| 1. |
- Belting, Mattias, et al.
(författare)
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Glypican-1 is a vehicle for polyamine uptake in mammalian cells. A pivotal role for nitrosothiol-derived nitric oxide.
- 2003
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Ingår i: Journal of Biological Chemistry. - 1083-351X. ; 278:47, s. 47181-47189
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Tidskriftsartikel (refereegranskat)abstract
- Polyamines (putrescine, spermidine, and spermine) are essential for growth and survival of all cells. When polyamine biosynthesis is inhibited, there is up-regulation of import. The mammalian polyamine transport system is unknown. We have previously shown that the heparan sulfate (HS) side chains of recycling glypican-1 (Gpc-1) can sequester spermine, that intracellular polyamine depletion increases the number of NO-sensitive N-unsubstituted glucosamines in HS, and that NO-dependent cleavage of HS at these sites is required for spermine uptake. The NO is derived from S-nitroso groups in the Gpc-1 protein. Using RNA interference technology as well as biochemical and microscopic techniques applied to both normal and uptake-deficient cells, we demonstrate that inhibition of Gpc-1 expression abrogates spermine uptake and intracellular delivery. In unperturbed cells, spermine and recycling Gpc-1 carrying HS chains rich in N-unsubstituted glucosamines were co-localized. By exposing cells to ascorbate, we induced release of NO from the S-nitroso groups, resulting in HS degradation and unloading of the sequestered polyamines as well as nuclear targeting of the deglycanated Gpc-1 protein. Polyamine uptake-deficient cells appear to have a defect in the NO release mechanism. We have managed to restore spermine uptake partially in these cells by providing spermine NONOate and ascorbate. The former bound to the HS chains of recycling Gpc-1 and S-nitrosylated the core protein. Ascorbate released NO, which degraded HS and liberated the bound spermine. Recycling HS proteoglycans of the glypican-type may be plasma membrane carriers for cargo taken up by caveolar endocytosis.
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| 5. |
- Belting, Mattias, et al.
(författare)
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Tumor attenuation by combined heparan sulfate and polyamine depletion.
- 2002
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Ingår i: Proceedings of the National Academy of Sciences. - 1091-6490. ; 99:1, s. 371-376
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Tidskriftsartikel (refereegranskat)abstract
- Cells depend on polyamines for growth and their depletion represents a strategy for the treatment of cancer. Polyamines assemble de novo through a pathway sensitive to the inhibitor, alpha-difluoromethylornithine (DFMO). However, the presence of cell-surface heparan sulfate proteoglycans may provide a salvage pathway for uptake of circulating polyamines, thereby sparing cells from the cytostatic effect of DFMO. Here we show that genetic or pharmacologic manipulation of proteoglycan synthesis in the presence of DFMO inhibits cell proliferation in vitro and in vivo. In cell culture, mutant cells lacking heparan sulfate were more sensitive to the growth inhibitory effects of DFMO than wild-type cells or mutant cells transfected with the cDNA for the missing biosynthetic enzyme. Moreover, extracellular polyamines did not restore growth of mutant cells, but completely reversed the inhibitory effect of DFMO in wild-type cells. In a mouse model of experimental metastasis, DFMO provided in the water supply also dramatically diminished seeding and growth of tumor foci in the lungs by heparan sulfate-deficient mutant cells compared with the controls. Wild-type cells also formed tumors less efficiently in mice fed both DFMO and a xylose-based inhibitor of heparan sulfate proteoglycan assembly. The effect seemed to be specific for heparan sulfate, because a different xyloside known to affect only chondroitin sulfate did not inhibit tumor growth. Hence, combined inhibition of heparan sulfate assembly and polyamine synthesis may represent an additional strategy for cancer therapy.
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| 6. |
- Cheng, Fang, et al.
(författare)
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APP/APLP2 expression is required to initiate endosome-nucleus-autophagosome trafficking of glypican-1-derived heparan sulfate.
