Glycobiology Advance Access originally published online on September 12, 2006
Glycobiology 2006 16(12):1251-1261; doi:10.1093/glycob/cwl045
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Constitutive and vitamin C-induced, NO-catalyzed release of heparan sulfate from recycling glypican-1 in late endosomes
Department of Experimental Medical Science, Section of Neuroscience, Glycobiology Group, Lund University, Biomedical Centre A13, SE-221 84, Lund, Sweden
1 To whom correspondence should be addressed; e-mail: lars-ake.fransson{at}med.lu.se
Received on June 1, 2006; revised on August 21, 2006; accepted on September 5, 2006
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Abstract |
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The recycling heparan sulfate (HS)-containing proteoglycan glypican-1 (Gpc-1) is processed by nitric oxide (NO)-catalyzed deaminative cleavage of its HS chains at N-unsubstituted glucosamines. This generates anhydromannose (anMan)-containing HS degradation products that can be detected by a specific antibody. Here we have attempted to identify the intracellular compartments where these products are formed. The anMan-positive degradation products generated constitutively in human bladder carcinoma cell line (T24) or fibroblasts appeared neither in caveolin-1-associated vesicles nor in lysosomes. In Niemann–Pick C-1 (NPC-1) fibroblasts, where deaminative degradation is abrogated, formation of anMan-positive products can be restored by ascorbate. These products colocalized with Rab7, a marker for late endosomes. When NO-catalyzed degradation of HS was depressed in mouse neuroblastoma cell line (N2a) by using 3-ß[2(diethylamino) ethoxy]androst-5-en-17-one (U18666A), both ascorbate and dehydroascorbic acid restored formation of anMan-positive products that colocalized with Rab7. In T24 cells, constitutively generated anMan-positive products colocalized with both Rab7 and Rab9, whereas Gpc-1 colocalized with Rab9, a marker for transporting endosomes. Inhibition of endosomal acidification, which blocks transfer from early (Rab5) to late (Rab7) endosomes, abrogated deaminative degradation of HS. This could also be overcome by the addition of ascorbate, which induced formation of anMan-positive degradation products that colocalized with Rab7. After 35S-sulfate labeling, similar degradation products were recovered in Rab7-positive vesicles. We conclude that NO-catalyzed degradation of HS may begin in early endosomes but is mainly taking place in late endosomes.
Key words: cholesterol / endosomes / glypican / heparan sulfate / nitric oxide
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Introduction |
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TopAbstractIntroductionResultsDiscussionMaterials and methodsAcknowledgmentsConflict of interest statementReferences |
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The plasma membrane of animal cells contains a multitude of glycoconjugates. Among the protein-based forms, the proteoglycans (PGs) have attracted considerable attention in recent years. Cell-surface-bound heparan sulfate (HS)-containing PGs (HSPGs) are widely expressed, from Caenorhabditis elegans to humans, and known to modulate the activity of a large number of extracellular ligands, including growth factors, cytokines, chemokines, and morphogens. The HSPGs thereby regulate a great diversity of biological processes involved in development and tumorigenesis (for reviews, see Bellin et al., 2003
; Fransson et al., 2004).
The two major cellular HSPG families are the syndecans and the glypicans. Their HS chains may be similar or identical, but their core proteins are different. The syndecan core proteins are all transmembrane proteins, whereas the glypicans are attached to membrane lipids, via a C-terminal glycosylphosphatidylinositol (GPI) anchor. The extracellular domains, which carry the HS chains, vary greatly in size within the syndecan family but display considerable homology among the glypicans. The latter share a characteristic pattern of 14 conserved Cys residues, and their HS attachment sites are concentrated to a short region near the C-terminal (for reviews, see Bellin et al., 2003; Fransson et al., 2004).
In recent years, several different portals of entry into cells have been revealed (for a review, see, e.g., Conner and Schmid, 2003). One of these involves cholesterol-rich lipid rafts that can occur singly or as clusters or organized by caveolins (Cav) into special invaginations called caveolae (for reviews, see, e.g., Nichols, 2003; Pelkmans, 2005). Both lipid raft-mediated uptake and caveolae-mediated uptake of cargo have been demonstrated. Cargo and Cav-associated raft components are transported to caveosomes, which are seen as intracellular distribution centers for individual caveolar vesicles (Figure 1). These vesicles transiently fuse with early endosomes under the control of the small GTPase Rab5. When early endosomes migrate toward the cell center, Rab5 is replaced by Rab7, forming late endosomes (Rink et al., 2005; for a review, see Russell et al., 2006). Transport from late endosomes to the Golgi is governed by Rab9 (Pfeffer, 2005).
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Cell-surface HSPGs are constitutively endocytosed and degraded. Their HS chains are first cleaved into oligosaccharides by heparanase in endosomal compartments and then terminally degraded in lysosomes (for a review, see Yogalingam and Hopwood, 2001
). Glypican-1 (Gpc-1), the most widely expressed member of the glypican family, is lipid raft-associated and can be endocytosed in many cell types, whereupon its HS chains are released and partially degraded in an endosomal compartment (Fransson et al., 2004). The remaining truncated Gpc-1 is recycled back to the cell surface via the Golgi, where new HS chains are added on the stubs (Edgren et al., 1997).
