Glycobiology Advance Access originally published online on April 27, 2006
Glycobiology 2006 16(8):711-718; doi:10.1093/glycob/cwj121
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Defective nitric oxide-dependent, deaminative cleavage of glypican-1 heparan sulfate in Niemann–Pick C1 fibroblasts
Department of Experimental Medical Science, Division of Neuroscience, Glycobiology Group, Lund University, Biomedical Center C13, SE-221 84 Lund, Sweden
1 To whom correspondence should be addressed; e-mail: lars-ake.fransson{at}medkem.lu.se
Received on January 18, 2006; revised on April 21, 2006; accepted on April 21, 2006
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Abstract |
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Exit of recycling cholesterol from late endosomes is defective in Niemann–Pick C1 (NPC1) and Niemann–Pick C2 (NPC2) diseases. The traffic route of the recycling proteoglycan glypican-1 (Gpc-1) may also involve late endosomes and could thus be affected in these diseases. During recycling through intracellular compartments, the heparan sulfate (HS) side chains of Gpc-1 are deaminatively degraded by nitric oxide (NO) derived from preformed S-nitroso groups in the core protein. We have now investigated whether this NO-dependent Gpc-1 autoprocessing is active in fibroblasts from NPC1 disease. The results showed that Gpc-1 autoprocessing was defective in these cells and, furthermore, greatly depressed in normal fibroblasts treated with U18666A (3-ß-[2-(diethylamino)ethoxy]androst-5-en-17-one), a compound widely used to induce cholesterol accumulation. In both cases, autoprocessing was partially restored by treatment with ascorbate which induced NO release, resulting in deaminative cleavage of HS. However, when NO-dependent Gpc-1 autoprocessing is depressed and heparanase-catalyzed degradation of HS remains active, a truncated Gpc-1 with shorter HS chains would prevail, resulting in fewer NO-sensitive sites/proteoglycan. Therefore, addition of ascorbate to cells with depressed autoprocessing resulted in nitration of tyrosines. Nitration was diminished when heparanase was inhibited with suramin or when Gpc-1 expression was silenced by RNAi. Gpc-1 misprocessing in NPC1 cells could thus contribute to neurodegeneration mediated by reactive nitrogen species.
Key words: cholesterol / glypican-1 / heparan sulfate / Niemann–Pick C / nitric oxide
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Introduction |
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TopAbstractIntroductionResultsDiscussionMaterials and MethodsConflict of interest statementAcknowledgmentsReferences |
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Niemann–Pick type C (NPC) is an autosomal, recessive neurovisceral lipid storage disease resulting in fatal neurodegeneration. At the cellular level, the disorder is characterized by the accumulation of cholesterol and glycosphingolipids in late endosomes and lysosomes. Approximately 95% of the patients have mutations in the NPC1 gene that encodes a large membrane glycoprotein primarily located to late endosomes. The remainder have mutations in the NPC2 gene that encodes a small, soluble, endosomal/lysosomal cholesterol-binding glycoprotein. Despite extensive investigations conducted in various animal models, the precise functions of NPC1 and NPC2 at the molecular level, as well as the pathophysiological effects of their dysfunction, have remained elusive (for reviews, see Vanier and Millat, 2003
; Liscum and Sturley, 2004; Mukherjee and Maxfield, 2004; Chang et al., 2005; Ioannou, 2005).
Cholesterol is delivered to cells by uptake of low-density lipoproteins via the low-density lipoprotein receptor and released in lysosomes (Brown and Goldstein, 1986). Exogenously as well as endogenously synthesized cholesterol, together with sphingolipids and glycosylphosphatidylinositol (GPI)-anchored proteins, is enriched in plasma membrane microdomains called lipid rafts and in the raft-derived, caveolin-coated membrane invaginations called caveolae. Clustering of lipid raft components can trigger caveolae-mediated endocytosis, whereby cholesterol-, sphingolipid-, and GPI-protein-enriched vesicles move along microtubules to caveosomes. Caveolin-free vesicles can exit from caveosomes and reach late endosomes and then return to the cell surface via the Golgi (for reviews, see Nichols, 2003; Parton and Richards, 2003; Pelkmans and Helenius, 2003; Soccio and Breslow, 2004). The sorting of GPI-linked, cell-surface proteins to recycling endosomes appears extremely sensitive to the extent of clustering (Sharma et al., 2004).
