The heparan sulfate–specific epitope 10E4 is NO-sensitive and partly inaccessible in glypican-1

Katrin Mani3, Fang Cheng3, Staffan Sandgren3, Jacob van den Born4, Birgitta Havsmark3, Kan Ding1 and Lars-Åke Fransson2,3

3 Department of Cell and Molecular Biology, Section for Cell and Matrix Biology, Lund University, BMC C13, SE-221 84, Lund, Sweden and 4 Department of Cell Biology, Free University of Amsterdam, Van der Boechorststraat 7, 1981 BT, Amsterdam, The Netherlands

Received on October 16, 2003; revised on February 11, 2004; accepted on February 23, 2004


    Abstract
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 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 References
 
The monoclonal antibody 10E4, which recognizes an epitope supposed to contain N-unsubstituted glucosamine, is commonly used to trace heparan sulfate proteoglycans. It has not been fully clarified if the N-unsubstituted glucosamine is required for antibody recognition and if all heparan sulfates carry this epitope. Here we show that the epitope can contain N-unsubstituted glucosamine and that nitric oxide–generated deaminative cleavage at this residue in vivo can destroy the epitope. Studies using flow cytometry and confocal immunofluorescence microscopy of both normal and transformed cells indicated that the 10E4 epitope was partially inaccessible in the heparan sulfate chains attached to glypican-1. The 10E4 antibody recognized mainly heparan sulfate degradation products that colocalized with acidic endosomes. These sites were greatly depleted of 10E4-positive heparan sulfate on suramin inhibition of heparanase. Instead, there was increased colocalization between 10E4-positive heparan sulfate and glypican-1. When both S-nitrosylation of Gpc-1 and heparanase were inhibited, detectable 10E4 epitope colocalized entirely with glypican-1. In nitric oxide–depleted cells, there was both an increased signal from 10E4 and increased colocalization with glypican-1. In suramin-treated cells, the 10E4 epitope was destroyed by ascorbate-released nitric oxide with concomitant formation of anhydromannose-containing heparan sulfate oligosaccharides. Immunoisolation of radiolabeled 10E4-positive material from unperturbed cells yielded very little glypican-1 when compared with specifically immunoisolated glypican-1 and total proteoglycan and degradation products. The 10E4 immunoisolates were either other heparan sulfate proteoglycans or heparan sulfate degradation products.

Key words: glypican-1 / heparanase / heparan sulfate / mAb 10E4 / nitric oxide


    Introduction
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 Abstract
 Introduction
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 Materials and methods
 References
 
Heparan sulfate (HS) is a common substituent of cell-surface proteoglycans (PG). These composite proteins are either transmembrane proteins (like the syndecans) or attached via a glycosylphosphatidylinositol (GPI) anchor (like the glypicans). Genetic and biochemical studies on invertebrate model organisms and dysmorphic syndromes in humans have demonstrated that HS PGs are critical for a wide spectrum of events during development (for reviews, see Esko and Selleck, 2002Go; Perrimon and Bernfield, 2000Go). The glypicans, which include six known mammalian forms, have attracted special interest because the two Drosophila glypicans, dally (division abnormally delayed) and dally-like play important roles in the orchestration of growth factor, cytokine, and morphogen signaling during various patterning events (for recent reviews, see Esko and Selleck, 2002Go; Filmus and Selleck, 2001Go; Fransson, 2003Go).

The HS side chains, which consist of an alternating glucosamine (GlcN)-hexuronic acid (HexUA) backbone, are long and linear and display a high degree of variability owing to different levels of sulfation and positions of the sulfate groups along the chain (Esko and Selleck, 2002Go; Fransson, 2003Go). A unique structural feature of HS is the variable substitution of the amino group of GlcN. It can be acetylated (GlcNAc), sulfated (GlcNSO3), or unsubstituted (). Although the occurrence of a few units in HS preparations was recognized early, they were generally regarded as artifacts. When a monoclonal antibody (mAb) JM-403, specifically directed against epitopes containing the unit had been developed, it was apparent that HS with this epitope had a specific tissue distribution (van den Born et al., 1995Go).

Another much used mAb against HS is 10E4, which reacts with an epitope that is destroyed by N-desulfation (David et al., 1992Go). This epitope was found to codistribute with the abnormal prion protein (PrPSc) in early brain lesions of scrapie-infected mice (Leteux et al., 2001Go). Competition experiments with defined HS oligosaccharides indicated that the 10E4 epitope comprised a tetrasaccharide motif, possibly including a unit.