In addition to the heparanase-catalyzed degradation, there is a nitric oxide (NO)-catalyzed, deaminative cleavage of HS at N-unsubstituted glucosamine (GlcNH3+) residues (Cheng et al., 2002; Ding et al., 2002). These residues are present in HS chains in a relatively limited number and usually near the core protein attachment (Ding, Jönsson et al., 2001; Westling and Lindahl, 2002). Deaminative cleavage catalyzed by nitrite or NO generates HS chains and oligosaccharides containing anhydromannose (anMan) as the reducing terminal sugar (Horton and Phillips, 1973; Vilar et al., 1997; Mani et al., 2000; Cheng et al., 2002; Ding et al., 2002). By using confocal immunofluorescence microscopy as well as immunochemical methods, we have shown that Cys residues in Gpc-1 can be S-nitrosylated (SNO) both in cell-free experiments and in cultured cells. NO derived from these SNO groups catalyzes deaminative release of HS chains from Gpc-1. Release of NO can be triggered by an unknown endogenous agent or by exogenously supplied ascorbate (Cheng et al., 2002; Ding et al., 2002). Owing to the short half-life of NO, other HSPGs should not be susceptible to cleavage unless they are in close proximity to Gpc-1-SNO (see Fransson et al., 2004). It is not known if other glypicans are processed in the same way.
To identify the subcellular localization of the NO-dependent Gpc-1 modification and degradation steps, we have previously arrested recycling by using brefeldin A. Brefeldin A interferes with the recruitment of coat protein complex I to transporting endosomes (Whitney et al., 1995). Brefeldin A-arrested Gpc-1 colocalizes with caveolin-1 (Cav-1) but not with Rab9. Gpc-1 is devoid of SNO groups but its HS chains contain GlcNH3+ residues. In unperturbed cells, Cav-1-associated Gpc-1 contains SNO groups but is still substituted with GlcNH3+-containing HS chains. This is in keeping with S-nitrosylation of Gpc-1 that already carries GlcNH3+-containing HS chains in the early part of the recycling pathway (Figure 1) followed by deaminative cleavage of HS at a later stage (Cheng et al., 2002). The NO-catalyzed deaminative cleavage of HS in Gpc-1 requires reductive release of NO from the SNO groups in the Gpc-1 core protein (Fransson et al., 2004). Late endosomes contain a strong reducing power (Fivaz et al., 2002), making this compartment a likely site for the deaminative cleavage of HS (Figure 1).
Transport of cholesterol from late endosomes to the Golgi is blocked in Niemann–Pick C (NPC) cells but restored by over-expression of Rab9 (for reviews, see Vanier and Millat, 2003; Ioannou, 2005). We have recently found that NO-catalyzed degradation of Gpc-1 HS is greatly depressed in NPC-1 fibroblasts but can be restored by the addition of ascorbate, which releases NO from Gpc-1-SNO and enables deaminative cleavage of HS (Mani et al., 2006). These results suggested that cholesterol and Gpc-1 have a common traffic route and that an endosome-based reducing agent was involved in Gpc-1 autoprocessing (Figure 1).
Here, we have used confocal immunofluorescence microscopy, radiolabeling, gel chromatography, and immunomagnet isolation of Rab5-, Rab7-, and Rab9-positive vesicles in order to locate anMan-positive HS degradation products in normal proliferating fibroblasts, ascorbate-treated proliferating NPC-1 fibroblasts, as well as in dehydroascorbic acid-treated mouse neuroblastoma cell line (N2a) and in human bladder carcinoma cell line (T24). Inhibition of transfer from early to late endosomes precluded formation of anMan-positive products. The results indicate that anMan-positive HS oligosaccharides are present primarily in Rab7-positive late endosomes.
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Results |
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Constitutive and ascorbate-induced deaminative cleavage of HS in fibroblasts
Deaminatively cleaved HS chains and oligosaccharides were visualized by confocal microscopy using a specific monoclonal antibody (mAb) directed against anMan-containing deaminative cleavage products of heparin (Pejler et al., 1988
; Liljeblad et al., 1998). Gpc-1 was detected by using a polyclonal antiserum raised against recombinant human Gpc-1 core protein (Mani et al., 2000). Proliferating normal human fetal lung fibroblasts (HFL-1) generated constitutively anMan-positive products which both colocalized with Gpc-1 (Figure 2A, yellow) and occurred separately (Figure 2A, green). The Gpc-1 staining was present both at the cell surface and throughout the cytoplasm, especially around the nucleus, and appeared both punctate and reticular. This is to be expected as Gpc-1 protein should be present in both the secretory and the endocytic pathways. Most of the anMan-positive staining did not colocalize with LysoTracker Red (Figure 2B, red), suggesting that the HS degradation products were not generated in lysosomes.