The GPI-anchored, heparan sulfate (HS)-substituted and recycling proteoglycan glypican-1 (Gpc-1) is abundant in the brain and potentially self-associating (for review, see Fransson et al., 2004). Gpc-1 localizes to lipid rafts (Watanabe et al., 2004) and is internalized via vesicles that contain caveolin-1 and react with cholera toxin B (Cheng et al., 2002). During recycling via endosomes and the Golgi, Gpc-1 undergoes modifications of both the core protein and the HS chains as well as degradations of the HS chains (Figure 1). Removal of the N-substituent on some of the glucosamine residues in HS, generating GlcNH3+, and S-nitrosylation (SNO) of conserved cysteines in the core protein by endogenously formed nitric oxide (NO) in a copper-dependent redox reaction (Figure 1B and C) initiates this processing (Mani et al., 2000; Ding et al., 2002). Later, presumably in late endosomes (Fivaz et al., 2002), NO is released from SNO and probably converted to nitroxyl (HNO) completing the copper redox cycle (Figure 1D). HNO then catalyzes deaminative cleavage at the GlcNH3+ residues of HS in Gpc-1 generating HS oligosaccharides with reducing terminal anhydromannose (anMan) residues. These oligosaccharides eventually separate from the truncated Gpc-1. Some of the oligosaccharides are transported to the nucleus, depending on the cell type; others are presumably degraded in lysosomes. The truncated Gpc-1 returns to the Golgi and new HS chains are extended on the stubs (Figure 1E). Preceding or concurrent with the deaminative cleavage of the HS chains in Gpc-1, heparanase-catalyzed degradation of HS can take place (Figure 1B and C). The deaminative cleavage of HS can also be induced by ascorbate, which is known to induce release of NO from SNO (Jaffrey et al., 2001). Both in a cell-free system—using purified Gpc-1, Cu(II) ions, and an NO donor—and when added to cells in culture, ascorbate can initiate deaminative cleavage of HS in Gpc-1 (Fransson et al., 2004).
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The traffic route of recycling Gpc-1 may involve late endosomes and could thus be affected in NPC1 disease. NPC1 fibroblasts also fail to oxidize cholesterol to oxysterols (Vanier and Millat, 2003; Liscum and Sturley, 2004; Mukherjee and Maxfield, 2004; Chang et al., 2005; Ioannou, 2005), possibly because cholesterol does not interact with the endosome-located redox system (Fivaz et al., 2002). NO- and copper-dependent processing of recycling Gpc-1 also requires an intracellular redox system (Fransson et al., 2004). This prompted us to examine whether NO-dependent Gpc-1 autoprocessing was affected in NPC1 fibroblasts and in normal cells treated with cationic amphiphiles such as imipramine and U18666A (3-ß-[2-(diethylamino)ethoxy]androst-5-en-17-one), which have been widely used to induce cholesterol accumulation (Liscum and Faust, 1989; Kobayashi et al., 1999; Lange et al., 2000; Ko et al., 2001).
We show here that NO-dependent deaminative cleavage of Gpc-1 HS is defective in NPC1 fibroblasts and severely depressed in cells treated with U18666A, while heparanase degradation of HS is unaffected. In both NPC1 and U18666A-treated cells, the inhibition of NO-dependent HS degradation is partially overcome by addition of ascorbate. When cells with arrested deaminative autoprocessing of Gpc-1 receive ascorbate, there is formation of nitrotyrosine. This can be diminished by inhibition of heparanase with suramin.
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Results |
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Defective or depressed NO-dependent, deaminative Gpc-1 HS autoprocessing in NPC1 fibroblasts and U18666A-treated normal fibroblasts
When Gpc-1 recycles through cells, it is processed by enzymatic (heparanase) as well as nonenzymatic (NO-catalyzed) de-glycanations. These constitute two competing degradative processes (Figure 2). Whereas heparanase is expected to degrade HS on both SNO-free and SNO-containing Gpc-1, deaminative de-glycanation can only take place on Gpc-1-SNO (Fransson et al., 2004
). As shown in Figure 3A and B, deaminative processing of Gpc-1 was active in growing human fetal lung fibroblasts (HFL-1) as indicated by the formation of anMan-containing HS oligosaccharides (Figure 3B), which was determined by an mAb raised against deaminative degradation products of heparin (Pejler et al., 1988). In contrast, formation of such oligosaccharides in growing NPC1 fibroblasts was reduced to 
10% of the level in normal fibroblasts (cf. Figure 3B and D, insets), although the expression of Gpc-1 appeared unaffected (cf. Figure 3A and C).
