heparitin-sulfate and Neuroblastoma

Initial studies described the significance of heparan sulfate proteoglycans of Balb/c 3T3 cells in their adhesion on fibronectin matrices, including their binding to multiple domains in FN, the importance of this binding in microfilament and close contact formation, and the cooperativity of both HS-PG and 140k glycoprotein integrin's binding to FN to achieve tight-focal contacts under cells. These analyses utilized model HS-binding proteins, such as platelet factor 4, and proteolytic fragments of FN with differing binding activities in both cell biological analyses of adhesion responses and in biochemical analyses of the HS-PG in the adhesion sites. In contrast, dermatan sulfate proteoglycans (DS-PG) inhibit 3T3 adhesion on FN but not on collagen; of special note is the discovery that certain integrin-binding fragments of FN also contain a third HS/DS-binding domain that is cryptic and that provides a more effective mechanism for inhibiting integrin: FN binding. Kirsten Ras oncogene-transformed 3T3 cells and their nude-mouse-derived primary or lung metastatic tumors are also being analyzed by similar approaches. HS-PGs in the adhesion sites of these tumor populations undergo extensive catabolism, resulting in alteration of their binding to FN affinity columns (and by implication alteration in adhesion responses of these tumor cells on FN matrices). Functions for HS-PG on the surface of neuronal cell derivatives, e.g., neuroblastoma cells derived from the neural crest of the embryo and potentially related in some ways to peripheral neurons, are also being explored. HS-binding fragments of FN or PF4 facilitate attachment and spreading of neuroblastoma cells but not neurite outgrowth, contrasting with the ability of dorsal root ganglion neurons to extend neurites on HS-binding substrata. The catabolism of HS-PG in neuroblastoma adhesion sites is minimal, indicating that this cannot be the explanation for incompetence in neurite extension. Neurite extension by neuroblastoma cells on FN results from three different and overlapping binding activities of non-PG receptors on the cell surface--RGDS-dependent binding to integrin, an RGDS-independent mechanism (perhaps a cell type-specific domain), and a ganglioside-dependent process. However, these neurite-extending reactions can be modulated either by exogenous addition of proteoglycans acting in a "trans" manner with the cell surface or by endogenous HG-PG acting in a "cis" manner with one or more of these recept

Topics: Axons; Cell Adhesion; Cell Line, Transformed; Chondroitin Sulfate Proteoglycans; Dermatan Sulfate; Fibroblasts; Genes, ras; Glycosaminoglycans; Heparan Sulfate Proteoglycans; Heparitin Sulfate; Kirsten murine sarcoma virus; Neuroblastoma; Proteoglycans; Tumor Cells, Cultured

The amyloid beta (Aβ) peptides (mainly Aβ40 and Aβ42), which are derived from the amyloid precursor protein (APP), can oligomerize into antibody A11-positive, neurotoxic species, believed to be involved in Alzheimer's disease. Interestingly, APP binds strongly to the heparan sulfate (HS) proteoglycan (PG) glypican-1 (Gpc-1) in vitro and both proteins are colocalized inside cells. In endosomes, APP is proteolytically processed to yield Aβ peptides. The HS chains of S-nitrosylated (SNO) Gpc-1 PG are cleaved into anhydromannose (anMan)-containing di- and oligosaccharides by an NO-dependent reaction in the same compartments. Here, we have studied the toxicity of oligomers/aggregates of Aβ40 and Aβ42, as well as Aβ40/42 mixtures that were formed in the presence of immobilized Gpc-1 PG or immobilized HS oligosaccharides. Afterwards, Aβ was displaced from the matrices, analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis and assayed for A11 immunoreactivity, for effects on growth of mouse N2a neuroblastoma cells and for membrane leakage in rat cortical neurons. HS generally promoted and accelerated Aβ multimerization into oligomers as well as larger aggregates that were mostly A11 positive and showed toxic effects. However, non-toxic Aβ was formed in the presence of Gpc-1 PG or when anMan-containing HS degradation products were simultaneously generated. Both toxic and non-toxic Aβ peptides were taken up by the cells but toxic forms appeared to enter the nuclei to a larger extent. The protection afforded by the presence of HS degradation products may reflect a normal intracellular function for the Aβ peptides.

