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
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Abstract |
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Key words: glypican-1 / heparanase / heparan sulfate / mAb 10E4 / nitric oxide
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Introduction |
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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, 2002; Fransson, 2003
). 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., 1995
).
Another much used mAb against HS is 10E4, which reacts with an epitope that is destroyed by N-desulfation (David et al., 1992). This epitope was found to codistribute with the abnormal prion protein (PrPSc) in early brain lesions of scrapie-infected mice (Leteux et al., 2001
). 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., 2000
). 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., 2001a
). The same arrangement has been observed in other HS preparations (Westling and Lindahl, 2002
). The number of
units in Gpc-1 HS is greatly increased when cells are exposed to
-difluoromethylornithine (DFMO), which inhibits endogenous polyamine synthesis and induces Gpc-1-assisted polyamine uptake from the environment (Belting et al., 1999
, 2003
; Ding et al., 2001b
). 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., 2002). 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., 2002
). 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., 2002
). 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., 2003
).
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., 2002
). 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., 2002
), 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.
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Results |
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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 2AC; 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 2DF). This shows that much of the 10E4-positive material in growing fibroblasts comprised HS degradation products generated by heparanase degradation.
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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., 2002; Ding et al., 2002
). 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., 2002). In fibroblasts treated with both BFA and suramin, the signal from the 10E4 epitope was much weakened but colocalized entirely with Gpc-1 (Figure 3AC). 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., 2002
; Ding et al., 2002
). 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., 2002
). Thus 10E4-positive HS material not colocalizing with Gpc-1 (green in Figure 3F) could partly represent such degradation products.
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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., 2002
; Ding et al., 2001b
). 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., 2002), 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., 2000
). 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 4050).
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T24 cells also contain HS degradation products (fractions 3555 in Figure 4F; see also Mani et al., 2000). The smaller HS oligosaccharides eluting between fractions 4555 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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Discussion |
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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., 2002) 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., 1988
). 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., 2002). 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., 2001
). The tumor-derived T24 cells also degrade HS nonenzymatically by NO (Cheng et al., 2002
; Ding et al., 2002
). Tumor cells usually express high levels of both heparanase (Eccles, 1999
; Vlodavsky et al., 1999
) and NO-synthase (Doi et al., 1996
; Thomsen and Miles, 1998
).
The 10E4-positive HS chains associated with scrapie lesions (Leteux et al., 2001) 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., 2003
). 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., 2002
). 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., 2002
). It is intriguing that copper deficiency has been demonstrated in brain tissue of animals or humans afflicted with neurodegenerative diseases (for reviews, see Brown, 2002
; Lehmann, 2002
). Furthermore, scrapie infection of neuroblastoma cells precludes NO production when the cells are challenged with lipopolysaccharide (Lindegren et al., 2003
). 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.
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Materials and methods |
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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., 2001a,b
; Mani et al., 2000
). 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., 2000
). Size was assessed by Superose 6 gel chromatography before and after release of HS chains by alkaline elimination and borohydride reduction (Ding et al., 2001a
,b
; Mani et al., 2000
;)
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., 2002; Ding et al., 2002
). 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.
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Acknowledgements |
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Footnotes |
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1 Present address: Department of Developmental and Cell Biology, School of Biological Sciences, University of California at Irvine, Irvine CA 92697-2300
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Abbreviations |
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References |
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