| 1. |
- Belting, Mattias, et al.
(författare)
-
Glypican-1 is a vehicle for polyamine uptake in mammalian cells. A pivotal role for nitrosothiol-derived nitric oxide.
- 2003
-
Ingår i: Journal of Biological Chemistry. - 1083-351X. ; 278:47, s. 47181-47189
-
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.
|
|
| 2. |
- Belting, Mattias, et al.
(författare)
-
Tumor attenuation by combined heparan sulfate and polyamine depletion.
- 2002
-
Ingår i: Proceedings of the National Academy of Sciences. - 1091-6490. ; 99:1, s. 371-376
-
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.
|
|
| 3. |
- Cheng, Fang, et al.
(författare)
-
Nitric oxide-dependent processing of heparan sulfate in recycling S-nitrosylated glypican-1 takes place in caveolin-1 containing endosomes.
- 2002
-
Ingår i: Journal of Biological Chemistry. - 1083-351X. ; 277:46, s. 44431-44439
-
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.
|
|
| 4. |
- Ding, Kan, et al.
(författare)
-
Copper-dependent autocleavage of glypican-1heparan sulfate by nitric oxide derived fromintrinsic nitrosothiols.
- 2002
-
Ingår i: Journal of Biological Chemistry. - 1083-351X. ; 277:36, s. 33353-33360
-
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+.
|
|
| 5. |
- Ding, Kan, et al.
(författare)
-
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
-
Ingår i: Journal of Biological Chemistry. - 1083-351X. ; 276:50, s. 46779-46791
-
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.
|
|
| 6. |
- Ding, Kan, et al.
(författare)
-
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
-
Ingår i: Journal of Biological Chemistry. - 1083-351X. ; 276:6, s. 3885-3894
-
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.
|
|
| 7. |
- Fransson, Liisa, et al.
(författare)
-
Parameterization and evaluation of sulfate adsorption in a dynamic soil chemistry model.
- 2003
-
Ingår i: Environmental Pollution. - 0269-7491. ; 124:1, s. 119-125
-
Tidskriftsartikel (refereegranskat)abstract
- Sulfate adsorption was implemented in the dynamic, multi-layer soil chemistry model SAFE. The process is modeled by an isotherm in which sulfate adsorption is considered to be fully reversible and dependent on sulfate concentration as well as pH in soil solution. The isotherm was parameterized by a site-specific series of simple batch experiments at different pH (3.8–5.0) and sulfate concentration (10–260 mol l−1) levels. Application of the model to the Lake Gårdsjön roof covered site shows that including sulfate adsorption improves the dynamic behavior of the model and sulfate adsorption and desorption delay acidification and recovery of the soil. The modeled adsorbed pool of sulfate at the site reached a maximum level of 700 mmol/m2 in the late 1980s, well in line with experimental data.
|
|
| 8. |
- Fransson, Liisa, et al.
(författare)
-
The effect of changes in natural and anthropogenic deposition on modelling recovery from acidification.
- 2003
-
Ingår i: Hydrology and Earth System Sciences. - 1607-7938. ; 7:5, s. 766-776
-
Tidskriftsartikel (refereegranskat)abstract
- The mufti-layer dynamic soil chemistry SAFE model was used to study the dynamics of recovery in the F1 catchment at Lake Gardsjon, Sweden. The influence of (F) sulphate adsorption, and (2) changes in marine deposition, oil model predictions of recovery was studied. Sulphate adsorption/desorption in SAFE is modeled by all isotherm in which sulphate adsorption is dependent oil both the sulphate concentration and the pH in the soil solution. This isotherm was parameterised for the B-horizon of F1 for the sulphate concentration range 10-260 mumol(-1) and the pH range 3.8-5.0. Sulphate adsorption/desorption as the only soil process involving sulphate is adequate to predict sulphate in run-off at F1. Adding the process caused time-delays in sulphate concentration in run-off of only 1-2 years. which was Much shorter than previously seen in the adjacent G1 catchment. The location of Lake Gardsjon. approximately 15 km inland from the Swedish west coast, ensures that the marine deposition to the area is high. Model Output showed that the temporal variation in marine deposition has a considerable impact oil the run-off-chemistry. Such changes in marine deposition are difficult to foresee and their influence on modelled run-off-chemistry, can be large when soils start to recover as the previously high concentrations of anthropogenic sulphate in the soil solution decrease.
|
|
| 9. |
- Fransson, Liisa, et al.
(författare)
-
The importance of including the pH dependence of sulfate adsorption in a dynamic soil chemistry model
- 2004
-
Ingår i: Water, Air and Soil Pollution. - 1573-2932. ; 154:1-4, s. 349-356
-
Tidskriftsartikel (refereegranskat)abstract
- Sulfate adsorption is an important process when modeling the dynamics of recovery from acidification. In the dynamic soil chemistry model SAFE sulfate adsorption is modeled by a pH and sulfate concentration dependent isotherm. This isotherm has been parameterized by fitting it to data from batch experiments on soil samples using multiple linear regression. The soil samples were from the B-horizon from three sites, two from the Lake Gardsjon area in south-west Sweden and one from southern Poland. The pH dependency of sulfate adsorption is important. The batch experiments show that the adsorbed amount, at a specific sulfate concentration, increases considerably with decreasing pH. This implies that the adsorbed pool of anthropogenic sulfate in soils may remain fairly high during the recovery phase. Although a decrease of sulfate concentration is commonly seen, the modeled pH increase is often slow, and thus delays in the modeled desorption of anthropogenic sulfate can be expected.
|
|
| 10. |
- Fransson, Lars-Åke, et al.
(författare)
-
Novel aspects of glypican glycobiology.