Topics: Amyloid beta-Peptides; Animals; Cell Survival; Cerebral Cortex; Deamination; Glypicans; Heparitin Sulfate; Humans; Mice; Neuroblastoma; Neurons; Rats; Rats, Wistar

The S-nitrosylated proteoglycan glypican-1 recycles via endosomes where its heparan sulfate chains are degraded into anhydromannose-containing saccharides by NO-catalyzed deaminative cleavage. Because heparan sulfate chains can be associated with intracellular protein aggregates, glypican-1 autoprocessing may be involved in the clearance of misfolded recycling proteins. Here we have arrested and then reactivated NO-catalyzed cleavage in the absence or presence of proteasome inhibitors and analyzed the products present in endosomes or co-precipitating with proteasomes using metabolic radiolabeling and immunomagnet isolation as well as by confocal immunofluorescence microscopy. Upon reactivation of deaminative cleavage in T24 carcinoma cells, [(35)S]sulfate-labeled degradation products appeared in Rab7-positive vesicles and co-precipitated with a 20 S proteasome subunit. Simultaneous inhibition of proteasome activity resulted in a sustained accumulation of degradation products. We also demonstrated that the anhydromannose-containing heparan sulfate degradation products are detected by a hydrazide-based method that also identifies oxidized, i.e. carbonylated, proteins that are normally degraded in proteasomes. Upon inhibition of proteasome activity, pronounced colocalization between carbonyl-staining, anhydro-mannose-containing degradation products, and proteasomes was observed in both T24 carcinoma and N2a neuroblastoma cells. The deaminatively generated products that co-precipitated with the proteasomal subunit contained heparan sulfate but were larger than heparan sulfate oligosaccharides and resistant to both acid and alkali. However, proteolytic degradation released heparan sulfate oligosaccharides. In Niemann-Pick C-1 fibroblasts, where deaminative degradation of heparan sulfate is defective, carbonylated proteins were abundant. Moreover, when glypican-1 expression was silenced in normal fibroblasts, the level of carbonylated proteins increased raising the possibility that deaminative heparan sulfate degradation is involved in the clearance of misfolded proteins.

Topics: Animals; Carcinoma; Cell Line; Cell Line, Tumor; Cell Membrane; Endosomes; Fibroblasts; Glypicans; Heparitin Sulfate; Humans; Mice; Microscopy, Confocal; Models, Biological; Neuroblastoma; Oxidation-Reduction; Proteasome Endopeptidase Complex; Protein Binding; Proteins; Sulfates

Heparan sulfate chains have been found to be associated with amyloid deposits in a number of diseases including transmissible spongiform encephalopathies. Diverse lines of evidence have linked proteoglycans and their glycosaminoglycan chains, and especially heparan sulfate, to the metabolism of the prion protein isoforms. Glypicans are a family of glycosylphosphatidylinositol-anchored, heparan sulfate-containing, cell-associated proteoglycans. Cysteines in glypican-1 can become nitrosylated by endogenously produced nitric oxide. When glypican-1 is exposed to a reducing agent, such as ascorbate, nitric oxide is released and autocatalyses deaminative cleavage of heparan sulfate chains. These processes take place while glypican-1 recycles via a non-classical, caveolin-associated pathway. We have previously demonstrated that prion protein provides the Cu2+ ions required to nitrosylate thiol groups in the core protein of glypican-1. By using confocal immunofluorescence microscopy and immunomagnetic techniques, we now show that copper induces co-internalization of prion protein and glypican-1 from the cell surface to perinuclear compartments. We find that prion protein is controlling both the internalization of glypican-1 and its nitric oxide-dependent autoprocessing. Silencing glypican-1 expression has no effect on copper-stimulated prion protein endocytosis, but in cells expressing a prion protein construct lacking the copper binding domain internalization of glypican-1 is much reduced and autoprocessing is abrogated. We also demonstrate that heparan sulfate chains of glypican-1 are poorly degraded in prion null fibroblasts. The addition of either Cu2+ ions, nitric oxide donors, ascorbate or ectopic expression of prion protein restores heparan sulfate degradation. These results indicate that the interaction between glypican-1 and Cu2+-loaded prion protein is required both for co-internalization and glypican-1 self-pruning.

Topics: Animals; Blotting, Western; Caveolin 1; Cell Line, Tumor; Copper; Cytosol; Fluorescent Antibody Technique; Green Fluorescent Proteins; Heparan Sulfate Proteoglycans; Heparitin Sulfate; Immunomagnetic Separation; Immunoprecipitation; Mice; Neuroblastoma; Nitric Oxide; Prions; Protein Transport; RNA, Small Interfering; Transfection

During prion diseases the normal prion protein PrP(C) is refolded into an abnormal conformer PrP(Sc). We have studied the PrP(Sc) inhibiting activity of a library of synthetic heparan mimetic (HM) biopolymers. HMs are chemically derived dextrans obtained by successive substitutions with carboxymethyl, benzylamide, and sulfate groups on glucose residues. Some HMs eliminated PrP(Sc) from prion-infected cells after a 5 day course at 100 ng/ml and were 15 x potent than pentosan sulfate in this system. The anti-PrP(Sc) activity of HMs correlated with the degree of sulfation but was increased by benzylamidation. HMs did not reduce the synthesis of PrP(C) nor its attachment to lipid rafts, but instead blocked its conversion into PrP(Sc). The anti-PrP(Sc) HMs also prevented the uptake of prion rods by cultured cells. HMs may thus block the interaction of PrP(Sc) with a putative cellular receptor, possibly heparan sulfate. HMs provide an attractive chemical approach for the synthesis of TSE therapeutic and prophylactic reagents.