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Constitutive generation of anMan-positive HS degradation products was undetectable in growth-quiescent fibroblasts (Figure 2C, no green). However, ascorbate can be taken up by fibroblasts (Corpe et al., 2005
; Wilson, 2005) and release NO from Gpc-1-SNO resulting in deaminative cleavage of HS (Ding et al., 2002; see also Table I). Hence, when growth-quiescent fibroblasts were treated with ascorbate, anMan-positive products were generated and appeared again both colocalized with Gpc-1 and at separate sites (Figure 2D, yellow and green). Some of the anMan-positive degradation products may have segregated from Gpc-1 into a different endosomal compartment and/or may be derived from other glypicans.
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NPC diseases are characterized by a transport block in late endosomes (Figure 1) resulting in the accumulation of cholesterol in the endosomal/lysosomal compartments (for a review, see, e.g., Vanier and Millat, 2003
; Ioannou, 2005). We have recently found that constitutive NO-catalyzed HS degradation is defective in proliferating fibroblasts from the major form (NPC-1) of these diseases (Mani et al., 2006). However, addition of ascorbate partially restored NO-dependent HS degradation. Quantitative estimates using flow cytometry indicated that the content of anMan-positive HS degradation products increased 
3-fold upon addition of ascorbate.
Late endosomes are known to be Rab7- and/or Rab9-positive (Pfeffer, 2005; Rink et al., 2005; Russell et al., 2006). To examine whether deaminative degradation products of HS appear in late endosomes of ascorbate-treated NPC-1 fibroblasts, we stained sparse cultures for anMan-positive degradation products, Gpc-1, and the two Rabs. As shown previously, anMan-positive products could not be detected in untreated cells (Figure 3A, no green). Furthermore, Gpc-1 displayed only limited colocalization with Rab9 (Figure 3B). The anMan-positive HS degradation products generated in ascorbate-treated NPC-1 cells colocalized with Rab7-positive compartments (Figure 3C, yellow arrow), and Gpc-1 displayed increased colocalization with Rab9 at paranuclear sites (Figure 3D, yellow arrow).
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There are two competing HS degradations, the heparanase-catalyzed hydrolytic degradation and the NO-catalyzed deaminative cleavage (Fransson et al., 2004
; see also Table I). When heparanase is inhibited by suramin, more HS is available for NO-dependent cleavage. Accordingly, when NPC-1 cells are first treated with suramin and then with ascorbate, more anMan-positive HS degradation products are generated. Previous quantitative estimates using flow cytometry indicated that the content of anMan-positive HS degradation products increases 4-fold (Mani et al., 2006). These products colocalized even more intensely with Rab7 (Figure 3E, yellow arrow). However, Gpc-1 did not colocalize with Rab9 (Figure 3F, green arrow) suggesting that inhibition of heparanase or some other effect of suramin on NPC-1 cells retards Gpc-1 recycling.
The protonophore carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone (FCCP), which inhibits endosomal acidification, abrogates protein trafficking from early (Rab5) to late (Rab7) endosomes (Rink et al., 2005; Hurtado-Lorenzo et al., 2006; see also Table I). However, NPC-1 fibroblasts treated with suramin and both FCCP and ascorbate still generated anMan-positive HS degradation products that colocalized entirely with Rab7 (Figure 3G, yellow arrow). Moreover, colocalization between Gpc-1 and Rab9 was partially restored (Figure 3H, yellow arrow). Apparently, FCCP and ascorbate counteracted the inhibitory effect of suramin on Gpc-1 recycling in these cells. Taken together, the results suggested that ascorbate may have two effects, that is, initiation of deaminative HS cleavage in early endosomes and transport of the anMan-positive degradation products to Rab7-positive late endosomal compartments.
Constitutive and ascorbic/dehydroascorbic acid-induced deaminative cleavage of HS in N2a cells
Cationic steroids, such as 3-ß[2(diethylamino) ethoxy]androst-5-en-17-one (U18666A), a compound widely used to induce cholesterol accumulation, inhibits deaminative HS degradation in non-NPC cells which results in an NPC-like phenotype (for references, see Mani et al., 2006; see also Table I). As shown in Figure 4A, N2a cells, which grow in colonies, generated constitutively anMan-positive HS degradation products. Staining for such products was reduced by 40% after treatment with U18666A as indicated by flow cytometry (Figure 4B, inset). Subsequent exposure to ascorbate restored formation of such products, which appeared mainly in a punctate pattern, in keeping with formation in a vesicular compartment (Figure 4C).
