Chemotherapy-induced mobilization of karyotypically normal PBSC for autografting in CML

Heparan sulfate species expressed by different cell and tissue types differ in their structural and functional properties. Limited information is available on differences in regulation of heparan sulfate biosynthesis within a single tissue or cell population under different conditions. We have approached this question by studying the effect of cell differentiation on the biosynthesis and function of heparan sulfate in human colon carcinoma cells (CaCo-2). These cells undergo spontaneous differentiation in culture when grown on semipermeable supports; the differentiated cells show phenotypic similarity to small intestine enterocytes. Metabolically labeled heparan sulfate was isolated from the apical and basolateral media from cultures of differentiated and undifferentiated cells. Compositional analysis of disaccharides, derived from the contiguous N-sulfated regions of heparan sulfate, indicated a greater proportion of 2-O-sulfated iduronic acid units and a smaller amount of 6-O-sulfated glucosamine units in differentiated than in undifferentiated cells. By contrast, the overall degree of sulfation, the chain length and the size distribution of the N-acetylated regions were similar regardless the differentiation status of the cells. The structural changes were found to affect the binding of heparan sulfate to the long isoform of platelet-derived growth factor A chain but not to fibroblast growth factor 2. These findings show that heparan sulfate structures change during cell differentiation and that heparan sulfate-growth factor interactions may be affected by such changes.     

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.     

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.     

Up-regulation of insulin/insulin-like growth factor-I hybrid receptors during differentiation of HT29-D4 human colonic carcinoma cells

To assess the autocrine function of insulin-like growth factor II (IGF-II) in the balance of proliferation and differentiation in HT29-D4 human colonic cancer cells, we studied the expression of IGF-I receptors (IGF-IR) and insulin receptors (IR) in relation to the state of cell differentiation. IGF-IR and IR were expressed in both undifferentiated and enterocyte-like differentiated HT29-D4 cells. IGF-IR had two isoforms with a 97-kDa and a 102-kDa beta-subunit. In addition, HT29-D4 cells expressed hybrid receptors (HR) formed by the association of two alphabeta heterodimers from both IR and IGF-IR. HR were evidenced through 1) inhibition of IGF-I binding by the B6 anti-IR antibody and 2) immunoprecipitation with the alpha-IR3 anti-IGF-IR antibody, which revealed an additional 95-kDa IR beta-subunit that disappeared when the heterotetrameric receptor was dissociated by disulfide reduction into alphabeta heterodimers before immunoprecipitation. Like IGF-IR, HR had a high affinity for IGF-I (Kd, approximately 1.5 nM), but did not bind insulin significantly; the latter interacted with the native IR only (Kd, approximately 4 nM). In the differentiated HT29-D4 cell monolayer, all receptor species were strongly polarized (>97%) toward the basolateral membrane. Moreover, HT29-D4 cell differentiation was accompanied by an approximately 2-fold increase in the number of IR, whereas the number of IGF-I-binding sites was unaltered. However, in differentiated HT29-D4 cells, approximately 55% of the latter were involved in HR vs. approximately 20% in undifferentiated HT29-D4 cells. Thus, HT29-D4 cell differentiation is characterized by an up-regulation (approximately 3-fold) of the level of HR coupled to a down-regulation (approximately 40%) of the level of native tetrameric IGF-IR. Alterations were induced early during the cell differentiation process, i.e. 5 days postconfluence, and remained unchanged for at least 21 days. Taken together, these results suggest that the IGF-II autocrine loop in HT29-D4 cells may trigger distinct signaling pathways if it activates native IGF-IR, which predominate in undifferentiated cells, or if it activates HR, which are up-regulated in differentiated cells.     

OCI-5/rat glypican-3 binds to fibroblast growth factor-2 but not to insulin-like growth factor-2

OCI-5 encodes the rat homologue of glypican-3, a membrane-bound heparan sulfate proteoglycan that is mutated in the Simpson-Golabi-Behmel overgrowth syndrome. OCI-5 and glypican-3 are 95% identical. It has been recently suggested that glypican-3 interacts with insulin-like growth factor-2 (IGF-2) and that this interaction regulates IGF-2 activity. We report here that we have transfected OCI-5 into two different cell lines, and we have not been able to detect an interaction between the OCI-5 proteoglycan produced by the transfected cells and IGF-2. On the other hand, we have found that OCI-5 interacts with FGF-2, as has already been shown for glypican-1. This interaction is mediated by the heparan sulfate chains of OCI-5 because it can be inhibited by heparin or by heparitinase.     