Topics: Animals; Biomimetic Materials; Biomimetics; Cells, Cultured; Dose-Response Relationship, Drug; Endocytosis; Heparitin Sulfate; Mice; Neuroblastoma; Peptide Library; PrPSc Proteins; Tumor Cells, Cultured

Gangliosides on the external side of the plasma membrane are important modulators of cellular functions. In previous work we had found that in cultured human SK-N-MC neuroblastoma cells a cell surface sialidase activity specifically cleaved terminal sialic acids from gangliosides, leading to a shift from higher sialylated species to GM1 and a decrease of GM3. To further elucidate the function of the enzyme, we have now examined the consequences of ganglioside sialidase inhibition. When present in the culture medium, the ganglioside sialidase inhibitors 2-deoxy-2,3-dehydro-N-acetylneuraminic acid (NeuAc2en), heparin, and heparan sulfate caused dramatic changes in cell behavior. Thus, the inhibitors uniformly led to a complete release from contact inhibition of growth, and to the loss of the differentiation markers neuron-specific enolase and neurofilaments, and a decrease of cyclic AMP. In presence of NeuAc2en, cells that normally were spread out evenly and were firmly attached, appeared smaller, rounded, and only loosely adherent to the culture vessel. Exogenous addition of vibrio cholerae sialidase mimicked the action of the plasma membrane ganglioside sialidase by retarding cell proliferation and increasing intracellular acetylcholinesterase. That the ganglioside sialidase inhibitors in the culture medium indeed affected solely the cell surface enzyme and not also a lysosomal sialidase, was demonstrated in an experiment where the desialylation of exogenously added radioactive gangliosides was determined in absence and presence of NeuAc2en and NH4Cl, an inhibitor of lysosomal function. Taken together, our results suggest that the ganglioside sialidase on the surface of SK-N-MC cells is responsible for growth control and differentiation in this neuronal cell line.

Topics: Antigens, Differentiation; Antigens, Surface; Cell Differentiation; Cell Division; Gangliosides; Heparin; Heparitin Sulfate; Humans; N-Acetylneuraminic Acid; Neuraminidase; Neuroblastoma; Tumor Cells, Cultured

Heparin sulfate proteoglycans are believed to play an important role in amyloidosis as pathologic chaperones. They bind to amyloidogenic proteins and may mediate the deposition and fibrillogenesis of amyloid at specific tissue sites. In the present study, we demonstrate that heparin sulfate glycosaminoglycan and proteoglycan both bind to the beta-amyloid peptide involved in Alzheimer's disease. The interaction of heparan sulfate proteoglycan and glycosaminoglycan can be inhibited by other sulfated compounds such as heparin, dextran sulfate and pentosan polysulfate. These polysaccharides which are currently used clinically, their derivatives or analogs may be effective as therapeutic agents to prevent or slow the progression of amyloidogenesis in Alzheimer's disease or other amyloidogenic disorders.

Topics: Amyloid beta-Peptides; Binding, Competitive; Chromatography, Affinity; Dextran Sulfate; Heparan Sulfate Proteoglycans; Heparin; Heparitin Sulfate; Neuroblastoma; Pentosan Sulfuric Polyester; Polysaccharides; Protein Binding; Proteoglycans; Structure-Activity Relationship; Sulfates

A human cell line committed to neuronal lineage was used to examine the influence of differentiation on proteoglycan synthesis and function. Where the LA-N-2 cells were stimulated to differentiate towards a phenotype of cholinergic neurons, proteoglycans of the heparan sulphate class increased relative to chondroitin sulphate proteoglycans and displayed more homogeneously shorter glycosaminoglycan chains with increasing degrees of sulphation. The changes were accompanied by increasing potency of the heparan sulphate proteoglycans in neurite growth-promoting activity when immobilized on a laminin substrate. These studies begin to address the role of activity-independent growth and differentiation on the synthesis and release by neurons of neurite growth-promoting proteoglycans. The observations have implications for understanding the role of proteoglycan overexpression and the production of dystrophic neurites in Alzheimer disease.