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Intracellular accumulation of vitamin C (ascorbic or dehydroascorbic acid) is mediated either by the specific, low-capacity Na-ascorbate transporters SVCT1 and SVCT2 or by the unspecific, high-capacity dehydroascorbic acid/glucose transporters GLUT1 and GLUT3 (Corpe et al., 2005
; Wilson, 2005). Dehydroascorbic acid can be converted to ascorbic acid by reducing agents present in the cytosol (Table I). Both transport mechanisms occur in many cells in the body, but fibroblasts have only SVCT-mediated ascorbate uptake (Corpe et al., 2005). We therefore compared vitamin C-induced, NO-dependent HS degradation in N2a cells and in HFL-1 fibroblasts. We first inhibited deaminative HS degradation by treating cells with U18666A (U) and then induced deaminative degradation of HS by exposing cells to either ascorbate or dehydroascorbic acid. As predicted, both ascorbate and dehydroascorbic acid induced formation of anMan-positive HS in N2a cells (Figure 4C and D), whereas only ascorbate could activate such HS degradation in HFL-1 fibroblasts (Figure 4G and H). Product formation was maximal after a 1 h exposure to ascorbic acid and after 4 h of exposure to dehydroascorbic acid.
In untreated N2a cells, the anMan-positive staining appeared both punctate and diffuse (Figure 4A), suggesting both vesicular and cytosolic localizations of the products. When U18666A-treated N2a cells were exposed to ascorbate for 1 h, staining was mostly punctate (Figure 4C), whereas the 4 h exposure to dehydroascorbic acid resulted in mostly diffuse localizations (Figure 4D). These results suggested that anMan-containing HS was formed in vesicular structures but then escaped into the cytosol.
To maximize formation of anMan-containing HS degradation products, we treated N2a cells first with suramin to inhibit heparanase, then with U18666A to inhibit deaminative cleavage and thereby induce accumulation of Gpc-1-SNO, and finally with dehydroascorbic acid to activate deaminative HS degradation. The anMan signal remained essentially unaffected (Figure 4E and F), indicating that NO-catalyzed HS degradation was the major route for Gpc-1 processing in untreated N2a cells. The HS products generated both constitutively and during dehydroascorbic acid treatment colocalized with Rab7, mostly near the cell surface (Figure 4E and F, yellow). However, the HS degradation products generated during dehydroascorbic acid treatment were also present separately, often diffusely located at sites forming bulges, suggesting escape into the cytosol (Figure 4F, green arrows). Overall, the effects observed were not critically dependent on cell density.
Identification of anMan-positive, radiolabeled HS degradation products in late endosomes of T24 cells
To study HSPG degradation, we incubated T24 cells with 35S-sulfate, treated them with difluoromethyl ornithine (DFMO), suramin, U18666A, and ascorbate, in various combinations, and chromatographed the total cell lysates on Superose 6 (Figure 5A–E). In addition, we treated cells with the same drugs and examined them for anMan-positive HS degradation products by confocal microscopy (Figure 5F–J). Previous studies have shown that treatment with DFMO increases the number of GlcNH3+ residues, that is, NO-sensitive sites, in HS of Gpc-1 (Ding, Sandgren et al., 2001). Hence, pretreatment with DFMO is another way to increase the yield of anMan-containing HS chains and oligosaccharides. To minimize heparanase degradation of HS, we treated cells with suramin. U18666A was used to inhibit NO-catalyzed deaminative cleavage of HS. Ascorbate was used to reactivate the latter cleavage (see also Table I). Both syndecans and glypicans can be internalized and degraded. Proteolytic degradation of HSPG can precede cleavage by heparanase (Bame, 1993). Therefore, inhibition of heparanase by suramin may result in the formation of free HS chains by proteolytic cleavage of HSPG.
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DFMO-treated and 35S-sulfate-labeled T24 cells generated radiolabeled macromolecules eluting from Superose 6 as shown (Figure 5A). The elution position of the peak marked PG coincided with that of Gpc-1 previously immunoisolated from T24 cells (Ding, Jönsson et al., 2001
). There was also a peak in the position of free HS chains as well as lower-molecular size material (see bar). Confocal immunofluorescence microscopy of DFMO-treated cells showed that anMan-positive HS degradation products were generated (Figure 5F). We then inhibited formation of such products by treating cells with U18666A (U). Staining for anMan-positive products was greatly reduced (Figure 5G) and the radiolabeled components eluting from Superose 6 around position 38–40 decreased, whereas the PG pool remained unchanged (Figure 5B). When heparanase-catalyzed degradation of HS was inhibited by suramin, formation of radiolabeled products eluting between positions 35 and 45 increased markedly (Figure 5C, filled symbols) and there was also increased staining for anMan-positive HS (Figure 5H). To isolate radiolabeled anMan-positive HS degradation products, we passed a lysate of DFMO- and suramin-treated, 35S-sulfate-labeled T24 cells through Protein G-Sepharose 4B precoated with the mAb specific for anMan-containing oligosaccharides. Bound material was displaced from the gel and chromatographed on Superose 6. The radiolabeled anMan-positive degradation products included one component eluting as free HS chains and another eluting in a more retarded position but earlier than free sulfate (Figure 5C, open symbols). However, a major portion of the radiolabeled degradation products was apparently not anMan-positive and possibly generated by proteolytic degradation of HSPGs (Figure 5C, filled symbols). Simultaneous inhibition of heparanase by suramin and of NO-catalyzed HS degradation by U18666A, followed by reactivation of the latter process by ascorbate, did not markedly affect the yield of radiolabeled (Figure 5D) or anMan-positive HS degradation products (Figure 5I), except that the latter appeared in the nuclei to some extent. Suramin treatment increased the size of the PG pool, in keeping with the inhibition of heparanase degradation (cf. Figure 5B with C and D). These results indicated that radiolabeled, anMan-positive HS chains and oligosaccharides were generated and that the major route for HS degradation in unperturbed T24 cells is via heparanase.
