Biomedical subjects

Lars-Ake Fransson

Publications and source records attributed to Lars-Ake Fransson.

13 recordsLinked to original sources

PubMed2006

Constitutive and vitamin C-induced, NO-catalyzed release of heparan sulfate from recycling glypican-1 in late endosomes.

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-beta[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 (35)S-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.

Androstenes↗

PubMed2006

Defective nitric oxide-dependent, deaminative cleavage of glypican-1 heparan sulfate in Niemann-Pick C1 fibroblasts.

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-beta-[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.

Androstenes↗

PubMed2005

The amyloid precursor protein (APP) of Alzheimer disease and its paralog, APLP2, modulate the Cu/Zn-Nitric Oxide-catalyzed degradation of glypican-1 heparan sulfate in vivo.

Processing of the recycling proteoglycan glypican-1 involves the release of its heparan sulfate chains by copper ion- and nitric oxide-catalyzed ascorbate-triggered autodegradation. The Alzheimer disease amyloid precursor protein (APP) and its paralogue, the amyloid precursor-like protein 2 (APLP2), contain copper ion-, zinc ion-, and heparan sulfate-binding domains. We have investigated the possibility that APP and APLP2 regulate glypican-1 processing during endocytosis and recycling. By using cell-free biochemical experiments, confocal laser immunofluorescence microscopy, and flow cytometry of tissues and cells from wild-type and knock-out mice, we find that (a) APP and glypican-1 colocalize in perinuclear compartments of neuroblastoma cells, (b) ascorbate-triggered nitric oxidecatalyzed glypican-1 autodegradation is zinc ion-dependent in the same cells, (c) in cell-free experiments, APP but not APLP2 stimulates glypican-1 autodegradation in the presence of both Cu(II) and Zn(II) ions, whereas the Cu(I) form of APP and the Cu(II) and Cu(I) forms of APLP2 inhibit autodegradation, (d) in primary cortical neurons from APP or APLP2 knock-out mice, there is an increased nitric oxide-catalyzed degradation of heparan sulfate compared with brain tissue and neurons from wild-type mice, and (e) in growth-quiescent fibroblasts from APLP2 knock-out mice, but not from APP knock-out mice, there is also an increased heparan sulfate degradation. We propose that the rate of autoprocessing of glypican-1 is modulated by APP and APLP2 in neurons and by APLP2 in fibroblasts. These observation identify a functional relationship between the heparan sulfate and copper ion binding activities of APP/APLP2 in their modulation of the nitroxyl anion-catalyzed heparan sulfate degradation in glypican-1.

Alzheimer Disease↗

PubMed2004

The heparan sulfate-specific epitope 10E4 is NO-sensitive and partly inaccessible in glypican-1.

The monoclonal antibody 10E4, which recognizes an epitope supposed to contain N-unsubstituted glucosamine, is commonly used to trace heparan sulfate proteoglycans. It has not been fully clarified if the N-unsubstituted glucosamine is required for antibody recognition and if all heparan sulfates carry this epitope. Here we show that the epitope can contain N-unsubstituted glucosamine and that nitric oxide-generated deaminative cleavage at this residue in vivo can destroy the epitope. Studies using flow cytometry and confocal immunofluorescence microscopy of both normal and transformed cells indicated that the 10E4 epitope was partially inaccessible in the heparan sulfate chains attached to glypican-1. The 10E4 antibody recognized mainly heparan sulfate degradation products that colocalized with acidic endosomes. These sites were greatly depleted of 10E4-positive heparan sulfate on suramin inhibition of heparanase. Instead, there was increased colocalization between 10E4-positive heparan sulfate and glypican-1. When both S-nitrosylation of Gpc-1 and heparanase were inhibited, detectable 10E4 epitope colocalized entirely with glypican-1. In nitric oxide-depleted cells, there was both an increased signal from 10E4 and increased colocalization with glypican-1. In suramin-treated cells, the 10E4 epitope was destroyed by ascorbate-released nitric oxide with concomitant formation of anhydromannose-containing heparan sulfate oligosaccharides. Immunoisolation of radiolabeled 10E4-positive material from unperturbed cells yielded very little glypican-1 when compared with specifically immunoisolated glypican-1 and total proteoglycan and degradation products. The 10E4 immunoisolates were either other heparan sulfate proteoglycans or heparan sulfate degradation products.

Antibodies, Monoclonal↗

PubMed2004

Tumor attenuation by 2(6-hydroxynaphthyl)-beta-D-xylopyranoside requires priming of heparan sulfate and nuclear targeting of the products.

We have previously reported that the heparan sulfate-priming glycoside 2-(6-hydroxynaphthyl)-beta-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)-beta-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.

Animals↗

PubMed2004

Involvement of glycosylphosphatidylinositol-linked ceruloplasmin in the copper/zinc-nitric oxide-dependent degradation of glypican-1 heparan sulfate in rat C6 glioma cells.

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.

Animals↗

PubMed2003

Glypican-1 is a vehicle for polyamine uptake in mammalian cells: a pivital role for nitrosothiol-derived nitric oxide.

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.

Ascorbic Acid↗

PubMed2003

Prion, amyloid beta-derived Cu(II) ions, or free Zn(II) ions support S-nitroso-dependent autocleavage of glypican-1 heparan sulfate.

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.

Amyloid beta-Peptides↗

PubMed2003

Initiation of the decorin glycosaminoglycan chain in the endoplasmic reticulum-Golgi intermediate compartment.

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 GalNAcbeta1-4GlcUAbeta1-3Galbeta1-3Galbeta1-4Xyl-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.

Alkaline Phosphatase↗

PubMed2003

Glypicans.

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.

Animals↗

PubMed2002

Nitric oxide-dependent processing of heparan sulfate in recycling S-nitrosylated glypican-1 takes place in caveolin-1-containing endosomes.

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.-A. (2002) J. Biol. Chem. 277, 33353-33360). 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 that 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.

Animals↗

PubMed2002

Copper-dependent autocleavage of glypican-1 heparan sulfate by nitric oxide derived from intrinsic nitrosothiols.

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.-A., 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.-A. (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 slightly charged glypican-1 glycoforms containing heparan sulfate chains rich in N-unsubstituted glucosamines. These glycoforms were converted to highly 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 autocatalytic process contained terminal anhydromannose residues. We conclude that the core protein of glypican-1 can slowly accumulate NO as nitrosothiols, whereas 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, whereas Cu(+) is oxidized to Cu(2+).

Catalysis↗

PubMed2001

Tumor attenuation by combined heparan sulfate and polyamine depletion.

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.

Animals↗