| 1. |
- Jönsson, Mats, et al.
(författare)
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Initiation of the decorin glycosaminoglycan chain in the endoplasmic reticulum-Golgi intermediate compartment
- 2003
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Ingår i: Journal of Biological Chemistry. - 1083-351X. ; 278:24, s. 21415-21420
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Tidskriftsartikel (refereegranskat)abstract
- We have transiently expressed decorin with a C- terminal KDEL endoplasmic reticulum retention signal peptide in COS- 7 cells to study initiation of galactosaminoglycan synthesis in the endoplasmic reticulum- Golgi intermediate compartment. All decorin- KDEL molecules were substituted with N- linked oligosaccharides sensitive to endoglycosidase H, indicating that the core protein was located proximal to the medial- Golgi. O-Linked glycosylation was only initiated in a minor fraction of the molecules. The O- linked saccharides were characterized by gel filtration after stepwise degradations using chondroitin ABC/ AC-I lyases, beta1 - 3- glycuronidase, beta-galactosidase, and alkaline phosphatase. The major O- linked saccharide was the linkage region pentasaccharide GalNAcbeta1-4GlcUAbeta1-3Galbeta1-3Galbeta1-4-Xyl- 2- phosphate, demonstrating initiation of chondroitin synthesis in the endoplasmic reticulum- Golgi intermediate compartment. In the presence of brefeldin A, partial elongation of a chondroitin chain took place, indicating retrieval of polymerases but not of sulfotransferases.
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| 2. |
- 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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| 3. |
- 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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| 4. |
- 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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| 5. |
- 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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| 6. |
- Ding, Kan, et al.
(författare)
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Modulations of glypican-1 heparan sulfate structure by inhibition of endogenous polyamine synthesis. Mapping of spermine-binding sites and heparanase, heparin lyase, and nitric oxide/nitrite cleavage sites
- 2001
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Ingår i: Journal of Biological Chemistry. - 1083-351X. ; 276:50, s. 46779-46791
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Tidskriftsartikel (refereegranskat)abstract
- Cell surface heparan sulfate proteoglycans facilitate uptake of growth-promoting polyamines (Belting, M., Persson, S., and Fransson, L.-A. (1999) Biochem. J. 338, 317-323; Belting, M., Borsig, L., Fuster, M. M., Brown, J. R., Persson, L., Fransson, L.-A., and Esko, J. D. (2001) Proc. Natl. Acad. Sci. U. S. A., in press). Here, we have analyzed the effect of polyamine deprivation on the structure and polyamine affinity of the heparan sulfate chains in various glypican-1 glycoforms synthesized by a transformed cell line (ECV 304). Heparan sulfate chains of glypican-1 were either cleaved with heparanase at sites embracing the highly modified regions or with nitrite at N-unsubstituted glucosamine residues. The products were separated and further degraded by heparin lyase to identify sulfated iduronic acid. Polyamine affinity was assessed by chromatography on agarose substituted with the polyamine spermine. In heparan sulfate made by cells with undisturbed endogenous polyamine synthesis, free amino groups were restricted to the unmodified, unsulfated segments, especially near the core protein. Spermine high affinity binding sites were located to the modified and highly sulfated segments that were released by heparanase. In cells with up-regulated polyamine uptake, heparan sulfate contained an increased number of clustered N-unsubstituted glucosamines and sulfated iduronic acid residues. This resulted in a greater number of NO/nitrite-sensitive cleavage sites near the potential spermine-binding sites. Endogenous degradation by heparanase and NO-derived nitrite in polyamine-deprived cells generated a separate pool of heparan sulfate oligosaccharides with an exceptionally high affinity for spermine. Spermine uptake in polyamine-deprived cells was reduced when NO/nitrite-generated degradation of heparan sulfate was inhibited. The results suggest a functional interplay between glypican recycling, NO/nitrite-generated heparan sulfate degradation, and polyamine uptake.
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| 7. |
- Ding, Kan, et al.
(författare)
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N-unsubstituted glucosamine in heparan sulfate of recycling glypican-1 from suramin-treated and nitrite-deprived endothelial cells. mapping of nitric oxide/nitrite-susceptible glucosamine residues to clustered sites near the core protein
- 2001
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Ingår i: Journal of Biological Chemistry. - 1083-351X. ; 276:6, s. 3885-3894
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Tidskriftsartikel (refereegranskat)abstract
- We have analyzed the content of N-unsubstituted glucosamine in heparan sulfate from glypican-1 synthesized by endothelial cells during inhibition of (a) intracellular progression by brefeldin A, (b) heparan sulfate degradation by suramin, and/or (c) endogenous nitrite formation. Glypican-1 from brefeldin A-treated cells carried heparan sulfate chains that were extensively degraded by nitrous acid at pH 3.9, indicating the presence of glucosamines with free amino groups. Chains with such residues were rare in glypican-1 isolated from unperturbed cells and from cells treated with suramin and, surprisingly, when nitrite-deprived. However, when nitrite-deprived cells were simultaneously treated with suramin, such glucosamine residues were more prevalent. To locate these residues, chains were first cleaved at linkages to sulfated l-iduronic acid by heparin lyase and released fragments were separated from core protein carrying heparan sulfate stubs. These stubs were then cleaved off at sites linking N-substituted glucosamines to d-glucuronic acid. These fragments were extensively degraded by nitrous acid at pH 3.9. When purified proteoglycan isolated from brefeldin A-treated cells was incubated with intact cells, endoheparanase-catalyzed degradation generated a core protein with heparan sulfate stubs that were similarly sensitive to nitrous acid. We conclude that there is a concentration of N-unsubstituted glucosamines to the reducing side of the endoheparanase cleavage site in the transition region between unmodified and modified chain segments near the linkage region to the protein. Both sites as well as the heparin lyase-sensitive sites seem to be in close proximity to one another.