Comparative acute toxicity and primary irritancy of the ethylidene and vinyl isomers of norbornene

Perlecan and aggrecan are proteoglycans that receive primarily heparan sulfate and chondroitin sulfate side chains, respectively. Their large multidomained core proteins have little or no homology to each other and their glycosaminoglycan (GAG) attachment sites are restricted to certain domains only. We examined the involvement of the non-GAG bearing domains in designating the GAG type added to the GAG attachment domain by preparing cDNA constructs that expressed perlecan/aggrecan chimeras as recombinant products in COS-7 cells and then determining the size and GAG composition of the recombinant products. The results showed that domain I of perlecan receives primarily (73-81%) heparan sulfate when coupled with domain II and III of perlecan, but when coupled with the G3 domain of aggrecan, it receives primarily (59-63%) chondroitin sulfate. Furthermore, the chondroitin sulfate attachment region of aggrecan received GAG side chains more readily when coupled to the G3 domain of aggrecan than when coupled to domains II and III of perlecan. The GAG side chains on all these recombinant products were small and similar in size. These findings indicate that the utilization of attachment sites for heparan and chondroitin sulfate or the sulfation of these GAGs can be influenced, in part, by non-GAG bearing domains.     

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.     

Heparan sulfate proteoglycans as adhesive and anti-invasive molecules. Syndecans and glypican have distinct functions

ARH-77 cells do not adhere to type I collagen and readily invade into collagen gels, but following expression of the transmembrane heparan sulfate proteoglycan syndecan-1, they bind collagen and fail to invade. We now show that cells transfected with syndecan-2 or syndecan-4 also bind collagen and are non-invasive. In contrast, cells transfected with the glycosylphosphatidylinositol-anchored proteoglycan glypican-1 do not bind to collagen and remain invasive, even though glypican- and syndecan-expressing cells have similar surface levels of heparan sulfate, and their proteoglycans have similar affinities for collagen. Analysis of cells expressing syndecan-1-glypican-1 chimeric proteoglycans reveals that inhibition of invasion requires the extracellular domain of syndecan but not its transmembrane or cytoplasmic domain. Surprisingly, cells bearing a chimera composed of the glypican extracellular domain fused to the syndecan transmembrane and cytoplasmic domains bind to collagen but remain invasive, implying that adhesion to collagen is not by itself sufficient to inhibit invasion. Apparently, the extracellular domain of syndecan-1, presumably by interacting with cell-surface signal transducing molecules, directly regulates complex cell behaviors such as motility and invasiveness. These results also show for the first time that syndecans and glypicans can have distinct functions, even when expressed by the same cell type.     

Age-dependent modulation of heparan sulfate structure and function

Heparan sulfate interacts with growth factors, matrix components, effectors and modulators of enzymatic catalysis as well as with microbial proteins via sulfated oligosaccharide domains. Although a number of such domains have been characterized, little is known about the regulation of their formation in vivo. Here we show that the structure of human aorta heparan sulfate is gradually modulated during aging in a manner that gives rise to markedly enhanced binding to isoforms of platelet-derived growth factor A and B chains containing polybasic cell retention sequences. By contrast, the binding to fibroblast growth factor 2 is affected to a much lesser extent. The enhanced binding of aorta heparan sulfate to platelet-derived growth factor is suggested to be due to an age-dependent increase of GlcN 6-O-sulfation, resulting in increased abundance of the trisulfated L-iduronic acid (2-OSO3)-GlcNSO3(6-OSO3) disaccharide unit. Such units have been shown to hallmark the platelet-derived growth factor A chain-binding site in heparan sulfate.     