We had earlier detected units in the HS chains of recycling glypican-1 (Gpc-1) (Mani et al., 2000Go). Normally, the HS chains of Gpc-1 contain only a few units, located to the unsulfated, GlcNAc-rich segments close to the linkage region to the protein core (Ding et al., 2001aGo). The same arrangement has been observed in other HS preparations (Westling and Lindahl, 2002Go). The number of units in Gpc-1 HS is greatly increased when cells are exposed to {alpha}-difluoromethylornithine (DFMO), which inhibits endogenous polyamine synthesis and induces Gpc-1-assisted polyamine uptake from the environment (Belting et al., 1999Go, 2003Go; Ding et al., 2001bGo). How the HS synthesizing machinery senses changes in polyamine concentration is unknown.

The glypican proteins are all of similar size and contain 14 conserved Cys residues in the central domain (for references, see Fransson, 2003). Cys residues in Gpc-1 can become S-nitrosylated (SNO) by nitric oxide (NO) in a Cu(II)-dependent reaction (Ding et al., 2002Go). When Gpc-1 is exposed to ascorbate, either in vivo or in purified form in vitro, NO is released and catalyzes deaminative cleavage of HS at the units. The result is a Gpc-1 molecule with truncated HS chains and free HS oligosaccharides terminating at the reducing end with anhydromannose (anMan). By using confocal immunofluorescence microscopy, we have visualized the modifications of the Gpc-1 protein and its HS chains at various intracellular sites of a recycling pathway (Cheng et al., 2002Go). At the cell surface, S-nitrosylation of Gpc-1 is generally low, and the HS chains contain few units. After internalization, there is formation of additional units followed by S-nitrosylation. The HS chains are then degraded either enzymatically by heparanase or nonenzymatically by NO derived from SNO. We have proposed that these degradations take place while Gpc-1 recycles via late endosomes to paranuclear, caveolin-1-positive compartments (Cheng et al., 2002Go). Deglycanated Gpc-1 and the various HS degradation products appear to separate at some stage and take different routes, but this has not been fully clarified. Recycling Gpc-1 can carry exogenous polyamines bound to its HS side chains and deliver them to intracellular sites. Unloading of the polyamines requires degradation of HS by SNO-derived NO (Belting et al., 2003Go).

During these studies we traced -containing HS by using mAb JM-403 rather than 10E4. The former mAb was expected to be more specific for units, because the JM-403 epitope was destroyed by N-acetylation of , whereas 10E4 was not (Cheng et al., 2002Go). Although this does not exclude the possibility that a unit is part of the 10E4 epitope, it may not be required for antibody recognition. During these studies we also observed that the JM-403 epitope colocalized well with Gpc-1 (Cheng et al., 2002Go), whereas the 10E4 epitope did not. We now report that in immunolocalization experiments using cultured cells, the 10E4 epitope can be inaccessible in Gpc-1 HS but accessible in HS fragments released from it by heparanase.


    Results
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 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 References
 
Effect of nitrite or ascorbate on the 10E4 and JM-403 epitopes
It has been shown that the 10E4 epitope contains N-sulfated glucosamine (GlcNSO3), and it has been proposed that a unit can also be part of it (David et al., 1992Go; Leteux et al., 2001Go). To further investigate whether a unit is an essential part of the epitope we carried out deaminative cleavages at units in vivo. As shown in Figure 1A–B, the signal from the 10E4 epitope (green) in fibroblast HS was sensitive to treatment with nitrite at pH 3.9, which specifically cleaves at units. Likewise, ascorbate-induced release of NO from SNO groups also reduced the signal (Figure 1C). The latter result was confirmed by flow cytometry (see inserts in Figure 1A and 1C). Hence, a unit may be part of the 10E4 epitope and cleavage at this site by NO destroys it.



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Fig. 1. Effect of nitrite or ascorbate on the immunolocalization of the HS-specific 10E4 epitope in fibroblasts (AC) and of the HS-specific epitopes 10E4 and JM-403 in T24 bladder carcinoma cells (DI). The panels show confocal laser immunofluorescence staining for the HS epitopes 10E4 or JM-403 (10E4, JM-403, both green) and for total Gpc-1 (GPC, red). Cells were treated with HNO2 at pH 3.9 to cleave at (B) or by treatment with ascorbate (Asc) to release NO and similarly cleave at (C, E, I). Bar, 20 µm. Inserts, results of flow cytometry of cells without or with added 10E4 (–/+ in A and D) and of 10E4-positive cells after treatment with 1 mM ascorbate for 10 min (in C and E). The median fluorescence intensity of untreated 10E4-positive cells is set at 100%, n = 6, and bars correspond to SD.