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NPC1 cells might fail to generate high amounts of anMan-containing HS oligosaccharides either because there are few NO-sensitive sites in Gpc-1 HS or because there is defective NO release from SNO groups in Gpc-1 or because Gpc-1 is poorly S-nitrosylated. Exogenous addition of ascorbate to cells can trigger deaminative cleavage of HS in SNO-containing Gpc-1 (Fransson et al., 2004). Moreover, by inhibition of heparanase degradation of HS by suramin, which does not interfere with NO-dependent degradation (Cheng et al., 2002), more Gpc-1 HS will be available for deaminative cleavage (Figure 2). Accordingly, when NPC1 fibroblasts were exposed to ascorbate, formation of anMan-containing HS degradation products increased (Figure 3E and F), especially when heparanase was inhibited by suramin (Figure 3F). Then, the amount of oligosaccharides generated by ascorbate increased 3-fold compared with untreated cells (cf. Figure 3D and F, insets) and reached 50% of the level in normal fibroblasts (cf. Figure 3B and F, insets).
The cationic amphiphile U18666A causes arrest of cholesterol recycling, yielding a cellular phenotype similar to that of NPC cells. We therefore examined whether NO-dependent Gpc-1 autoprocessing could be abrogated by treating normal fibroblasts with U18666A. Although treatment with 3 µg/mL of U18666A for 8 h was sufficient to cause cholesterol accumulation in HFL-1 cells as determined by staining with filipin (result not shown), inhibition of HS degradation required treatment with 10 µg/mL of U18666A for 16 h (Figure 3H), presumably because growing fibroblasts have an exceptionally active NO-dependent HS degradation (Figure 3B; Cappai et al., 2005). When normal fibroblasts were treated with U18666A, the formation of anMan-containing HS oligosaccharides was reduced to 20% of the level in untreated cells (cf. Figure 3B and H, insets). By exposing U18666A-treated normal fibroblasts to ascorbate, the level of anMan-containing oligosaccharides increased to 35% of the level in untreated fibroblasts (cf. Figure 3B and J, insets).
Further experiments showed that the cationic amphiphile imipramine (80 µM for 18 h) did not inhibit formation of anMan-containing degradation products, although it generated accumulation of cholesterol (results not shown). Estrone (3 µg/mL for 16 h), which is not cationic but has a C-17 keto group similar to that of U18666A, caused neither cholesterol accumulation nor inhibition of NO-dependent HS degradation (results not shown). Depletion of total cellular cholesterol by treatment with 10 mM methyl-ß-cyclodextrin for 2 h did not affect the deaminative HS degradation (result not shown).
We then tested whether inhibition of deaminative HS degradation by U18666A in a neuronal cell-line could be overcome by exogenous addition of ascorbate.
Ascorbate restores NO-dependent Gpc-1 autoprocessing in U18666A-treated N2a cells
N2a neuroblastoma cells generated constitutively anMan-containing HS oligosaccharides. The staining appeared as a punctate pattern in the periphery of the cytoplasm (Figure 4A and E). Staining for such oligosaccharides was reduced by 40% after treatment with 3 µg/mL of U18666A (Figure 4B and F). Subsequent treatment with ascorbate restored oligosaccharide formation in some of the cells (Figure 4C and G). If heparanase degradation of HS is inhibited by suramin, more HS should be available for NO-dependent deaminative cleavage (Figure 2). Hence, treatment with both suramin and U18666A, followed by triggering of deaminative cleavage by ascorbate, increased the level of anMan-containing HS oligosaccharides in all of the cells, by 2.5-fold compared with U18666A-treated cells and by 1.5-fold compared with untreated cells (cf. Figure 4H with Figure 4F and Figure 4H with Figure 4E, respectively). These results indicated that heparanase-catalyzed degradation of HS was unaffected by U18666A. The oligosaccharide staining generated by exposure to ascorbate also assumed a punctate pattern but was more widely distributed in the cytoplasm.