Glypican and biglycan in the nuclei of neurons and glioma cells: presence of functional nuclear localization signals and dynamic changes in glypican during the cell cycle

We have investigated the expression patterns and subcellular localization in nervous tissue of glypican, a major glycosylphosphatidylinositol-anchored heparan sulfate proteoglycan that is predominantly synthesized by neurons, and of biglycan, a small, leucine-rich chondroitin sulfate proteoglycan. By laser scanning confocal microscopy of rat central nervous tissue and C6 glioma cells, we found that a significant portion of the glypican and biglycan immunoreactivity colocalized with nuclear staining by propidium iodide and was also seen in isolated nuclei. In certain regions, staining was selective, insofar as glypican and biglycan immunoreactivity in the nucleus was seen predominantly in a subpopulation of large spinal cord neurons. The amino acid sequences of both proteoglycans contain potential nuclear localization signals, and these were demonstrated to be functional based on their ability to target beta-galactosidase fusion proteins to the nuclei of transfected 293 cells. Nuclear localization of glypican beta-galactosidase or Fc fusion proteins in transfected 293 cells and C6 glioma cells was greatly reduced or abolished after mutation of the basic amino acids or deletion of the sequence containing the nuclear localization signal, and no nuclear staining was seen in the case of heparan sulfate and chondroitin sulfate proteoglycans that do not possess a nuclear localization signal, such as syndecan-3 or decorin (which is closely related in structure to biglycan). Transfection of COS-1 cells with an epitope-tagged glypican cDNA demonstrated transport of the full-length proteoglycan to the nucleus, and there are also dynamic changes in the pattern of glypican immunoreactivity in the nucleus of C6 cells both during cell division and correlated with different phases of the cell cycle. Our data therefore suggest that in certain cells and central nervous system regions, glypican and biglycan may be involved in the regulation of cell division and survival by directly participating in nuclear processes.     

Eukaryotic expression of recombinant biglycan. Post-translational processing and the importance of secondary structure for biological activity

Biglycan is a small chondroitin sulfate proteoglycan found in many tissues and is structurally related to decorin, fibromodulin, and lumican. The biological function of biglycan is poorly understood, although several studies have indicated interaction with other extracellular matrix components. We have initiated studies of structural and functional domains of biglycan by transient eukaryotic expression using the vaccinia virus/T7 bacteriophage expression system. A recombinant vaccinia virus, vBGN4 encoding the mature biglycan core protein as a polyhistidine fusion protein under control of the T7 phage promoter was expressed in HT-1080 cells and UMR106 cells. The structure of the recombinant biglycan secreted by these cells was defined by analyzing molecules labeled in the presence of [35S]sulfate, [3H]glucosamine, and [35S]methionine. Glycoforms of biglycan were separated by imidazole gradient elution, under non-denaturing conditions, and comprised: a large proteoglycan form substituted with two chondroitin sulfate chains of molecular mass approximately 34 kDa (HT-1080 cells) or approximately 40 kDa (UMR106 cells); a small proteoglycan form substituted with two chondroitin sulfate chains with a median molecular mass approximately 28 kDa; and a core protein form secreted devoid of glycosaminoglycan chains. All the glycoforms were substituted with two N-linked oligosaccharides, and the disaccharide composition of the two glycosaminoglycan populations were identical. Approximately 70% of the recombinant biglycan secreted by HT-1080 cells was substituted with chondroitin sulfate chains, whereas about 50% of the biglycan expressed by UMR106 cells was substituted with chondroitin sulfate chains. Infection with vBGN4 in both HT-1080 and UMR106 cells resulted in the production of approximately 10 mg of biglycan/10(9) cells per 24 h. The native recombinant biglycan was shown to bind to collagen type V and the complement protein, C1q. However, when the secondary structure of recombinant biglycan was disrupted by exposure to 4 M guanidine hydrochloride, the affinity for collagen type V was dramatically reduced. These data demonstrate the importance of secondary structure to the function of this small proteoglycan.     

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.     