 
In T24 cells there was no effect on the 10E4 epitope by ascorbate as indicated both by flow cytometry and confocal microscopy analysis (Figure 1D–E). This result suggested that the 10E4 epitope may not always contain a unit or that it was not accessible to NO derived from SNO. Moreover, the 10E4 epitope did not colocalize with Gpc-1, except at a few paranuclear sites in subconfluent (Figure 1F) as well as confluent cell cultures (data not shown). Also in fibroblasts, the HS-specific 10E4 epitope colocalized poorly with Gpc-1 both in subconfluent and confluent cell cultures (data not shown). In contrast, the HS-specific -containing JM-403 epitope colocalized extensively with Gpc-1 in T24 cells (Figure 1G, yellow) and was completely abolished by ascorbate (Figure 1H–I).

Subcellular localization of 10E4-positive HS and the effects of suramin, brefeldin A, NO deprivation, and DFMO
In fibroblasts, 10E4-positive HS was present at the cell surface and in cytoplasmic vesicles where it colocalized extensively with LysoTracker Red, a marker for acidic endosomes and lysosomes (Figure 2A–C; LTR, red). Because this 10E4-positive material could represent products generated by heparanase degradation of HS, we tested the effect of suramin, a known inhibitor of heparanase. After suramin treatment, signal from the 10E4 epitope was weakened as indicated by flow cytometry (see inserts in Figure 2A and D) and essentially absent from the cytoplasmic vesicles (Figure 2D–F). This shows that much of the 10E4-positive material in growing fibroblasts comprised HS degradation products generated by heparanase degradation.



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Fig. 2. Effect of suramin on the immunolocalization of the HS-specific 10E4 epitope in fibroblasts (AF) and in T24 bladder carcinoma cells (GL). The panels show confocal laser immunofluorescence staining for the HS epitope 10E4 (10E4), Gpc-1 (GPC), and LysoTracker Red (LTR). Bar, 20 µm. Inserts, results of flow cytometry of untreated 10E4-positive cells (in A and G) and of 10E4-positive cells after treatment with 0.2 mM suramin for 24 h (in D and J).

 
Previous studies have shown that T24 cells contain both a pool of HS oligosaccharides and a pool of Gpc-1 with truncated HS side chains (Mani et al., 2000Go). In growing T24 cells, 10E4-positive HS was also mainly present in acidic vesicles, as demonstrated by colocalization with LysoTracker Red (Figure 2G–I, yellow in I). When heparanase-catalyzed degradation was inhibited in subconfluent T24 cells by the addition of suramin, the signal from the 10E4 epitope decreased as indicated by flow cytometry (see inserts in Figure 2G and J), although some cells still displayed a strong signal (compare Figure 2J and K). In this case, partial colocalization with Gpc-1 remained at paranuclear sites (yellow in Figure 2L; compare Figure 1F). These results indicated that heparanase was partly responsible for HS degradation also in proliferating T24 cells. In confluent T24 cultures, suramin treatment completely abrogated reactivity with the 10E4 epitope (results not shown). Apparently, in confluent T24 cultures HS degradation is mainly due to heparanase. It should be added that at the suramin concentration used (0.2 mM) there is no inhibition of cell proliferation (Sandgren and Belting, 2003Go).

We have previously shown that HS in Gpc-1 from T24 cells can also be subject to deaminative cleavage by NO derived from SNO groups present in the Gpc-1 core protein (Cheng et al., 2002Go; Ding et al., 2002Go). Deaminative and heparanase-catalyzed cleavage can take place simultaneously. Therefore, much of the 10E4-positive material generated in unperturbed, proliferating T24 cells (Figure 2G) could also have been generated by partial deaminative cleavage.