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Nitration of tyrosine upon exposure to U18666A and ascorbate
When deaminative cleavage of HS in Gpc-1 is depressed, as in NPC1 fibroblasts and in U18666A-treated cells, Gpc-1 continues to be de-glycanated by heparanase. Hence, an SNO-rich Gpc-1 with truncated HS chains should be obtained (Figure 2, bottom left). When this Gpc-1 is subsequently exposed to a reducing agent, such as ascorbate, the deaminative cleavage process should be initiated. However, the capacity to deaminate may exceed the number of remaining cleavage sites in the HS stubs (Figure 2, bottom right). NO radicals, generated in excess, can then react with molecular oxygen/superoxide anion, forming peroxynitrite (ONOO–) or nitrogen dioxide radical (·NO2), both of which can lead to nitration of tyrosine residues in proteins (Ischiropoulos, 2003). As this is believed to contribute to neurodegeneration, we tested whether nitrotyrosine could be detected in N2a cells after the arrest of NO-dependent HS degradation followed by restoration using ascorbate.
N2a cells that had been treated with 1 mM ascorbate for 1 h displayed no significant increase in nitrotyrosine formation over the low level detected in untreated cells (Figure 5A). Treatment with U18666A to inhibit NO-dependent Gpc-1 processing followed by treatment with ascorbate for 10 min did not significantly affect the nitrotyrosine content that remained undetectable by confocal microscopy (Figure 5A and B). After treatment of U18666A pre-exposed cells with ascorbate for 1 h, there was a 4- to 5-fold increase in nitrotyrosine formation, which was also easily detectable by confocal microscopy (Figure 5C). The nitrotyrosine staining was diffuse but mostly colocalizing with Gpc-1 (Figure 5C, yellow in inset). Pretreatment with suramin to inhibit heparanase reduced nitrotyrosine formation by almost 50% (Figure 5D).
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As NO-dependent HS degradation was defective in NPC1 fibroblasts, we also examined nitrotyrosine formation in these cells and in normal fibroblasts after exposure to ascorbate. In normal fibroblasts (HFL-1), a 1-h ascorbate treatment generated insignificant amounts of nitrotyrosine staining (Figure 5E). However, in untreated NPC1 fibroblasts, there was a small but significantly higher level of nitrotyrosine formation generating a weak punctate staining around the cell surface and in the cytoplasm (Figure 5F). When NPC1 fibroblasts were exposed to ascorbate for 1 h, there was an almost 3-fold increase in nitrotyrosine content and an increased punctate staining colocalizing with Gpc-1 (Figure 5G). Pretreatment with suramin to inhibit heparanase reduced the ascorbate-generated nitrotyrosine formation by almost 50% (Figure 5H).
To show that Gpc-1 was involved in the generation of nitrotyrosine, Gpc-1 expression was silenced using RNAi. The expression level was reduced to 30% of normal, as estimated by SDS–PAGE and western blotting. When NPC1 fibroblasts were mock-transfected with a vector encoding a scrambled sequence, both Gpc-1 and nitrotyrosine could be detected after treatment with ascorbate (Figure 6A and B), but when the same cells were transfected with a vector expressing human Gpc-1-specific short-interfering RNA (siRNA), expression of Gpc-1 and formation of ascorbate-induced nitrotyrosine were both reduced (Figure 6C and D).
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Discussion |
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TopAbstractIntroductionResultsDiscussionMaterials and MethodsConflict of interest statementAcknowledgmentsReferences |
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During intracellular recycling, Gpc-1 is processed via two independent de-glycanation steps, heparanase-catalyzed endoglycosidic and NO-dependent, deaminative HS degradation, respectively (Figure 2). The deaminative degradation requires prior S-nitrosylation of the Gpc-1 core protein and generation of GlcNH3+ residues in the HS chains. To initiate deaminative cleavage of HS, the participation of a reducing agent, probably located to late endosomes, is required. Exogenously supplied ascorbate can be taken up by fibroblasts via the sodium ascorbate symport (Corpe et al., 2005
) and trigger deaminative cleavage when the endogenous mechanism fails (Fransson et al., 2004). The present results show that constitutive, NO-dependent, deaminative HS degradation is defective in cells with arrested cholesterol recycling, that is, NPC1 fibroblasts and non-NPC cell lines treated with the cationic amphiphile U18666A. However, the heparanase-catalyzed HS degradation is not affected. Deaminative HS degradation is partially restored by ascorbate in both NPC1 fibroblasts and U18666A-treated non-NPC cells and further augmented when heparanase is simultaneously inhibited by suramin. These findings suggest that Gpc-1 and cholesterol have a common traffic route.