Polarity influences the efficiency of recombinant adenoassociated virus infection in differentiated airway epithelia

To better understand mechanisms that limit rAAV transduction in the lung, we have evaluated several unique features of rAAV infection in polarized primary airway epithelial cultures. rAAV was found to transduce the basolateral surface of airway epithelia 200-fold more efficiently than the apical membrane. These differences in membrane infection correlated with the abundance of apical heparan sulfate proteoglycan (AAV-2 receptor) and virus binding. UV irradiation augmented rAAV transduction greater than 20-fold, only when virus was applied to the apical membrane. Ultrastructural analysis of UV-irradiated primary cultures demonstrated significant changes in microvilli architecture following exposure to 25 J/m2 UV. Although virus binding and the abundance of heparan sulfate proteoglycan were not increased at the apical membrane following UV irradiation, increased receptor-independent endocytosis of fluorescent beads was seen at the apical membrane following UV irradiation. We hypothesize that endocytotic processes associated with apical membrane-specific pathways of viral entry, and/or processing of virus to the nucleus, may be altered following UV irradiation. Interestingly, UV irradiation had an inhibitory effect on rAAV transduction from the basolateral membrane, which correlated with a decrease in the abundance of heparan sulfate proteoglycan at the basal membrane. In summary, these findings suggest that independent pathways of viral transduction may occur in the apical and basolateral compartments of polarized airway epithelia.     

Central administration of dopamine D3 receptor antisense to rat: effects on locomotion, dopamine release and [3H]spiperone binding

Interleukin-5 (IL-5) is the major cytokine regulating eosinophil production. In allergic disease tissue damage is primarily caused by eosinophils. Heparan sulfate proteoglycans are components of the bone marrow stroma, which supports hemopoietic cell differentiation and proliferation. We show that at low IL-5 concentrations heparan sulfate enhances the proliferation of an IL-5-dependent cell line. To investigate a mechanism for this effect we used an artificial proteoglycan to establish an enzyme-linked immunosorbent assay for the binding of heparin to proteins. Using this assay we demonstrate that IL-5 binds to heparin. The IL-5/heparin interaction is inhibited by ethylenediaminetetraacetate and enhanced by low concentrations of zinc ions. IL-5 interacts with iduronic acid containing glycosaminoglycans, and heparan sulfate preparations that have numerous N-sulfated domains per chain are especially efficient at inhibiting heparin binding. Both IL-5/heparin binding and the synergistic effect of IL-5 and heparan sulfate on cell proliferation were inhibited by an anti-IL-5 monoclonal antibody. These data suggest that the binding of IL-5 to heparan sulfate modulates IL-5 activity.     

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.     

Foam cell conversion of macrophages alters the biosynthesis of heparan sulfate

Heparan sulfate is thought to regulate the biological activities of several proteins implicated in the pathogenesis of atherosclerosis. While the interactions of heparan sulfate with lipoprotein lipase and various growth factors have been actively studied, little is known of the cellular regulation of heparan sulfate biosynthesis in response to lipid accumulation. We have investigated heparan sulfate biosynthesis during conversion of murine J774 macrophages into lipid-laden foam cells. Such conversion is shown to accelerate the rate of glycosaminoglycan synthesis and the transport of newly synthesized proteoglycans into the medium. Moreover, the structure of heparan sulfate is specifically altered due to an approximately 30% increase in the 6-O-sulfation of glucosamine residues within the N-sulfated heparan sulfate domains, whereas the sulfation of chondroitin sulfate remains unaffected. These results suggest a selective effect of foam cell conversion on the biosynthesis of heparan sulfate.     

Differential effects of deoxycholic acid on proliferation of neoplastic and differentiated colonocytes in vitro