Topics: Brain Neoplasms; Cell Differentiation; Choline O-Acetyltransferase; Glycosaminoglycans; Heparitin Sulfate; Humans; Immunohistochemistry; Microscopy, Fluorescence; Neurites; Neuroblastoma; Neurons; Proteoglycans; Tumor Cells, Cultured

PrPSc is the only known component of the scrapie prion. The difference between PrPSc and its normal isoform PrPc is probably conformational, since no difference has been found in the amino acid sequence or postranslational modifications between both proteins. Heparan sulfate (HS) has been shown to be a component of amyloid plaques in a number of diseases including the prion diseases. We now present evidence that PrP can specifically bind to heparin-like compounds and that this interaction might have a physiological significance. HS can increase the concentration of PrP in normal neuroblastoma cells, whereas low molecular weight heparin (LMWH) does not. In contrast, LMWH and other heparin-like molecules, excluding HS, can inhibit the synthesis of PrPSc in scrapie infected cells and reverse their phenotype back to normal as judged by measurement of PrPSc by immunoblotting and by infectivity experiments. Whether an interaction between PrP and glycosaminoglycans plays a direct role in the conversion of PrPc into PrPSc remains to be established.

Topics: Amino Acids; Amyloid; Animals; Chlorates; Glycosaminoglycans; Heparin; Heparitin Sulfate; Immunohistochemistry; Isomerism; Mice; Neuroblastoma; Phenotype; Prions; Protein Conformation; PrPSc Proteins; Time Factors; Tumor Cells, Cultured

FN-C/H II is a heparin binding synthetic peptide from the C-terminal cell and heparin binding domain of fibronectin (FN) that mediates neuronal cell adhesion, spreading, and neurite outgrowth. Cellular interactions with FN-C/H II are inhibited by soluble heparin, suggesting that a cell-surface proteoglycan may mediate interactions with FN-C/H II (Haugen et al., 1990). To test this hypothesis further, heparan sulfate (HS) or chondroitin sulfate (CS) was removed from the cell surface by enzyme treatment. Heparitinase but not chondroitinase treatment of cells inhibited rat B104 neuroblastoma cell adhesion and spreading on FN-C/H II. Additionally, heparitinase treatment decreased the spreading of cells on the 33/66 kDa fragments containing the C-terminal heparin binding domain of FN. Furthermore, antibodies generated against a mouse melanoma HS proteoglycan (HSPG) inhibited B104 cell adhesion to FN-C/H II and the 33/66 kDa FN fragments. 35S-HSPG isolated from B104 cells directly bound to FN-C/H II both in solid phase assays and by affinity chromatography, but failed to bind to a control peptide from this region, CS1. The binding of 35S-HSPG was predominantly mediated by the HS and not the core protein of the HSPG. SDS-PAGE of iodinated HSPG demonstrated a single 78 kDa core protein following heparitinase digestion, which migrated at 51 kDa under nonreducing conditions. Anti-HSPG antibodies recognized the 78 kDa core protein by immunoblotting, and stained the surface of rat B104 neuroblastoma cells and cells of the primary neonatal rat nervous system. These results identify a cell-surface HSPG that likely mediates neuronal cell binding interactions with FN-C/H II.

Topics: Amino Acid Sequence; Animals; Binding Sites; Cell Adhesion; Cell Line; Cell Membrane; Cells, Cultured; Chromatography, Affinity; Fibronectins; Ganglia, Spinal; Heparan Sulfate Proteoglycans; Heparin; Heparitin Sulfate; Kinetics; Molecular Sequence Data; Molecular Weight; Neuroblastoma; Neurons; Peptide Fragments; Polysaccharide-Lyases; Proteoglycans; Rats; Spinal Cord

Subclones of F11 neuronal hybrid cells (neuroblastoma x dorsal root ganglion neurons) have segregated differing and/or overlapping neuritogenic mechanisms on three substrata--plasma fibronectin (pFN) with its multiple receptor activities, cholera toxin B subunit (CTB) for binding to ganglioside GM1, and platelet factor-4 (PF4) for binding to heparan sulfate proteoglycans. In this study, specific cell surface receptor activities for the three substrata were tested for their modulation during neuritogenesis by several experimental paradigms, using F11 subclones representative of three differentiation classes (neuritogenic on pFN only, on CTB only, or on all three substrata). When cycloheximide was included in the medium to inhibit protein synthesis during the active period, neurite formation increased significantly for all subclones on all three substrata, virtually eliminating substratum selectivity for differentiation mediated by cell surface integrin, ganglioside GM1, or heparan sulfate proteoglycans. Therefore, one or more labile proteins (referred to as disintegrins) must modulate functions of matrix receptors (e.g., integrins) mediating neurite formation. To verify whether cycloheximide-induced neuritogenesis was also regulated by integrin interaction with cell surface GM1, two approaches were used. When (Arg-Gly-Asp-Ser)-containing peptide A was added to the medium, it completely inhibited cycloheximide-induced neuritogenesis on all three substrata of all subclones, indicating stringent requirement for cell surface integrin function in these mechanisms. In contrast, when CTB or a monoclonal anti-GM1 antibody was also added to the medium, cycloheximide-induced neuritogenesis was amplified further on pFN and sensitivity to peptide A inhibition was abolished. Therefore, in some contexts ganglioside GM1 must complex with integrin receptors at the cell surface to modulate their function. These results also indicate that (a) cycloheximide treatment leads to loss of substratum selectivity in neuritogenesis, (b) this negative regulation of neurite outgrowth is affected by integrin receptor association with labile regulatory proteins (disintegrins) as well as with GM1, and (c) complexing of GM1 by multivalent GM1-binding proteins shifts neuritogenesis from an RGDS-dependent integrin mechanism to an RGDS-independent receptor mechanism.