Cholesterol trafficking via multivesicular endosomes is regulated by the NPC1 and NPC2 proteins. It is not clear how NPC1 and NPC2 work in concert to transport cholesterol (Ioannou, 2005). Therefore, the disease pathogenesis remains obscure. One possibility is that a functional NPC1 protein is required for Rab9-mediated vesicular transport from late endosomes to the trans-Golgi network (Seachrist and Ferguson, 2003). Another possibility is that NPC1 is a lipid flippase that collaborates with the obligatory cholesterol transporter NPC2 (Ioannou, 2005). This collaboration could generate the lipid asymmetry required to induce proper membrane curvature. Thereby, fusion of cholesterol- and GPI-anchored protein-containing internal vesicles with the delimiting membrane of late endosomes and the subsequent generation of transport vesicles may be facilitated (Figure 1D). In addition, when NPC1 or NPC2 is defective, cholesterol is not oxidized to 25-hydroxycholesterol (Frolov et al., 2003; Zhang et al., 2004). If GPI-anchored Gpc-1 is transported by the same mechanism, this may explain why the redox-dependent deaminative processing of Gpc-1-HS is also affected in NPC1 fibroblasts.
U18666A as well as several other cationic amphiphiles is known to interfere with vesicular cholesterol traffic between late endosomes and the Golgi and thereby mimic the cholesterol traffic block and the lack of oxysterol formation characteristic of the NPC diseases. However, inhibition of NO-dependent deaminative processing of Gpc-1-HS in normal fibroblasts required higher concentrations of U18666A than was required to induce cholesterol accumulation. U18666A has a reducible keto group at C-17 that could participate in redox reactions and thereby consume the endogenous reducing power needed to initiate deaminative processing of Gpc-1. Estrone, which also has a C-17 keto group but no cationic group, interferes neither with deaminative HS degradation nor with cholesterol trafficking. Therefore, the cationic head group of U18666A should be important for its localization to late endosomes. Imipramine, which also has a cationic head group and causes cholesterol accumulation, has no reducible group and does not affect deaminative HS degradation.
When NO-dependent, deaminative autoprocessing of Gpc-1 is depressed, as in NPC1 fibroblasts and U18666A-treated normal cells, exogenously supplied ascorbate can partially restore NO-dependent Gpc-1 autoprocessing. The anMan-containing HS oligosaccharides, generated both constitutively and after exposure to ascorbate, appeared to be present largely in vesicular structures, possibly endosomes, of N2a cells. Constitutive formation of oligosaccharides may be restricted to a subset of endosomes. Studies are in progress to determine the precise localization of the HS oligosaccharides.
Unfortunately, restoration of deaminative HS degradation by ascorbate leads to tyrosine nitration. The mechanism appears to be as follows. When deaminative cleavage of HS is temporarily inhibited or defective, while the heparanase-catalyzed de-glycanation of Gpc-1 proceeds undisturbed, the HS chains on Gpc-1 should become truncated while the core protein should remain fully S-nitrosylated (Figure 2). Moreover, many of the NO-sensitive GlcNH3+ units in HS are situated in the HS oligosaccharides that are liberated by heparanase. These segregate from the SNO-containing and HS-truncated Gpc-1 during recycling and will not be cleaved by NO released from Gpc-1-SNO (Fransson et al., 2004). When an excess of NO is released from HS-truncated Gpc-1-SNO, tyrosines in the Gpc-1 core protein as well as in other adjacent proteins can become nitrated, probably by ONOO– or ·NO2 generated from ·NO or HNO. This could be destructive and contribute to neurodegeneration. Although tyrosine nitration was profoundly inhibited by silencing of Gpc-1 expression, it cannot be excluded that other glypicans contribute to the observed effects.
In summary, NO-dependent, deaminative degradation of HS is severely depressed in NPC1 fibroblasts and in U18666A-treated normal fibroblasts and N2a neuroblastoma cells, while heparanase-catalyzed HS degradation is unaffected. Upon exposure to ascorbate, deaminative HS degradation is partially restored, but an excess of reactive nitrogen species generates nitrotyrosines, a potentially destructive process. The present results thus suggest that misprocessing of Gpc-1 in NPC1 cells leads to nitrosative stress and cell damage. As nitrosation is reduced by suramin inhibition of heparanase, this could be a useful treatment in NPC diseases.