The secondary bile acid deoxycholic acid is believed to be a promoter of large bowel cancer, in part by inducing colonic epithelial proliferation. The effects of deoxycholic acid on [3H]thymidine incorporation by the human colon cancer cell line HT29 and two differentiated subclones were measured and compared. The subclone HT29-C1 has features of mature absorptive cells and HT29-N2 cells secrete mucus under cholinergic control. The three cell lines were treated with deoxycholic acid (DCA) at concentrations of 0, 5, 10, 50, 100, 150, and 300 microM for 3, 6, 9, 15, 24, and 48 hr. A significant increase in proliferation was noted in HT29 cells only at 6 hr with 5 and 10 microM deoxycholic acid. Neither the subclone HT29-C1, nor HT29-N2 cells exhibited significant change in [3H]thymidine incorporation with DCA at these concentrations or time points. Higher doses of deoxycholic acid above 50 microM and duration of exposure greater than 24 hr were cytotoxic to all three cell lines. The proliferative effects of DCA in HT29 cells were not paralleled by changes in protein kinase C activity or protein kinase C isoform expression. Quantitative and qualitative differences in PKC isoform expression were not noted in the three cell lines used in this study. The proliferative effects of DCA on HT29 cells appear to be independent of the PKC signal transduction pathway.     

Herpes simplex virus infection and propagation in a mouse L cell mutant lacking heparan sulfate proteoglycans

We have isolated a variant line of mouse L cells, termed gro2C, which is partially resistant to infection by herpes simplex virus type 1 (HSV-1). Characterization of the genetic defect in gro2C cells revealed that this cell line harbors a specific defect in the heparan sulfate synthesis pathway. Specifically, anion-exchange high-performance liquid chromatography of metabolically radiolabeled glycosaminoglycans indicated that chondroitin sulfate moieties were synthesized normally in the mutant cells, whereas heparin-like chains were absent. Because of these properties, we have used these cells to investigate the role of heparan sulfate proteoglycans in the HSV-1 life cycle. In this report, we demonstrate that the partial block to HSV-1 infection in gro2C cells occurs in the virus entry pathway. Virus adsorption assays using radiolabeled HSV-1 (KOS) revealed that the gro2C cell surface is a relatively poor target for HSV-1 in that virus attachment was 85% lower in the mutant cells than in the parental L cell controls. A portion of the 15% residual virus adsorption was functional, however, insofar as gro2C cells were susceptible to HSV-1 infection in plaque assays and in single-step growth experiments. Moreover, although the number of HSV-1 plaques that formed in gro2C monolayers was reduced by 85%, the plaque morphology was normal, and the virus released from the mutant cells was infectious. Taken together, these results provide strong genetic evidence that heparan sulfate proteoglycans enhance the efficiency of HSV attachment to the cell surface but are otherwise not essential at any stage of the lytic cycle in culture. Moreover, in the absence of heparan sulfate, other cell surface molecules appear to confer susceptibility to HSV, leading to a productive viral infection.     

Hydroxyl radicals depolymerize glomerular heparan sulfate in vitro and in experimental nephrotic syndrome

Heparan sulfate, the polysaccharide side chain of heparan sulfate proteoglycan, is important for the permselective properties of the glomerular basement membrane. In this report, we show a role for hydroxyl radicals in heparan sulfate degradation and an enhanced glomerular basement membrane permeability. First, in enzyme-linked immunosorbent assay, exposure of coated heparan sulfate (proteoglycan) to reactive oxygen species resulted in a +/-50% decrease of binding of a monoclonal antibody against heparan sulfate, whereas binding of an antibody against the core protein remained unaltered. Second, on polyacrylamide gel electrophoresis, the molecular weight of heparan sulfate exposed to radicals was reduced which indicates depolymerization. Both in enzyme-linked immunosorbent assay and gel electrophoresis, hydroxyl radicals are instrumental for heparan sulfate degradation as shown by the addition of various radical scavengers. Third, in an experimental model for human nephrotic syndrome (Adriamycin nephropathy in rats), glomerular basement membrane staining of two recently described anti-heparan sulfate antibodies (JM403 and KJ865) was reduced by 24 and 43%. Treatment of Adriamycin-exposed rats with the hydroxyl radical scavenger dimethylthiourea both reduced albuminuria by 37% (p < 0.01) and partly prevented loss of heparan sulfate staining by 53% (JM403) and 39% (KJ865) (p < 0.03). In contrast to the heparan sulfate side chains, the core protein expression and the extent of glycanation did not change in Adriamycin nephropathy. We conclude that glomerular basement membrane heparan sulfate is susceptible to depolymerization by hydroxyl radicals leading to loss of glomerular basement membrane integrity and albuminuria.