NO-dependent HS degradation can be prevented by brefeldin A (BFA), which both inhibits recycling of Gpc-1 and precludes SNO formation (Cheng et al., 2002Go). In fibroblasts treated with both BFA and suramin, the signal from the 10E4 epitope was much weakened but colocalized entirely with Gpc-1 (Figure 3A–C). NO-dependent HS degradation can also be suppressed by a combined inhibition of NO-synthase, prevention of SNO formation and NO release by chelation of Cu(I) ions with neocuproine and by quenching of NO by sulfamate (Cheng et al., 2002Go; Ding et al., 2002Go). Accordingly, in T24 cells deprived of NO, there was an increased 10E4 signal (see inserts in Figure 3D and E) that colocalized partially with Gpc-1 (Figure 3F). It should be added that heparanase-catalyzed degradation is unaffected by NO depletion (Cheng et al., 2002Go). Thus 10E4-positive HS material not colocalizing with Gpc-1 (green in Figure 3F) could partly represent such degradation products.



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Fig. 3. Effect of BFA, suramin, NO deprivation, DFMO, and ascorbate in various combinations on the immunolocalization of the HS-specific 10E4 epitope in fibroblasts (AC) and in T24 bladder carcinoma cells (DL). The panels show confocal laser immunofluorescence staining for the HS epitope 10E4 (10E4), Gpc-1 (GPC), and anMan-containing HS oligosaccharides (AM, green). Bar, 20 µm. Inserts, results of flow cytometry of untreated 10E4-positive cells (in D) and of 10E4-positive cells after NO-deprivation (in E).

 
To be cleaved by SNO-derived NO, HS chains have to be attached to the Gpc-1 core protein (Ding et al., 2002Go). Therefore, ascorbate-induced and NO-dependent degradation of HS is not expected to engage heparanase-released HS chain fragments, unless they are in close contact with Gpc-1. However, when suramin inhibits enzymatic release of HS chains from Gpc-1 (Cheng et al., 2002Go; Ding et al., 2001aGo), this should increase the opportunities for nonenzymatic, NO-dependent cleavage of HS in Gpc-1. Accordingly, when suramin-treated T24 cells were exposed to ascorbate, the 10E4-reactivity (Figure 3G) disappeared completely (Figure 3H), whereas anMan-containing HS oligosaccharides were generated (Figure 3K). The amount of Gpc-1 protein was unaffected (Figure 3I), and neither untreated (Figure 3J) nor suramin-treated growing T24 cells (data not shown) reacted with the anMan-specific antibody.

We have previously reported that treatment of cells with DFMO results in increased formation of units in Gpc-1 HS. This can be detected both by greater sensitivity to nitrite at pH 3.9 and by an increased signal from the JM-403 epitope (Cheng et al., 2002Go; Ding et al., 2001bGo). However, DFMO treatment did not much change the 10E4 reactivity (Figure 3L; compare Figure 3D), suggesting that the JM-403 and 10E4 epitopes are different.

The results obtained thus far strongly suggested that the 10E4-epitope in Gpc-1 HS was inaccessible in vivo. We therefore compared the yields of immunoisolated 10E4-positive PG with that of Gpc-1 and total PG from cell extracts.

Immunoisolation of radiolabeled 10E4-positive HS products
Characterization of 10E4-positive HS products, immunoisolated Gpc-1 (Ding et al., 2002Go), and total PG was performed by gel permation chromatography on Superose 6 of [3H]glucosamine- or [33S]sulfate-labeled material from extracts of parallell cultures of confluent cells. Extraction was performed with radioimmunoprecipitation buffer containing sodium dodecyl sulfate, Triton X-100, and sodium deoxycholate to minimize coprecipitations (Mani et al., 2000Go). 10E4-Reactive products from fibroblasts eluted in or near the void volume, indicating that they were mainly composed of large PG forms (Figure 4A). There was very little material in the position corresponding to immunoisolated Gpc-1 (peak position around fraction 25; see Figure 4B). In the pool of total PG (Figure 4C), the yield of Gpc-1 was severalfold greater than in the pool of 10E4-reactive material (Figure 4A). The more retarded peaks in Figure 4C consist of decorin/biglycan (peak position around fraction 30) and glycosaminoglycan chains and degradation products (fractions 40–50).



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Fig. 4. Superose 6 chromatography of [3H]glucosamine- or [35S]sulfate-labeled PGs, glycosaminoglycans, and oligosaccharides either immunoisolated by using mAb 10E4 (A, D) or by using antiglypican-1 serum (B, E) or isolated by ion exchange chromatography on DEAE-cellulose (C, F) from fibroblasts (AC) or T24 cells (DF). Confluent cultures (75-cm2 dishes) were incubated with radiolabeled precursors for 24 h and material isolated from detergent extracts of the cells by the different methods was chromatographed on Superose 6. Open circles, 3H; filled circles, 35S. Vo, void volume; Vt, total volume.