Topics: Amino Acid Sequence; Animals; Axons; Cell Differentiation; Cell Line; Cholera Toxin; Chondroitin Sulfate Proteoglycans; Cycloheximide; Fibronectins; G(M1) Ganglioside; Ganglia, Spinal; Heparan Sulfate Proteoglycans; Heparitin Sulfate; Hybrid Cells; Integrins; Mice; Molecular Sequence Data; Neuroblastoma; Neurons; Oligopeptides; Platelet Factor 4; Rats; Receptors, Fibronectin; Receptors, Immunologic

Human neuroblastoma cells (Platt and La-N1) adhere and extend neurites on a ganglioside GM1-binding substratum provided by cholera toxin B (CTB). These adhesive responses, similar to those on plasma fibronectin (pFN), require the mediation of one or more cell-surface proteins [G. Mugnai and L. A. Culp (1987) Exp. Cell Res. 169, 328]. The involvement of two pFN receptor molecules in ganglioside GM1-mediated responses on CTB have now been tested. In order to test the role of cellular FN binding to its glycoprotein receptor integrin, a soluble peptide containing the Arg-Gly-Asp-Ser (RGDS) sequence was added to the medium. It did not inhibit attachment on CTB but completely inhibited formation of neurites; in contrast, the RGDS peptide minimally inhibited attachment or neurite formation on pFN. Once formed, neurites on CTB became resistant to the peptide. In order to test the role of cell-surface heparan sulfate proteoglycan (HS-PG), two approaches were used. First, the HS-binding protein platelet factor-4 (PF4) was used to dilute CTB or pFN on the substratum or, alternatively, added to the medium. Diluting the substratum ligand with PF4 had no effects on attachment on either CTB or pFN. However, neurite formation on CTB was readily inhibited and on pFN partially inhibited; the effects of PF4 were far greater than a similar dilution with nonbinding albumin. When PF4 was added to the medium of cells, attachment on either substratum was unaffected as was neurite outgrowth on pFN, revealing differences in PF4's inhibition as the substratum-bound or medium-borne component. In contrast, PF4 in the medium at low concentrations (1 microgram/ml) was highly inhibitory for neurite formation on CTB. The second approach utilized the addition of bovine cartilage dermatan sulfate proteoglycan (DS-PG), shown to bind to pFN as well as to substratum-bound CTB by ELISA, or cartilage chondroitin sulfate/keratan sulfate proteoglycan (CS/KS-PG) to the substratum or to the medium. At low concentrations, DS-PG but not CS/KS-PG actually stimulated neurite formation on CTB while at higher concentrations DS-PG completely inhibited attachment and neurite formation. While DS-PG partially inhibited attachment on pFN, it had no effect on neurite formation of the attached cells. Neuroblastoma cells adhered to some extent to substrata coated only with DS-PG, indicating "receptors" for PGs that permit stable interaction.(ABSTRACT TRUNCATED AT 400 WORDS)

Topics: Axons; Cell Adhesion; Cholera Toxin; Chondroitin Sulfate Proteoglycans; Dermatan Sulfate; Enzyme-Linked Immunosorbent Assay; Fibronectins; G(M1) Ganglioside; Heparan Sulfate Proteoglycans; Heparitin Sulfate; Humans; Neuroblastoma; Neurons; Oligopeptides; Platelet Aggregation Inhibitors; Proteoglycans; Receptors, Fibronectin; Receptors, Immunologic; Tumor Cells, Cultured