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| 8. |
- Fransson, Lars-Åke
(författare)
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Glypicans.
- 2003
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Ingår i: International Journal of Biochemistry & Cell Biology. - 1878-5875. ; 35:2, s. 125-129
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Tidskriftsartikel (refereegranskat)abstract
- A family of lipid-linked heparan sulfate (HS) proteoglycans, later named glypicans, were identified some 15 years ago. The discoveries that mutations in genes involved in glypican assembly cause developmental defects have brought them into focus. Glypicans have a characteristic pattern of 14 conserved cysteine residues. There are also two–three attachment sites for HS side-chains near the membrane anchor. The HS side-chains consist of a repeating disaccharide back-bone that is regionally and variably modified by epimerization and different types of sulfations, creating a variety of binding sites for polycationic molecules, especially growth factors. Recycling forms of glypican-1 are potential vehicles for transport of cargo into and through cells. The glypican-1 core protein is S-nitrosylated and nitric oxide released from these sites cleave the HS chains at glucosamine units lacking N-substitution. This processing is necessary for polyamine uptake.
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| 9. |
- Fransson, Lars-Åke, et al.
(författare)
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Novel aspects of glypican glycobiology.
- 2004
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Ingår i: Cellular and Molecular Life Sciences. - : Springer Science and Business Media LLC. - 1420-9071 .- 1420-682X. ; 61:9, s. 1016-1024
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Forskningsöversikt (refereegranskat)abstract
- Mutations in glypican genes cause dysmorphic and overgrowth syndromes in men and mice, abnormal development in flies and worms, and defective gastrulation in zebrafish and ascidians. All glypican core proteins share a characteristic pattern of 14 conserved cysteine residues. Upstream from the C-terminal membrane anchorage are 3–4 heparan sulfate attachment sites. Cysteines in glypican-1 can become nitrosylated by nitric oxide in a copper-dependent reaction. When glypican-1 is exposed to ascorbate, nitric oxide is released and participates in deaminative cleavage of heparan sulfate at sites where the glucosamines have a free amino group. This process takes place while glypican-1 recycles via a nonclassical, caveolin-1-associated route. Glypicans are involved in growth factor signalling and transport, e.g. of polyamines. Cargo can be unloaded from heparan sulfate by nitric oxide-dependent degradation. How glypican and its degradation products and the cargo exit from the recycling route is an enigma.
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| 10. |
- Mani, Katrin, et al.
(författare)
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Involvement of GPI-linked ceruloplasmin in the Cu/Zn-NO-dependent degradation of glypican-1 heparan sulfate in Rat C6 glioma cells.
- 2004
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Ingår i: Journal of Biological Chemistry. - 1083-351X. ; 279:13, s. 12918-12923
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Tidskriftsartikel (refereegranskat)abstract
- The core protein of glypican-1, a glycosylphosphatidylinositol-linked heparan sulfate proteoglycan, can bind Cu(II) or Zn(II) ions and undergo S-nitrosylation in the presence of nitric oxide. Cu(II)-to-Cu(I)-reduction supports extensive and permanent nitrosothiol formation, whereas Zn(II) ions appear to support a more limited, possibly transient one. Ascorbate induces release of nitric oxide, which catalyzes deaminative degradation of the heparan sulfate chains on the same core protein. Although free Zn(II) ions support a more limited degradation, Cu(II) ions support a more extensive self-pruning process. Here, we have investigated processing of glypican-1 in rat C6 glioma cells and the possible participation of the copper-containing glycosylphosphatidylinositol-linked splice variant of ceruloplasmin in nitrosothiol formation. Confocal microscopy demonstrated colocalization of glypican-1 and ceruloplasmin in endosomal compartments. Ascorbate induced extensive, Zn(II)-supported heparan sulfate degradation, which could be demonstrated using a specific zinc probe. RNA interference silencing of ceruloplasmin expression reduced the extent of Zn(II)-supported degradation. In cell-free experiments, the presence of free Zn(II) ions prevented free Cu(II) ion from binding to glypican-1 and precluded extensive heparan sulfate autodegradation. However, in the presence of Cu(II)-loaded ceruloplasmin, heparan sulfate in Zn(II)-loaded glypican-1 underwent extensive, ascorbate-induced degradation. We propose that the Cu(II)-to-Cu(I)-reduction that is required for S-nitrosylation of glypican-1 can take place on ceruloplasmin and thereby ensure extensive glypican-1 processing in the presence of free Zn(II) ions.
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