 
10E4-Reactive products from T24 cells also mainly comprised PGs larger than Gpc-1 (fractions 18–30 in Figure 4D). The immunoisolated Gpc-1 from T24 cells eluted in fractions 25–35 (Figure 4E). The yield of Gpc-1 in the total PG pool (Figure 4F; see also Ding et al., 2001aGo; Mani et al., 2000Go) was again severalfold greater than in the 10E4-reactive material (Figure 4D), confirming that recovery of Gpc-1 was low when using mAb 10E4. However, HS chains derived from immunoisolated Gpc-1 by alkaline elimination were quantitatively recovered by 10E4 immunoisolation (data not shown).

T24 cells also contain HS degradation products (fractions 35–55 in Figure 4F; see also Mani et al., 2000Go). The smaller HS oligosaccharides eluting between fractions 45–55 were not recovered by mAb 10E4 (compare Figure 4D and F), probably because they were generated by deaminative cleavage and thus devoid of the 10E4 epitope.


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 References
 
The 10E4 epitope
We propose that the 10E4 HS epitope can be inaccessible in the HS chains of Gpc-1 probably because the 10E4 epitope interacts with the core protein, possibly via the SNO groups (Figure 5). Accordingly, when heparanase is inhibited by suramin, the signal from the 10E4 epitope in Gpc-1 is weak. Conversely, the 10E4 signal is greatly increased when the HS chains are liberated by heparanase degradation. However, the JM-403 epitope appears to be fully accessible in either case.



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Fig. 5. Schematic model of a glypican (GPC) with two HS chains containing a cryptic 10E4 epitope (square) and an accessible JM-403 epitope (ellipse). Ascorbate releases NO from SNO in the core protein and NO cleaves at residues in both epitopes (solid arrow). A heparanase cleavage site is indicated between the two epitopes. A potential noncovalent interaction between the core protein and HS is indicated within parenthesis.

 
Apparently, the 10E4 epitope may contain units, which can be acetylated without affecting antibody recognition (Cheng et al., 2002Go). However, cleavage at this unit by NO released by ascorbate from intrinsic SNO groups in Gpc-1 results in inactivation (Figure 5). Hence, a unit in the 10E4 epitope is tolerated but not necessary for recognition. However, the flanking units appear to be essential and must be joined via the unit. In contrast, is an essential part of the JM-403 epitope, because it is destroyed by N-acetylation (Cheng et al., 2002Go). Therefore, mAb JM-403 can be used to trace all units in Gpc-1 as well as in other HS PGs, whereas mAb 10E4 primarily detects the heparanase-generated degradation products of Gpc-1 HS. Of course, 10E4 is also a potential marker for HS in other PG (syndecans, perlecan, etc.). However, in cells with excessive NO-dependent HS degradation, the 10E4 epitope will be low or undetectable.

Past and present results thus demonstrate that the commonly used mAbs S1 and 10E4 have restricted specificities and utilities. The mAb S1 identifies only S-nitrosylated Gpc-1 (Cheng et al., 2002Go) and the mAb 10E4 identifies an HS epitope that may not always by accessible and that is sensitive to endogenous cleavage by NO derived from SNO in Gpc-1. Therefore, when using these mAbs, neither the Gpc-1 molecules that have not become S-nitrosylated nor those that have exhausted their SNO content nor the deaminative HS degradation products will be detected. However, the anMan-specific mAb is a useful indicator of NO-dependent HS degradation, at least when the products are of tetrasaccharide size or larger (Pejler et al., 1988Go). After further degradation of these products, in, for example, lysosomes, they should no longer be detectable.

Heparanase versus NO-dependent degradation of HS
Modification and degradation of recycling Gpc-1 in T24 cells begins after internalization from the cell surface (Cheng et al., 2002Go). Formation of units in HS and S-nitrosylation of the core protein are prerequisites for NO-dependent autodegradation of HS. HS degradation can be accomplished enzymatically by heparanase, which takes place in caveolin-containing perinuclear compartments, possibly caveosomes, and human heparanase has indeed been located to perinuclear granules (Goldshmidt et al., 2001Go). The tumor-derived T24 cells also degrade HS nonenzymatically by NO (Cheng et al., 2002Go; Ding et al., 2002Go). Tumor cells usually express high levels of both heparanase (Eccles, 1999Go; Vlodavsky et al., 1999Go) and NO-synthase (Doi et al., 1996Go; Thomsen and Miles, 1998Go).