Tissue culture substratum adhesion sites from EGTA-detached Platt human neuroblastoma cells were extracted with a buffer containing ocytlglucoside, NaCl, guanidine hydrochloride, and a variety of protease inhibitors, an extraction which resulted in quantitative solubilization of the 35SO4 = -radiolabeled proteoglycans and 3H-leucine-radiolabeled proteins. Of the sulfate-radiolabeled material, the vast majority was heparan sulfate proteoglycan (Kav = 0.15 on Sepharose C14B columns) and the remainder was chondroitin sulfate chains (no single chains of heparan sulfate were observed). This extract was then fractionated on DEAE-Sephadex columns under two different buffer elution conditions. Under DEAE-I conditions in low ionic strength acetate buffer, two major peaks of 35SO4 = -radiolabeled material (A,B) and a minor peak (C) could be resolved in the NaCl gradient; however, three-fourths of the material required 4 M guanidine hydrochloride to elute it from the column (peak D). Under DEAE-II conditions in acetate buffer supplemented with 8 M urea, the vast majority of the proteoglycan material could be eluted in the NaCl gradient as peak AB. Peak D material was shown to contain aggregated proteoglycan, along with nonproteoglycan protein, which high concentrations of urea or guanidine could dissociate, but not nonionic or zwitterionic detergents. Three different affinity chromatography systems were used to further characterize these components. Approximately 60% of peak A heparan sulfate proteoglycan from DEAE-I binds to the hydrophobic matrix, octyl-Sepharose, while 80% of the proteoglycan in DEAE-I peak D binds to this hydrophobic column. A sizable fraction of peak A proteoglycan fails to bind to plasma fibronectin but does bind to platelet factor-4 affinity columns. In contrast, peak AB proteoglycan from DEAE-II columns yields a much higher proportion of molecules which do bind to fibronectin. To examine the basis for these differences in affinity binding, nonproteoglycan protein from these adhesion sites was mixed with peak AB proteoglycan prior to affinity chromatography; proteoglycan binding to fibronectin decreased markedly while binding to platelet factor-4 was unaffected. This modulating activity involves the binding of nonproteoglycan protein in adhesion site extracts to both fibronectin on the column, as well as to heparan sulfate proteoglycan itself, and it could not be mimicked by a number of known proteins in adhesion site extracts or several othe

Topics: Cell Adhesion; Cell Line; Chondroitin Sulfate Proteoglycans; Chromatography, Affinity; Chromatography, Gel; Fibronectins; Glycosaminoglycans; Heparan Sulfate Proteoglycans; Heparitin Sulfate; Humans; Neuroblastoma; Proteoglycans

We have examined the size, charge, and sulfation pattern of heparan sulfate in the cell-soluble fraction, membranes, and culture medium of PC12 pheochromocytoma cells cultured in the presence and absence of nerve growth factor (NGF) and compared the structural features of PC12 cell heparan sulfate to that of rat brain at several stages of early postnatal development. Nitrous acid degradation studies revealed significant differences in the distribution of N-sulfate and N-acetyl groups in heparan sulfate present in the PC12 cell-soluble fraction, membranes, and medium and demonstrated that NGF treatment led to an increased proportion of N-sulfated segments in the cell-associated heparan sulfate, although no such change was seen in that released into the culture medium. There was very little change in the N-sulfation of brain heparan sulfate during the first 30 days after birth. In brain, most of the heparan sulfate glucosamine residues are N-sulfated and yield predominantly di- and tetrasaccharide nitrous acid degradation products, whereas PC12 cell heparan sulfate contains large blocks of N-acetylglucosamine residues. There was very little difference in the overall charge or size (approximately 15,000 Da) of heparan sulfate chains between the different PC12 cell fractions or brain, although NGF treatment led to a decrease in the proportion of less-charged chains in the PC12 cell membranes and a small increase in molecular size. Our studies therefore demonstrate the presence in PC12 cells of several pools of heparan sulfate having different structural properties, and that significant alterations in the charge, size, and sulfation pattern of PC12 cell heparan sulfate accompany NGF-induced differentiation and neurite outgrowth.

Topics: Adrenal Gland Neoplasms; Age Factors; Animals; Brain; Cell Adhesion; Cell Differentiation; Cell Line; Glycosaminoglycans; Heparitin Sulfate; Molecular Weight; Nerve Growth Factors; Neuroblastoma; Nitrous Acid; Pheochromocytoma; Rats

Topics: Carbohydrate Sequence; Electrophoresis, Polyacrylamide Gel; Glycosaminoglycans; Heparitin Sulfate; Humans; Neuroblastoma; Proteoglycans

The chemical heterogeneity of radiolabelled neuroblastoma heparan sulfate has been studied by ion exchange chromatography and by affinity chromatography on heparan sulfate-agarose. Although the entire population of chains shows considerable homogeneity in charge density, the deaminative cleavage products ranged in size from disaccharides to eicosasaccharides. Under appropriate conditions neuroblastoma heparan sulfate could be separated into two pools of low or high affinity for lung heparan sulfate-agarose. Analyses of periodate oxidation-alkaline elimination indicated that the high affinity chains contained larger proportions of heparin-like segments, i.e. iduronate-rich and N-sulfated ones.

