heparin binding region at the N-terminal end of the thrombospondin molecule proved to be essential for cell surface binding. Gold-conjugated thrombospondin fragments devoid of the heparin binding region were not internalized. After 60 min incubation at 37 degrees C, thrombospondin-gold particles accumulated in the lysosomal compartment close to the nucleus. In the presence of monensin and ammonium chloride, vesicles in this area are swollen and the concentration of particulate marker is reduced. Binding and uptake of thrombospondin by vascular endothelial cells appears to require linkage of the heparin binding region of the thrombospondin molecule to coated pits and heparan sulfate-rich molecules as receptors. Colloidal gold conjugation of thrombospondin fragments proved to be useful for cytochemical characterization of molecular domains.     

Detection of cell type and marker specificity of nuclear binding sites for anionic carbohydrate ligands

The emerging functionality of glycosaminoglycan chains engenders interest in localizing specific binding sites using cytochemical tools. We investigated nuclear binding of labeled heparin, heparan sulfate, a sulfated fucan, chondroitin sulfate, and hyaluronic acid in epidermal keratinocytes, bone marrow stromal cells, 3T3 fibroblasts and glioma cells using chemically prepared biotinylated probes. Binding of the markers was cell-type specific and influenced by extraction of histones, but was not markedly affected by degree of proliferation, differentiation or malignancy. Cell uptake of labeled heparin and other selected probes and their transport into the nucleus also was monitored. Differences between keratinocytes and bone marrow stromal cells were found. Preincubation of permeabilized bone marrow stromal cells with label-free heparin reduced the binding of carrier-immobilized hydrocortisone to its nuclear receptors. Thus, these tools enabled binding sites for glycosaminoglycans to be monitored in routine assays.     

Detection of cell type and marker specificity of nuclear binding sites for anionic carbohydrate ligands

The emerging functionality of glycosaminoglycan chains engenders interest in localizing specific binding sites using cytochemical tools. We investigated nuclear binding of labeled heparin, heparan sulfate, a sulfated fucan, chondroitin sulfate, and hyaluronic acid in epidermal keratinocytes, bone marrow stromal cells, 3T3 fibroblasts and glioma cells using chemically prepared biotinylated probes. Binding of the markers was cell-type specific and influenced by extraction of histones, but was not markedly affected by degree of proliferation, differentiation or malignancy. Cell uptake of labeled heparin and other selected probes and their transport into the nucleus also was monitored. Differences between keratinocytes and bone marrow stromal cells were found. Preincubation of permeabilized bone marrow stromal cells with label-free heparin reduced the binding of carrier-immobilized hydrocortisone to its nuclear receptors. Thus, these tools enabled binding sites for glycosaminoglycans to be monitored in routine assays.     

Detection of cell type and marker specificity of nuclear binding sites for anionic carbohydrate ligands.

The emerging functionality of glycosaminoglycan chains engenders interest in localizing specific binding sites using cytochemical tools. We investigated nuclear binding of labeled heparin, heparan sulfate, a sulfated fucan, chondroitin sulfate, and hyaluronic acid in epidermal keratinocytes, bone marrow stromal cells, 3T3 fibroblasts and glioma cells using chemically prepared biotinylated probes. Binding of the markers was cell-type specific and influenced by extraction of histones, but was not markedly affected by degree of proliferation, differentiation or malignancy. Cell uptake of labeled heparin and other selected probes and their transport into the nucleus also was monitored. Differences between keratinocytes and bone marrow stromal cells were found. Preincubation of permeabilized bone marrow stromal cells with label-free heparin reduced the binding of carrier-immobilized hydrocortisone to its nuclear receptors. Thus, these tools enabled binding sites for glycosaminoglycans to be monitored in routine assays.