When T24 cells were not preexposed to DFMO, treatment with suramin, U18666A, and ascorbate also yielded a major pool of radiolabeled degradation products (Figure 5E), albeit less polydisperse than those obtained from DFMO-treated cells (Figure 5C and D). However, when DFMO treatment was omitted, accumulation of anMan-positive HS in the nuclei was more pronounced (Figure 5J).
To identify the endosomal localization of the anMan-positive HS degradation products generated in DFMO- and suramin-treated T24 cells, we used confocal immunofluorescence microscopy. The results showed that the anMan-positive products colocalized mostly with Rab7 (Figure 6A, same as Figure 5H) but also partly with Rab9 (Figure 6B). There was extensive colocalization between Gpc-1 and Rab9, mostly to one side of the nuclei (Figure 6C, yellow), but there was no colocalization between anMan-positive products and Cav-1 (Figure 6D, separate red and green staining). Inhibition of transfer from early to late endosomes by FCCP abrogated formation of anMan-positive products (Figure 6F), without affecting Gpc-1 expression (Figure 6E). Again, ascorbate could overcome the inhibitory effect of FCCP, albeit to a somewhat variable extent (Figure 6G). In cells where a substantial amount of anMan-positive HS degradation products were generated, they colocalized with Rab7 (Figure 6H, yellow arrow), but in other cells, the anMan-positive products were sometimes diffusely located at sites separate from Rab7 (Figure 6H, green arrow). Previous results indicated that heparanase degradation of Gpc-1 HS could take place in Cav-1-associated compartments of T24 cells (Cheng et al., 2002). The present results thus indicated that deaminative cleavage of HS in T24 cells is downstream of heparanase and generates products that appear in Rab7-positive endosomes and that suramin treatment does not interfere with transfer of Gpc-1 to Rab9-positive endosomes in these cells.
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To further study HS degradation products in the various endosomal compartments and in the cytosol, we incubated T24 cells with 35S-sulfate followed by mild homogenization and ultracentrifugation or immunomagnet isolation of Rab5-, Rab7-, and Rab9-positive vesicles. The cells were pretreated with DFMO and suramin to maximize deaminative HS degradation and to minimize heparanase-catalyzed degradation, respectively. The isolated Rab-positive vesicles were lysed, and the extracted radiolabeled material was subjected to gel chromatography on Superose 6 or Superdex peptide. Rab7-positive and, also to some extent, Rab5-positive vesicles contained degradation products that were well retarded on Superose 6 (Figure 7A and B), thus eluting in the same position as the anMan-positive radiolabeled oligosaccharides obtained from the total cell extract (Figure 5C). The oligosaccharides obtained from the Rab7-positive vesicles eluted shortly after the void volume on Superdex peptide (inset in Figure 7B), suggesting that they were relatively large, probably around 20mers. Rab9-positive vesicles contained Gpc-1-like PG and material that eluted in the position of HS chains or large chain fragments (Figure 7C).
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We also examined a postmicrosomal fraction obtained from DFMO- and suramin-treated, 35S-sulfate-labeled T24 cells by centrifugation of a cell homogenate at 150,000 x g. As shown in Figure 7D, the supernatant contained, in addition to unincorporated radioactivity, a major component that eluted around position 45, that is, between the anMan-positive HS chains and oligosaccharides obtained from the total cell extract (Figure 5C, open symbols). The microsomal fraction (Figure 7E) contained radiolabeled macromolecules eluting in the positions of PG and HS chains as well as a major peak in the same position as the oligosaccharides recovered with the Rab7-positive vesicles (Figure 7B) and the anMan-positive oligosaccharides derived from the total cell extract (Figure 5C, open symbols).
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Discussion |
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Previous results indicated that recycling Gpc-1 is S-nitrosylated in Cav-1-associated compartments, such as caveolae or caveosomes, where degradation by heparanase may also take place (Ding et al., 2002
). S-nitrosylation requires Cu(II) ions (Ding et al., 2002), which can be provided by the prion protein in fibroblasts or N2a cells (Mani et al., 2003; Cheng et al., 2006), by the brain-specific, GPI-linked splice variant of ceruloplasmin in glial cells (Mani, Cheng et al., 2004) or by the amyloid precursor protein in neural cells (Cappai et al., 2005). The present results indicate that Gpc-1-SNO is then transported to late endosomes where an unknown reducing agent induces NO-release from the SNO groups and initiates deaminative cleavage of HS, generating HS chains and oligosaccharides that react with an anMan-specific mAb (Figure 8). Constitutive formation of anMan-positive degradation products was seen in proliferating normal fibroblasts but not in growth-quiescent ones. However, both sparse and confluent cultures of transformed cells generated these products. AnMan-positive degradation products may also be derived from other minor glypicans.