The 10E4-positive HS chains associated with scrapie lesions (Leteux et al., 2001Go) could be derived from Gpc-1. This PG, which is common in the brain, is abundantly expressed in amyloid deposits found in Alzheimer's disease as well as in other neurodegenerative disorders (van Horssen et al., 2003Go). The enzymatic and nonenzymatic degradations of HS appear to be independent, that is, inhibition of heparanase with suramin does not prevent deaminative cleavage of HS and, conversely, NO deprivation does not prevent degradation by heparanase (Cheng et al., 2002Go). However, units in a heparanase-released HS fragment segregated from Gpc-1 could be inaccessible to NO derived from Gpc-1-SNO. An HS fragment with remaining units, and thus potentially both JM-403 and 10E4 positive, could thus be generated when heparanase is active, whereas there is insufficient generation of NO to sustain NO-dependent HS degradation. The SNO-level in Gpc-1 is regulated by a Cu(II)/Cu(I) redox cycle (Ding et al., 2002Go). It is intriguing that copper deficiency has been demonstrated in brain tissue of animals or humans afflicted with neurodegenerative diseases (for reviews, see Brown, 2002Go; Lehmann, 2002Go). Furthermore, scrapie infection of neuroblastoma cells precludes NO production when the cells are challenged with lipopolysaccharide (Lindegren et al., 2003Go). Studies on the extent of S-nitrosylation of Gpc-1 and formation of anMan-containing degradation products in healthy and diseased brain tissue may provide for a better understanding of the pathogenesis of neurodegenerative diseases.


    Materials and methods
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 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 References
 
Materials
The human bladder carcinoma cell-line T24, human embryonic lung fibroblasts, culture media, antisera or mAbs to human Gpc-1, to HS (JM-403), anMan (AM), and various compounds like LysoTracker Red, BFA, suramin, DFMO, radioactive precursors, enzymes, prepacked columns, Centriplus tubes, and chemicals were obtained as described previously (Arroyo-Yanguas et al., 1997Go; Cheng et al., 2002Go; Ding et al., 2001aGo,bGo, 2002Go; Mani et al., 2000Go). The 10E4 mAb was purchased from the Seikagaku (Tokyo).

Cell treatments
Cell cultures were either left untreated or treated with BFA (10 µg/ml), suramin (0.2 mM), or subjected to NO/nitrite deprivation and depression of S-nitrosylation by using 10 mM N-nitroarginine, 10 mM ammonium sulfamate, and 0.01 mM neocuproine all for 24 h. Polyamine deprivation was achieved by treatment with 5 mM DFMO for 48 h.

Isolation and degradation of Gpc-1
Cells were incubated with 20 µCi/ml D-[6-3H]glucosamine and/or 50 µCi/ml [35S]sulfate as described (Ding et al., 2001aGo,bGo; Mani et al., 2000Go). Cells were extracted with radioimmunoprecipitation buffer, and all Gpc-1 glycoforms were immunoisolated from the cell extract by using polyclonal anti-Gpc-1 antiserum (10). 10E4-Positive material was isolated by passage over a 1-ml column of antimouse IgM-agarose (Sigma, St. Louis, MO) presaturated with mAb 10E4. After extensive washing with loading buffer, bound material was eluted with 4 M guanidine. Total PGs and glycosaminoglycans were isolated by ion exchange chromatography from Triton X-100 cell extracts as described (Mani et al., 2000Go). Size was assessed by Superose 6 gel chromatography before and after release of HS chains by alkaline elimination and borohydride reduction (Ding et al., 2001aGo,bGo; Mani et al., 2000Go;)