Topics: Cell Line; Chromatography, Affinity; Chromatography, Ion Exchange; Glycosaminoglycans; Heparitin Sulfate; Humans; Neuroblastoma

Human and rat neuroblastoma cells extend neurites over plasma fibronectin (pFN)-coated substrata. For resolution of which fibronectin binding activities (the cell-binding domain (CBD), the heparan sulfate-binding domains, or a combination of the two) are responsible for neurite outgrowth, CBD was prepared free of heparan sulfate-binding activity as described by Pierschbacher et al. (Cell 26 (1981) 259-267). Neuroblastoma cells attached and extended neurites as stably and as effectively on CBD-coated substrata as on intact pFN, while cytoplasmic spreading was more extensive on pFN-coated substrata. The structures of growth cones on CBD or pFN were virtually identical. On substrata coated with the model heparan sulfate-binding protein, platelet factor 4 (PF4), cells attached and spread somewhat but never extended neurites. When cells were challenged with substrata coated with various ratios of CBD and PF4, PF4 was found to be an effective inhibitor of CBD-mediated neurite extension. Similarly, cells grown on substrata coated at different locations with CBD or PF4 in order to evaluate topographical dependence of growth cone formation extended neurites only onto the CBD-coated region or along the interface between these two proteins, but never onto the PF4 side of cells that bridged the interface. These studies indicate that (a) the CBD activity of pFN, and not its heparan sulfate-binding activity, is the critical determinant in neurite extension of these neural tumor cells from the central nervous system; (b) under some circumstances, heparan sulfate-binding activity can be antagonistic to neurite extension; (c) the chemical nature of the substratum controls the direction of neurite extension; (d) these neuroblastoma cells respond to these binding proteins very differently than fibroblasts or neurons from the peripheral nervous system.

Topics: Animals; Cell Adhesion; Chymotrypsin; Egtazic Acid; Fibronectins; Fluorescent Antibody Technique; Glycosaminoglycans; Heparitin Sulfate; Humans; Neuroblastoma; Neurons; Platelet Factor 4; Rats

Biosynthetically radiolabelled heparan sulphate proteoglycans have been isolated from the growth medium and the cell lysate of a human neuroblastoma cell line (CHP100). Chromatography on Sepharose CL-4B identified two heparan sulphate proteoglycans in the medium (Kav 0.220 and 0.389), whereas in the cell lysate the major proteoglycan species were more heterogenous and of a smaller overall molecular size (Kav 0.407) than the medium-derived counterparts. Chromatography on Sepharose CL-6B of free heparan sulphate glycosaminoglycan chains showed that the majority of cell-layer-derived material heparan sulphate 2, Kav = 0.509) was smaller than medium heparan sulphates (heparan sulphate 1 and heparan sulphate 2, Kav 0.230 and 0.317). Analysis of the patterns of polymer sulphation by nitrous acid treatment, gel chromatography and high-voltage electrophoresis established that in each heparan sulphate fraction there was on average 1.1 sulphate residues per disaccharide with an N:O sulphate ratio of 1.1. Heparan sulphate in the medium had a high proportion of di-O-sulphated disaccharides in regions of the chain with repeat disaccharide sequences of structure GlcA-GlcNSO3, whereas cell-associated material was enriched in di-O-sulphated tetrasaccharides of alternating sequences GlcA-GlcNAc-GlcA-GlcNSO3. The identification of several populations of heparan sulphate proteoglycans differing in molecular size and glycosaminoglycan fine structure may reflect the functional diversity of this family of macromolecules in the nervous system.

Topics: Cell Line; Centrifugation, Density Gradient; Chondroitin Sulfate Proteoglycans; Chromatography, Gel; Glucosamine; Glycosaminoglycans; Heparan Sulfate Proteoglycans; Heparitin Sulfate; Humans; Neuroblastoma; Proteoglycans; Sulfates; Sulfur Radioisotopes; Tritium