- 2014
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Ingår i: Journal of Biological Chemistry. - 1083-351X. ; 289:30, s. 20871-20878
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Tidskriftsartikel (refereegranskat)abstract
- Anhydromannose (anMan)-containing heparan sulfate (HS) derived from the proteoglycan glypican-1 (Gpc-1) is generated in endosomes by an endogenously or ascorbate induced SNO-catalyzed reaction. Processing of the amyloid precursor protein (APP) and APP-like protein 2 (APLP2) by β- and γ-secretases into amyloid beta (Aβ) and Aβ-like peptides also takes place in these compartments. Moreover, anMan-containing HS suppresses the formation of toxic Aβ assemblies in vitro. We show by using deconvolution immunofluorescence microscopy with an anMan-specific monoclonal antibody as well as 35S-labeling experiment that expression of APP/APLP2 is required for ascorbate-induced transport of HS from endosomes to the nucleus. Nuclear translocation was observed in wild-type mouse embryonic fibroblasts (Wt-MEF), Tg2576 MEF and N2a neuroblastoma cells but not in APP-/- and APLP2-/- MEF. Transfection of APP-/- cells with a vector encoding APP restored nuclear import of anMan-containing HS. In Wt-MEF and N2a neuroblastoma cells exposed to β- or γ-secretase inhibitors, nuclear translocation was greatly impeded, suggesting involvement of APP/APLP2 degradation products. In Tg2576 MEF, the β-inhibitor blocked transport but the γ- inhibitor did not. During chase in ascorbate-free medium, anMan-containing HS disappeared from the nuclei of Wt-MEF. Confocal immunofluorescence microscopy showed that they appeared in acidic, LC3-positive vesicles in keeping with an autophagosomal location. There was increased accumulation of anMan-containing HS in nuclei and cytosolic vesicles upon treatment with chloroquine indicating that HS was degraded in lysosomes. Manipulations of APP expression and processing may have deleterious effects upon HS function in the nucleus.
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| 7. |
- Cheng, Fang, et al.
(författare)
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Hypoxia induces NO-dependent release of heparan sulfate in fibroblasts from the Alzheimer mouse Tg2576 by activation of nitrite reduction.
- 2016
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Ingår i: Glycobiology. - : Oxford University Press (OUP). - 1460-2423 .- 0959-6658. ; 26:6, s. 623-634
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Tidskriftsartikel (refereegranskat)abstract
- There is a functional relationship between the heparan sulfate proteoglycan glypican-1 and the amyloid precursor protein of Alzheimer disease. In wild-type mouse embryonic fibroblasts, expression and processing of the amyloid precursor protein is required for endosome-to-nucleus translocation of anhydromannose-containing heparan sulfate released from S-nitrosylated glypican-1 by ascorbate-induced, nitrosothiol-catalyzed deaminative cleavage. In fibroblasts from the transgenic Alzheimer mouse Tg2576 there is increased processing of the amyloid precursor protein to amyloid-β peptides. Simultaneously, there is spontaneous formation of anhydromannose-containing heparan sulfate by an unknown mechanism. We have explored the effect of hypoxia on anhydromannose-containing heparan sulfate formation in wild-type and Tg2576 fibroblasts by deconvolution immunofluorescence microscopy and flow cytometry using an anhydromannose-specific monoclonal antibody and by (35)SO4-labeling experiments. Hypoxia prevented ascorbate-induced heparan sulfate release in wild-type fibroblasts, but induced an increased formation of anhydromannose-positive and (35)S-labeled heparan sulfate in Tg2576 fibroblasts. This appeared to be independent of glypican-1 S-nitrosylation as demonstrated by using a monoclonal antibody specific for S-nitrosylated glypican-1. In hypoxic wild-type fibroblasts, addition of nitrite to the medium restored anhydromannose-containing heparan sulfate formation. The increased release of anhydromannose-containing heparan sulfate in hypoxic Tg2576 fibroblasts did not require addition of nitrite. However, it was suppressed by inhibition of the nitrite reductase activity of xanthine oxidoreductase/aldehyde oxidase or by inhibition of p38 mitogen-activated protein kinase or by chelation of iron. We propose that normoxic Tg2576 fibroblasts maintain a high level of anhydromannose-containing heparan sulfate production by a stress-activated generation of nitric oxide from endogenous nitrite. This activation is enhanced by hypoxia.
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| 8. |
- Cheng, Fang, et al.
(författare)
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Nitric oxide-dependent processing of heparan sulfate in recycling S-nitrosylated glypican-1 takes place in caveolin-1 containing endosomes.
- 2002
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Ingår i: Journal of Biological Chemistry. - 1083-351X. ; 277:46, s. 44431-44439
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Tidskriftsartikel (refereegranskat)abstract
- We have previously demonstrated intracellular degradation of the heparan sulfate side-chains in recycling glypican-1 by heparanase and by deaminative cleavage at N-unsubstituted glucosamine with nitric oxide derived from intrinsic nitrosothiols [see Ding, K., Mani, K., Cheng, F., Belting, M. and Fransson, L.-. (2002) J. Biol. Chem., 277, xxx-xxx; prepub M203383200]. To determine where and in what order events take place, we have visualized, by using confocal laser-scanning immunofluorescence microscopy, glypican-1 variants in unperturbed cells or arrested at various stages of processing. In unperturbed proliferating cells, glypican-1 was partly S-nitrosylated. Intracellular glypican-1 was enriched in endosomes, colocalized significantly with GM-1 ganglioside, caveolin-1 and Rab9-positive endosomes, and carried side-chains rich in N-unsubstituted glucosamine residues. However, such residues were scarce in cell-surface glypican-1. Brefeldin A-arrested glypican-1, which was non-S-nitrosylated and carried side-chains rich in N-unsubstituted glucosamines, colocalized extensively with caveolin-1 but not with Rab9. Suramin, which inhibits heparanase, induced the appearance of S-nitrosylated glypican-1 in caveolin-1-rich compartments. Inhibition of deaminative cleavage did not prevent heparanase from generating heparan sulfate oligosaccharides that colocalized strongly with caveolin-1. Growth-quiescent cells displayed extensive NO-dependent deaminative cleavage of heparan sulfate generating anhydromannose-terminating fragments which were partly associated with acidic vesicles. Proliferating cells generated such fragments during polyamine uptake. We conclude that recycling glypican-1 that is associated with caveolin-1-containing endosomes undergoes sequential N-desulfation/N-deacetylation, heparanase cleavage, S-nitrosylation, NO-release and deaminative cleavage of its side-chains in conjunction with polyamine uptake.