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In NPC-1 fibroblasts, transport of cholesterol via late endosomes and formation of anMan-positive HS degradation products are both impeded (Mani et al., 2006
). The anMan-positive products generated after ascorbate treatment of NPC-1 cells colocalized with Rab7, a marker for late endosomes, whereas Gpc-1 colocalized with Rab9, a marker for endosomes that transport cargo to the Golgi. In suramin- and U18666A-treated N2a cells, where both heparanase- and NO-catalyzed degradations of HS are inhibited, reactivation of the latter process by dehydroascorbic acid generated anMan-positive degradation products that also colocalized with Rab7. Likewise, in suramin-treated T24 cells, anMan-positive degradation products colocalized with Rab7, whereas Gpc-1 colocalized with Rab9. Furthermore, the protonophore FCCP, which inhibits transport from Rab5-positive early endosomes to Rab7-positive late endosomes (Rink et al., 2005; Hurtado-Lorenzo et al., 2006), prevented constitutive deaminative HS degradation in T24 cells. In both T24 cells and NPC-1 fibroblasts, ascorbate could induce deaminative HS degradation in the presence of FCCP. Surprisingly, the anMan-positive products colocalized with Rab7 in both cases. These results suggested that ascorbate also stimulates transfer of Gpc-1 from Rab5- to Rab7-positive endosomes in the presence of FCCP. It has been shown that intra-endosomal acidification can recruit cytosolic transport mediators via a transmembrane pH-sensoring complex (Hurtado-Lorenzo et al., 2006). Incidentally, NO-catalyzed, deaminative cleavage of linkages between GlcNH3+ and uronic acid in HS is in itself acidifying, generating H3O+ when the GlcN=N+ intermediate is formed and consuming OH– when the glycosidic bond of the anMan carbocation intermediate is cleaved (Horton and Phillips, 1973). Hence, NO-catalyzed deaminative cleavage of HS may be initiated in early endosomes and completed in late endosomes. In N2a cells, prolonged reactivation of deaminative HS cleavage using dehydroascorbic acid resulted in a diffuse distribution of anMan-positive products suggestive of a cytosolic localization.
The 35S-labeled, anMan-positive degradation products generated in DFMO- and suramin-treated T24 cells included both HS chains and oligosaccharides. As shown previously, the NO-sensitive GlcNH3+ residues are usually concentrated to clustered sites near the core protein attachment, whereas DFMO induces increased GlcNH3+ formation at other sites (Ding, Jönsson et al., 2001; Ding, Sandgren et al., 2001). Accordingly, both HS chains and oligosaccharides should be generated. A major portion of the radiolabeled glycan chains and oligosaccharides generated when heparanase was inhibited by suramin were not recovered in the immunoisolation procedure using the anMan-specific antibody. There may be several reasons for this. Nonreactive glycan chains may have been released from PGs by proteolysis. Oligosaccharides may have arisen via another degradative pathway catalyzed by an endo-N-acetylhexosaminidase (Fuller et al., 2006) The anMan residue of deaminatively generated HS oligosaccharides may subsequently be lost or modified and therefore unrecognizable. The immunoisolation procedure may not be quantitative.
Radiolabeled oligosaccharides, probably around 20mers, and generated in T24 cells when heparanase was inhibited by suramin, were present in the microsomal fraction and in the Rab7-positive vesicles. These oligosaccharides were of the same size as the anMan-positive radiolabeled oligosaccharides isolated from the total cell extract. HS oligosasaccharides in Rab7-positive endosomes may be delivered to the lysosomes for final degradation to monosaccharides (Yogalingam and Hopwood, 2001).
Somewhat larger, anMan-negative degradation products were present in the postmicrosomal fraction suggesting escape into the cytosol. The escape mechanisms as well as the origin, function, and ultimate fate of these degradation products remain to be understood.
anMan-Positive HS degradation products were also observed in the nuclei of T24 cells, especially when DFMO was omitted and the degradation products therefore were less polydisperse. The presence of HS in the nuclei of cells has been reported previously. Conrad and coworkers (Fedarko and Conrad, 1986; Ishihara et al., 1986) showed that HSPG internalized by hepatocytes is degraded and that the HS chains or chain fragments are transported to the nucleus. The nuclear HS was enriched in 2-O-sulfated glucuronic acid, suggesting that the HS fragments had also been processed by heparanase, an endoglucuronidase that only cleaves at unsulfated glucuronic acid (Pikas et al., 1998; Okada et al., 2002). Gpc-1, which contains a functional nuclear localization signal in the core protein, has been identified in the nuclei of neurons and C6 glioma cells (Liang et al., 1997), suggesting that also the Gpc-1 core protein can escape from the endosomes. Incidentally, xyloside-primed HS chains are also targeted to the nuclei of T24 cells (Mani, Belting et al., 2004).