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Materials and Methods |
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Materials
HFL-1 and mouse N2a neuroblastoma cells were obtained from ATCC and NPC1 fibroblasts (GM03123) from the Corielle Institute and maintained in minimal essential medium (MEM) supplemented with 10% fetal calf serum. Polyclonal antisera against human Gpc-1, an mAb-recognizing anMan-terminating HS oligosaccharides (Pejler et al., 1988
), suitably tagged secondary antibodies, as well as suramin, 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, 2004). U18666A was obtained from Biomol (Plymouth Meeting, PA) estrone, filipin, imipramine, and methyl-ß-cyclodextrin from Sigma (Stockholm, Sweden), and an mAb to nitrotyrosine from Abcam (Cambridge, UK).
siRNA preparation and transfection
The vector pSilencer 2.0-U6 (Ambion, Austin, TX) containing the sequence GCTGGTCTACTGTGCTCAC (corresponding to nucleotides 977–995 in human Gpc-1) followed by a hairpin sequence TTCAAGAGA, then the reversed complementary Gpc-1 sequence with an additional C in the 5' end and a stretch of six T for RNA polymerase III termination followed by GGAA in the 3' end was synthesized by Genscript Corporation (Edison, NJ). Negative control vectors comprising scrambled sequences were also prepared. Transfection was accomplished by using Lipofectamine (Life Technologies, Stockholm, Sweden) according to the description of the manufacturer.
Confocal laser scanning immunofluorescence 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 either with acetone/H2O2/methanol or with 2% (w/v) paraformaldehyde, 0.1% (v/v) Triton X-100. Mouse N2a cells were first precoated with 10% anti-mouse immunoglobulin G (IgG) and then exposed to primary antibodies. The second antibody used was either goat anti-mouse total Ig when the primary antibody was monoclonal or goat anti-rabbit IgG when the primary antibody was polyclonal. The second antibodies were tagged with either fluorescein isothiocyanate or Texas Red and appropriately combined for colocalization studies. In the controls, the primary antibody was omitted. Before fixation and confocal microscopy, cells were inspected by light microscopy. Images shown were obtained at a focal plane that was at the center of the cell and of 0.3–0.5 µm thickness. Identical exposure settings were used for image capture. Images were digitized and transferred to Adobe PhotoShop for merging, annotation, and printing. Filipin staining for cholesterol was performed with 50 µg/mL for 2 h. Image capturing of the blue fluorescence was obtained by using a regular fluorescence microscope.
Flow cytometry
Cells were seeded in 24-well plates and grown to near confluence, rinsed with medium, and detached using trypsin [0.5 mL of 0.05% w/v 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 of medium supplemented with 10% fetal bovine serum (FBS). Cells were recovered by gentle suspension and transferred to tubes, adding 1 volume 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 of PBS after removal of the supernatant. Cells were fixed for 30 min in 1 mL of 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.
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Conflict of interest statement |
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TopAbstractIntroductionResultsDiscussionMaterials and MethodsConflict of interest statementAcknowledgmentsReferences |
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None declared.
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Acknowledgments |
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TopAbstractIntroductionResultsDiscussionMaterials and MethodsConflict of interest statementAcknowledgmentsReferences |
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We thank Prof. Catharina Svanborg and Prof. Hakon Leffler at the Department of Microbiology, Immunology, and Glycobiology, Lund University, for use of microscope facilities and Dr Gunnar Peijler, Uppsala University, Sweden, for a generous gift of monoclonal antibodies, and Dr Mattias Belting for advice. The work was supported by grants from the Swedish Science Council (VR-M), the Bergvall, Crafoord, Hedborg, Kock, Zoega and Österlund Foundations, and the Medical Faculty of Lund University.
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Abbreviations |
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anMan, anhydromannose; Gpc-1, glypican-1; GPI, glycosylphosphatidylinositol; HFL-1, human fetal lung fibroblasts; HNO, nitroxyl; HS, heparan sulfate; Ig, immunoglobulin; N2a, mouse neuroblastoma cell line; NO, nitric oxide; NPC1, Niemann–Pick type C1; NPC2, Niemann–Pick type C2; ONOO–, peroxynitrite; PBS, phosphate-buffered saline; siRNA, short-interfering RNA; SNO, S-nitrosylation; U18666A, 3-ß-[2-(diethylamino) ethoxy]androst-5-en-17-one
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References |
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