Human neuroblastoma cells (Platt) were detached from tissue culture substrata with a Ca2+ chelating agent, and then the suspended cells were extracted with a sodium dodecyl sulfate (SDS)-containing buffer to maximally solubilize their sulfate-radiolabeled proteoglycans. The majority of the high-molecular-weight material in these dissociative extracts was heparan sulfate proteoglycan, which resolves into two heterodisperse size classes upon gel filtration on columns of Sepharose CL4B. After removal of SDS from these extracts by hydrophobic chromatography on Sep-Pak C18 cartridges, extracts were further fractionated on various affinity matrices. All of the sulfate-radiolabeled material eluted as one peak from DEAE-Sephadex ion-exchange columns. In contrast, affinity fractionation on Sepharose columns derivatized with the heparan sulfate-binding protein, platelet factor-4, resolved three major and one minor subsets of these components. The nonbinding fraction contained some heparan sulfate proteoglycan and some chondroitin sulfate. The weak-binding fraction contained principally heparan sulfate proteoglycan, as well as a small amount of chondroitin sulfate proteoglycan; the gel-filtration properties of these proteoglycans before or after alkaline borohydride treatment indicated that they were small in size, containing perhaps 2 to 4 glycosaminoglycan chains. The high-affinity fraction eluted from platelet factor 4-Sepharose was composed entirely of "single-chain" heparan sulfate. A portion of the heparan sulfate proteoglycan of the original extract bound to the hydrophobic affinity matrix, octyl-Sepharose, and this hydrophobic proteoglycan partitioned into the nonbinding and weak-binding fractions of the platelet factor 4-Sepharose affinity columns. These studies reveal that the majority of the proteoglycan made by these neuronal cells in culture is of the heparan sulfate class, is small in size when compared to other characterized proteoglycans, and can be resolved into several overlapping subsets when fractionated on affinity matrices.

Topics: Calcium; Cell Line; Chromatography, Affinity; Chromatography, Gel; Chromatography, Ion Exchange; Glycosaminoglycans; Heparitin Sulfate; Humans; Neuroblastoma; Neurons; Proteoglycans

Confluent cultures of a human neuroblastoma cell line (CHP100) were incubated for 48 h with D-[1-3H]glucosamine and sodium [35S]sulphate. Radioactive glycosaminoglycans were analysed in the growth medium, rapid trypsin digest of the cell monolayer and a 1% (w/v) Triton/0.5 M NaOH extract of the final cell pellet. Sulphated glycosaminoglycans co-chromatographed when eluted by NaCl gradient from DEAE-cellulose. The medium contained mainly chondroitin sulphates, whereas the cell surface was enriched in heparan sulphate. Heparan sulphate was isolated as chondroitinase ABC-resistant material and treated with nitrous acid. Analysis of the scission products on Bio-Gel P-10 yielded fragments varying in size from single disaccharides to glycans consisting of nine disaccharide units. Cell-surface and medium heparan sulphate had respectively 52% and 54% N-sulphated glucosamine residues distributed in similar patterns along the polymer chain. The N:O-sulphate ratio of neuroblastoma heparan sulphate was 1.1:1. Analysis by high-voltage electrophoresis of di- and tetrasaccharide products produced by nitrous acid treatment showed that the distribution of 'O'-sulphate groups differed strikingly between heparan sulphates from the medium and cell-surface compartments. A di-O-sulphated tetrasaccharide was identified in both heparan sulphate species. The absence of detectable amounts of 35[S]sulphate associated with fragments larger than tetrasaccharide supports the close topographical association of N-sulphate and O-sulphate groups.

Topics: Cells, Cultured; Chondroitin Lyases; Chromatography, Gel; Glycosaminoglycans; Heparitin Sulfate; Humans; Molecular Weight; Neuroblastoma; Sulfates; Tissue Distribution

Beta-Xylosides stimulate 2- to 6-fold the synthesis of glycosaminoglycans by three types of nonconnective tissue cells (RG-C6, NB41A, and rat hepatoma cells, and normal and simian virus 40 (SV40)-transformed normal human skin fibroblasts. The effect, which is specific for the anomeric linkage and the glycone, is observed in the presence and absence of puromycin. Beta-Xylosides may substitute for xylosylated core protein as initiators of synthesis of chondroitin sulfate chains. No stimulation of synthesis of heparan sulfate was observed. With the use of a fluorogenic xyloside, 4-methylumbelliferyl-beta-D-xyloside, it was demonstrated that the free chondroitin sulfate chains secreted into the medium bear the xyloside at the reducing end, and have an average molecular weight of 16,500.

Topics: Animals; Carcinoma, Hepatocellular; Cells, Cultured; Chondroitin; Chromatography, Gel; Dermatan Sulfate; Fibroblasts; Galactose; Glycosaminoglycans; Glycosides; Heparitin Sulfate; Humans; Hyaluronic Acid; Hymecromone; Liver Neoplasms; Mice; Neuroblastoma; Neuroglia; Nitrophenols; Rats; Simian virus 40; Skin; Spectrometry, Fluorescence; Sulfur Radioisotopes; Sulfuric Acids; Tritium; Xylose