Flow cytometry
Cells were sparsely seeded in 24-well plates and allowed to adhere for 24 h. Growth medium was then replaced with fresh medium with no additions or medium containing ascorbate, suramin or inhibitors of NO production (see previous methods description). Cells were rinsed with medium and detached using trypsin (0.5 ml 0.05% w/v of trypsin in phosphate buffered saline [PBS] for 1 min) in the case of fibroblats or ethylenediamine tetra-acetic acid (EDTA) (0.5 ml 5 mM EDTA in PBS for 20 min) in the case of T24 cells. Trypsinization was terminated by replacing the trypsin solution with 0.5 ml medium supplemented with 10% fetal bovine serum. In both cases, cells were recovered by gentle suspension and transfered to tubes, adding 1 volume of PBS 1% BSA (w/v). Cells were then pelleted by centrifugation and resuspended in 0.2 ml PBS after removal of the supernatant. Cells were fixed for 30 min in 1 ml PBS containing 4% paraformaldehyde (w/v) while initially vortexing. Permeabilization was performed by incubation with 0.2% TritonX-100 in PBS (v/v) for 20 min. Immunostaining of the cells with the mAb 10E4 as the primary antibody and goat anti-mouse total Ig as the secondary antibody was performed as described for confocal microscopy. In the controls, mAb 10E4 was omitted. After each step, cells were recovered by centrifugation at 350 x g for 5 min. The cells were finally suspended in PBS containing 1% BSA and analyzed for fluorescence in a fluorescence assisted cell sorting instrument (Calibur, Becton Dickinson Biosciences) operated by Cell-Quest software.

Confocal laser scanning immunofluorescence microscopy
The various procedures, including seeding of cells, fixation, application of antibodies, generation of images, and data processing, were the same as those used previously (Cheng et al., 2002Go; Ding et al., 2002Go). The secondary antibodies used were either goat anti-mouse total Ig (when the primary antibody was a monoclonal) or goat antirabbit IgG (when the primary antibody was polyclonal). They were tagged with either fluorescein isothiocyanate or Texas Red and appropriately combined for colocalization studies.


    Acknowledgements
 
We thank Catharina Svanborg and Klaus Edvardsen, both at Lund University, for use of microscope and flow cytometry facilities. The technical assistance of Ms. Birgitta Havsmark is greatly appreciated. The work was supported by grants from the Swedish Science Council (V.R.-M.), the Cancer Fund, the Apotekare Hedberg, Thelma Zoega, Kock, Wiberg and Österlund Foundations, Polysackaridforskning i Uppsala AB, and the Medical Faculty of Lund University.


    Footnotes
 
2 To whom correspondence should be addressed; e-mail: lars-ake.fransson{at}medkem.lu.se

1 Present address: Department of Developmental and Cell Biology, School of Biological Sciences, University of California at Irvine, Irvine CA 92697-2300 Back


    Abbreviations
 
anMan, anhydromannose; BFA, brefeldin A; DFMO, {alpha}-difluoromethyl ornithine; EDTA, ethylenediamine tetra-acetic acid; Gpc-1, glypican-1; GPI, glycosylphosphatidylinositol; HS, heparan sulfate; mAb, monoclonal antibody; NO, nitric oxide; NOS, nitric oxide synthase; PBS, phosphate buffered saline; PG, proteoglycan; SNO, S-nitroso group


    References
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 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 References
 
Arroyo-Yanguas, Y., Cheng, F., Isaksson, A., Fransson, L.-Å., Malmström, A., and Westergren-Thorsson, G. (1997) Binding, internalization and degradation of antiproliferative heparan sulfate by human embryonic lung fibroblasts. J. Cell. Biochem., 64, 595–604.[CrossRef][ISI][Medline]

Belting, M., Persson, S., and Fransson, L.-Å. (1999) Proteoglycan involvement in polyamine uptake. Biochem. J., 338, 317–323.[CrossRef][ISI][Medline]

Belting, M., Mani, K., Jönsson, M., Cheng, F., Sandgren, S., Jonsson, S., Ding, K., Delcros, J.-G., and Fransson, L.-Å. (2003) Glypican-1 is a vehicle for polyamine uptake in mammalian cells. A pivotal role for nitrosothiol-derived nitric oxide. J. Biol. Chem., 278, 47181–47189.[Abstract/Free Full Text]

Brown, D.R. (2002) Copper and prion diseases. Biochem. Soc. Trans., 30, 742–745.[ISI][Medline]

Cheng, F., Mani, K., van den Born, J., Ding, K., Belting, M., and Fransson, L.-Å. (2002) Nitric oxide-dependent processing of heparan sulfate in recycling S-nitrosylated glypican-1 takes place in caveolin-1-containing endosomes. J. Biol. Chem., 277, 44431–44439.[Abstract/Free Full Text]

David, G., Bai, X.M., Van der Schueren, B., Cassiman, J.-J. and Van den Berghe, H. (1992) Developmental changes in heparan sulfate expression: in situ detection with mAbs. J. Cell Biol., 119, 961–975.[Abstract]

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