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| 9. |
- Cheng, Fang, et al.
(författare)
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Nucleolin is a nuclear target of heparan sulfate derived from glypican-1
- 2017
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Ingår i: Experimental Cell Research. - : Elsevier BV. - 0014-4827. ; 354:1, s. 31-39
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Tidskriftsartikel (refereegranskat)abstract
- The recycling, S-nitrosylated heparan sulfate (HS) proteoglycan glypican-1 releases anhydromannose (anMan)-containing HS chains by a nitrosothiol-catalyzed cleavage in endosomes that can be constitutive or induced by ascorbate. The HS-anMan chains are then transported to the nucleus. A specific nuclear target for HS-anMan has not been identified. We have monitored endosome-to-nucleus trafficking of HS-anMan by deconvolution and confocal immunofluorescence microscopy using an anMan-specific monoclonal antibody in non-growing, ascorbate-treated, and growing, untreated, wild-type mouse embryonic fibroblasts and hypoxia-exposed Alzheimer mouse Tg2576 fibroblasts and human U87 glioblastoma cells. In all cells, nuclear HS-anMan targeted a limited number of sites of variable size where it colocalized with DNA and nucleolin, an established marker for nucleoli. HS-anMan also colocalized with ethynyl uridine-tagged nascent RNA and two acetylated forms of histone H3. Acute hypoxia increased the formation of HS-anMan in both Tg2576 and U87 cells. A portion of HS-anMan colocalized with nucleolin at small discrete sites, while most of the nucleolin and nascent RNA was dispersed. In U87 cells, HS-anMan, nucleolin and nascent RNA reassembled after prolonged hypoxia. Nucleolar HS may modulate synthesis and/or release of rRNA.
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| 10. |
- Ding, Kan, et al.
(författare)
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Copper-dependent autocleavage of glypican-1heparan sulfate by nitric oxide derived fromintrinsic nitrosothiols.
- 2002
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Ingår i: Journal of Biological Chemistry. - 1083-351X. ; 277:36, s. 33353-33360
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Tidskriftsartikel (refereegranskat)abstract
- Cell-surface heparan sulfate proteoglycans facilitate uptake of growth-promoting polyamines [ [Belting, M., Borsig, L., Fuster, M.M., Brown, J.R., Persson, L., Fransson,L.-. and Esko, J.D. (2002) Proc. Natl. Acad. Sci. U.S.A., 99, 371-376] ]. Increased polyamine uptake correlates with an increased number of positively charged N-unsubstituted glucosamine units in the otherwise polyanionic heparan sulfate chains of glypican-1. During intracellular recycling of glypican-1 there is an NO-dependent deaminative cleavage of heparan sulfate at these glucosamine units, which would eliminate the positive charges [ [Ding, K., Sandgren, S., Mani, K., Belting, M. and Fransson, L.-. (2001) J. Biol. Chem., 276, 46779-46791] ]. Here, using both biochemical and microscopic techniques, we have identified and isolated S-nitrosylated forms of glypican-1 as well as low-charged glypican-1 glycoforms containing heparan sulfate chains rich in N-unsubstituted glucosamines. The latter were converted to high-charged species upon treatment of cells with 1 mM L-ascorbate, which releases NO from nitrosothiols, resulting in deaminative cleavage of heparan sulfate at the N-unsubstituted glucosamines. S-nitrosylation and subsequent deaminative cleavage were abrogated by inhibition of a Cu 2+ /Cu + -redox cycle. Under cell-free conditions, purified, S-nitrosylated glypican-1 was able to autocleave its heparan sulfate chains when NO-release was triggered by L-ascorbate. The heparan sulfate fragments generated in cells during this auto-catalytic process contained terminal anhydromannose residues. We conclude that the core protein of glypican-1 can slowly accumulate NO as nitrosothiols while Cu 2+ is reduced to Cu +. Subsequent release of NO results in efficient deaminative cleavage of the heparan sulfate chains attached to the same core protein while Cu + is oxidized to Cu 2+.
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