Other recent studies from this laboratory have shown that the prion protein is co-transported with Gpc-1 via a Cav-1-associated route (Cheng et al., 2006). Recycling proteins, such as the prion protein, may misfold in late endosomes (Campana et al., 2005). It is possible that HS oligosaccharides, either generated by heparanase cleavage or by NO-dependent deaminative cleavage, or some other process, serve to maintain aggregation-prone peptides and proteins in a soluble form. HS oligosaccharides may then accompany proteins to the lysosomes or escort them through the endosomal membrane barrier for transport to the cytosol and ultimately to the nucleus.
Several different neurological illnesses illustrate the importance of adequate HS turnover, notably NPC disease, where deaminative HS cleavage is impeded (Mani et al., 2006), and the Sanfilippo storage diseases, where HS degradation in the lysosomes is defective because of mutations in different HS-specific degradative enzymes. Both disorders result in severe central nervous system degeneration (Yogalingam and Hopwood, 2001; Vanier and Millat, 2003).
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Cells, antibodies, and reagents
HFL-1, N2a cells, and T24 cells were obtained from American Type Culture Collection and NPC-1 fibroblasts (GM03123) from the Corielle Institute and were maintained in minimal essential medium supplemented with 10% fetal calf serum. Polyclonal antisera against human Gpc-1, a mAb recognizing anMan-terminating HS oligosaccharides (Pejler et al., 1988
; Liljeblad et al. 1998), suitably tagged secondary antibodies, as well as suramin, DFMO, sodium L-ascorbate, enzymes, prepacked columns, and other chemicals were generated or obtained as described previously (Cheng et al., 2002; Ding et al., 2002; Belting et al., 2003; Mani et al., 2003; Cappai et al., 2005). U18666A was obtained from Biomol (Plymouth Meeting, PA), LysoTracker Red from Molecular Probes (Leiden, The Netherlands), polyclonal antibodies to Cav-1 from Transduction Laboratories (Becton Dickinson AB, Stockholm, Sweden), polyclonal antibodies to Rab7 and Rab9 from Santa Cruz (Santa Cruz, CA), and a mAb against Rab9 from Affinity BioReagents (Golden, CO). The commercially obtained antibodies were generally tested by western blotting according to the manufacturer details. Dynabeads M-280 sheep anti-rabbit immunoglobulin G (IgG) was obtained from Dynal ASA (Oslo, Norway). Carbonyl cyanide FCCP was from Sigma (Stockholm, Sweden). Dehydroascorbic acid was generated by leaving a 1 M stock solution of ascorbate in contact with air for 24 h.
Confocal microscopy
The various procedures including seeding of cells, the use of primary and secondary antibodies, generation of images by sequential scans, and data processing were the same as those used previously (Cheng et al., 2002; Ding et al., 2002; Belting et al., 2003; Mani et al., 2003, 2004) or as recommended by the manufacturers. Cells were fixed and permeabilized with acetone or H2O2 or methanol. Mouse N2a cells were first precoated with 10% anti-mouse IgG and then exposed to primary antibodies. The second antibody used was either goat anti-mouse total Ig when the primary antibody was a mouse monoclonal or goat anti-rabbit IgG when the primary antibody was a rabbit polyclonal. The second antibodies were tagged with either fluoresceine isothiocyanate or Texas Red and appropriately combined for colocalization studies. In the controls, the primary antibody was omitted. Prior to fixation and confocal microscopy, cells were inspected by light microscopy. For confocal microscopy, we used a Nikon Eclipse E800 microscope (Bergström Instrument AB, Lund, Sweden) equipped with a 100x objective and a Bio-Rad MRC 1024 confocal laser scanning system (Sundbyberg, Sweden). Images shown were obtained at a focal plane that was at the center of the cell and of 0.3–0.5 µm thickness, depending on the intensity of the staining. Identical focal plane thickness and exposure settings were used for image capture in the colocalization experiments. Images were digitized and transferred to Adobe PhotoShop (Adobe Inc., Lund, Sweden) for merging, annotation, and printing.
Flow cytometry
Cells were seeded in 24-well plates and grown to near confluence, rinsed with medium, and detached using trypsin (0.5 mL 0.05% w/v of trypsin, 0.5 mM ethylenediaminetetraacetic acid [EDTA] in growth medium for 3–4 min). Trypsinization was terminated by replacing the trypsin solution with 0.5 mL medium supplemented with 10% fetal bovine serum. Cells were recovered by gentle suspension and transferred to tubes, adding 1 vol of phosphate-buffered saline (PBS) containing 1% bovine serum albumin (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) whilst initially vortexing. Permeabilization was performed by incubation with 0.2% Triton X-100 in PBS (v/v) for 20 min. Immunostaining of the cells with the mAb specific for anMan-containing HS degradation products as the primary antibody and goat anti-mouse total Ig as the secondary antibody was performed as described for confocal microscopy. In the controls, the primary antibody 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, Stockholm, Sweden) operated by Cell-Quest software (Franklin Lakes, NJ).