- 2004
-
Ingår i: Cellular and Molecular Life Sciences. - : Springer Science and Business Media LLC. - 1420-9071 .- 1420-682X. ; 61:9, s. 1016-1024
-
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.
|
|
| 11. |
- Fransson, Mattias, et al.
(författare)
-
A role for neutrophils in intermittent allergic rhinitis
- 2004
-
Ingår i: Acta Otolaryngol. - : Informa UK Limited. - 0001-6489 .- 1651-2251. ; 124:5, s. 616-20
-
Tidskriftsartikel (refereegranskat)abstract
- OBJECTIVE: In patients with intermittent allergic rhinitis, allergen challenge may induce both early- and late-phase responses. The aim of this study was to examine the correlation between inflammatory cells in the nasal lavage fluid and clinical parameters following pollen challenge. MATERIAL AND METHODS: Nasal lavage fluids were obtained from 29 patients with intermittent allergic rhinitis before and 1 and 6 h after allergen provocation, representing the control, early and late phases, respectively. Symptom and rhinoscopic scores were registered on the same occasions. Inflammatory cells were determined in the nasal fluid. RESULTS: The early phase was characterized by increased symptom scores, rhinoscopic signs of oedema and secretion and neutrophilia. In the late phase, symptom scores had diminished, but the signs of ongoing secretion remained. Both the total nasal symptom score and the secretion score correlated with the number of neutrophils in lavage fluids at 1 h. The eosinophil count did not increase during the early or late phases. CONCLUSION: A single allergen provocation induces an early-phase response dominated by neutrophils, with secretion being the only clinical sign remaining during the late phase. The increase in neutrophil numbers correlated with the registration of secretory symptoms. The presented data indicate a role for neutrophils in intermittent allergic rhinitis and their relation with secretory parameters makes it intriguing to speculate that neutrophils may function as promoters of nasal secretion.
|
|
| 12. |
- Mani, Katrin, et al.
(författare)
-
Prion or amyloid-b-derived Cu(II)- or free Zn(II)-ions support S-nitroso-dependent autocleavage of glypican-1 heparan sulfate.
- 2003
-
Ingår i: Journal of Biological Chemistry. - 1083-351X. ; 278:40, s. 38956-38965
-
Tidskriftsartikel (refereegranskat)abstract
- Copper are generally bound to proteins, e.g. the prion and the amyloid beta proteins. We have previously shown that copper ions are required to nitrosylate thiol groups in the core protein of glypican-1, a heparan sulfate-substituted proteoglycan. When S-nitrosylated glypican-1 is then exposed to an appropriate reducing agent, such as ascorbate, nitric oxide is released and autocatalyzes deaminative cleavage of the glypican-1 heparan sulfate side chains at sites where the glucosamines are N-unsubstituted. These processes take place in a stepwise manner, whereas glypican-1 recycles via a caveolin-1-associated pathway where copper ions could be provided by the prion protein. Here we show, by using both biochemical and microscopic techniques, that (a) the glypican-1 core protein binds copper(II) ions, reduces them to copper(I) when the thiols are nitrosylated and reoxidizes copper(I) to copper(II) when ascorbate releases nitric oxide; (b) maximally S-nitrosylated glypican-1 can cleave its own heparan sulfate chains at all available sites in a nitroxyl ion-dependent reaction; (c) free zinc(II) ions, which are redox inert, also support autocleavage of glypican-1 heparan sulfate, probably via transnitrosation, whereas they inhibit copper(II)-supported degradation; and (d) copper(II)-loaded but not zinc(II)-loaded prion protein or amyloid beta peptide support heparan sulfate degradation. As glypican-1 in prion null cells is poorly S-nitrosylated and as ectopic expression of cellular prion protein restores S-nitrosylation of glypican-1 in these cells, we propose that one function of the cellular prion protein is to deliver copper(II) for the S-nitrosylation of recycling glypican-1.
|
|
| 13. |
- Mani, Katrin, et al.
(författare)
-
Tumor attenuation by 2(6-hydroxynaphthyl)-{beta}-D-xylopyranoside requires priming of heparan sulfate and nuclear targeting of the products.
- 2004
-
Ingår i: Glycobiology. - : Oxford University Press (OUP). - 1460-2423. ; 14:5, s. 387-397
-
Tidskriftsartikel (refereegranskat)abstract
- We have previously reported that the heparan sulfate-priming glycoside 2-(6-hydroxynaphthyl)-ß-D-xylopyranoside selectively inhibits growth of transformed or tumor-derived cells. To investigate the specificity of this xyloside various analogs were synthesized and tested in vitro. Selective growth inhibition was dependent on the presence of a free 6-hydroxyl in the aglycon. Because cells deficient in heparan sulfate synthesis were insensitive to the xyloside, we conclude that priming of heparan sulfate synthesis was required for growth inhibition. In growth-inhibited cells, heparan sulfate chains primed by the active xyloside were degraded to products that contained anhydromannose and appeared in the nuclei. Hence the degradation products were generated by nitric oxide–dependent cleavage. Accordingly, nitric oxide depletion reduced nuclear localization of the degradation products and counteracted the growth-inhibitory effect of the xyloside. We propose that 2-(6-hydroxynaphthyl)-ß-D-xylopyranoside entered cells and primed synthesis of heparan sulfate chains that were subsequently degraded by nitric oxide into products that accumulated in the nucleus. In vivo experiments demonstrated that the xyloside administered subcutaneously, perorally, or intraperitoneally was adsorbed and made available to tumor cells located subcutaneously. Treatment with the xyloside reduced the average tumor load by 70–97% in SCID mice. The present xyloside may serve as a lead compound for the development of novel antitumor strategies.
|
|