Metabolic labeling, separation, and isolation of radiolabeled anMan-positive HS products
T24 cells were grown in 25 or 75 cm2 plates until confluent, treated as described in the legend to Figure 5, incubated with [35S]sulfate (50 µCi/mL), and extracted, and products were isolated as described previously (Mani et al., 2000). In short, radiolabeled cells were lysed with Triton X-100 and aliquots (200 µL) were mixed with an equal volume of 8 M guanidine-HCl and directly chromatographed on Superose 6 eluted with 4 M guanidine-HCl/0.2% Triton X-100. Fractions were analyzed for radioactivity by ß-scintillation.
Isolation of [35S]sulfate-labeled anMan-positive oligosaccharides were performed as follows. Cells, grown, treated, and labeled as described above, were lysed in 0.1% (w/v) sodium dodecyl sulfate (SDS), 0.5% (v/v) Triton X-100, 0.5% (w/v) sodium deoxycholate, PBS supplemented with 0.5 mM phenylmethyl sulfonyl fluoride (Mani et al., 2000). The lysate was mixed with 150 µL Protein G-Sepharose 4B saturated with the mAb specific for anMan-containing oligosaccharides, shaken overnight in the cold, and spun at 150 x g in a benchtop centrifuge for 1 min. The pelleted gel was washed five times with lysis buffer and then eluted with 4 M guanidine-HCl and chromatographed on Superose 6 as described above.
Isolation of radiolabeled oligosaccharides from Rab7-positive vesicles and the cytosol
Confluent T24 cells were preincubated with 5 mM DFMO for 24 h and then with 0.2 mM suramin and [35S]sulfate for another 24 h as described previously (Mani et al., 2000). Cells were homogenized in PBS containing 0.25 M sucrose, 0.1 M EDTA, 0.5 mM phenylmethyl sulfonyl fluoride. Rab5-, Rab7-, or Rab9-positive vesicles were isolated from cell homogenates by using polyclonal antibodies against the various Rabs as primary antibodies and Dynabeads M-280 sheep anti-rabbit IgG as magnetic secondary antibody. The magnetic particles were recovered using a magnetic particle concentrator and extensively washed with PBS (at least 10 times). The radiolabeled, magnetically isolated endosomes were lysed in 0.15 M NaCl, 10 mM EDTA, 2% Triton X-100, 10 mM KH2PO4, pH 7.5, and mixed with an equal volume of 8 M guanidinium chloride. All extracts were then subjected to gel filtration chromatography on Superose 6 or Superdex peptide in 4 M guanidinium chloride/0.2% (v/v) Triton X-100 (Ding, Jönsson et al., 2001; Ding, Sandgren et al., 2001).
Cell homogenates of cells treated and radiolabeled as above were also subjected to ultracentrifugation at 150,000 x g for 1 h in a Beckman Coulter Optima XL centrifuge (Beckman Instruments, Inc., Fullerton, CA) using rotor 70.1 Ti. The supernatant was mixed with an equal volume of 8 M guanidinium chloride and the sedimented microsomes were dissolved in 4 M guanidinium chloride, and both were chromatographed on Superose 6.
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Acknowledgments |
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TopAbstractIntroductionResultsDiscussionMaterials and methodsAcknowledgmentsConflict of interest statementReferences |
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We thank Prof. Catharina Svanborg at the Department of Microbiology, Immunology, and Glycobiology, Lund University, for use of microscope facilities and Dr Gunnar Peijler, Uppsala University, and Dr Peter Påhlsson, Linköping University, Sweden for a generous gift of mAbs. The work was supported by grants from the Swedish Science Council (VR-M), the Bergvall, Crafoord, Hedborg, Kock, Segerfalk, Zoega, and Österlund Foundations, and the Medical Faculty of Lund University.
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Conflict of interest statement |
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TopAbstractIntroductionResultsDiscussionMaterials and methodsAcknowledgmentsConflict of interest statementReferences |
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None declared.
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Abbreviations |
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anMan, anhydromannose; BSA, bovine serum albumin; Cav, caveolin; DFMO, difluoromethyl ornithine; EDTA, ethylenediaminetetraacetic acid; FCCP, carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone; GlcNH3+, N-unsubstituted glucosamine; Gpc-1, glypican-1; GPI, glycosylphosphatidylinositol; HFL-1, human fetal lung fibroblasts; HS, heparan sulfate; HSPG, HS-containing proteoglycan; IgG, immunoglobulin G; mAb, monoclonal antibody; N2a, mouse neuroblastoma cell line; NO, nitric oxide; NPC-1, Niemann–Pick type C-1; PBS, phosphate-buffered saline; PG, proteoglycan; SNO, nitrosothiol; T24, human bladder carcinoma cell line; U18666A, 3-ß[2(diethylamino) ethoxy]androst-5-en-17-one
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