Biomedical subjects

M Belting

Publications and source records attributed to M Belting.

11 recordsLinked to original sources

PubMed2004

Novel aspects of glypican glycobiology.

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.

Animals↗

PubMed2001

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.

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.

Animals↗

PubMed2001

Expression of a functional proteinase inhibitor capable of accepting xylose: bikunin.

Bikunin is a Kunitz-type proteinase inhibitor, which is cross-linked to heavy chains via a chondroitin sulfate chain, forming inter-alpha-inhibitor and related molecules. Rat bikunin was produced by baculovirus-infected insect cells. The protein could be purified with a total yield of 20 mg/liter medium. Unlike naturally occuring bikunin the recombinant protein had no galactosaminoglycan chain. Endoglycosidase digestion also suggested that the recombinant form lacked N-linked oligosaccharides. Bikunin is translated as a part of a precursor, alpha1-microglobulin/bikunin, but the functional significance of the cotranslation is unknown. Our results indicate that the proteinase inhibitory function of bikunin is not regulated by the alpha1-microglobulin-part of the alpha1-microglobulin/bikunin precursor since recombinant bikunin had the same trypsin inhibitory activity as the recombinant precursor. Both free bikunin and the precursor were also functional as a substrate in an in vitro xylosylation system. This demonstrates that the alpha1-microglobulin-part is not necessary for the first step of galactosaminoglycan assembly.

Animals↗

PubMed2000

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.

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.

Cell Line↗

PubMed2000

Biosynthesis of decorin and glypican.

Decorin and glypican are two examples of exclusively chondroitin/dermatan sulfate and heparan sulfate-substituted proteoglycans, respectively. Decorin is a secretory product, whereas glypican is linked to membrane lipids via a glycosyl-phosphatidyl-inositol (GPI) anchor. The nascent decorin protein enters the lumen of the ER, whereas that of glypican is transferred to the preformed GPI-anchors. Both types of glycosaminoglycuronans are initiated on Ser residues located in special consensus sequences, and the first glycosylation steps constitute a common pathway: the generation of the linkage region GlcA-Gal-Gal-Xyl-Ser<. The nature of the enzymes involved will be reviewed with special emphasis on the recently discovered transient 2-phosphorylation of xylose. The initiation enzymes (betaGalNAc-T1 and alphaGlcNAc-T1) then use these tetrasaccharide primers for either chondroitin or heparan sulfate assembly. The selection mechanism is not yet fully understood. The transferases that form the linkage-region and add the first hexosamine, as well as the uronosyl C-5 epimerases, appear to be products of single genes, but many isoforms of the copolymerases and sulfotransferases forming the repetitive part of the glycan chains are currently being discovered. When these enzymes work together, the fine structure of the glycosaminoglycuronans appears to be generated through the selective expression of isoforms that only operate in certain structural contexts. During heparan sulfate assembly, generation of GlcNH(2) as a permanent feature is now well recognised. Studies on glypican-1 glycoforms that recycle suggest that heparan sulfate chains are degraded by endoheparanase at or near GlcNH(2) residues, followed by deaminative cleavage catalysed by NO-derived nitrite. Chain-truncated glypican-1 can serve as a precursor for the reformation of a proteoglycan with full-size chains. Regulation of biosynthesis can be exercised at several levels, such as expression of the core protein, selection for chondroitin or heparan sulfate assembly, expression of modifying enzymes, and degradation and remodelling. Cytokines, growth factors, NO and polyamines may have regulatory roles.

Animals↗

PubMed2000

A novel role for nitric oxide in the endogenous degradation of heparan sulfate during recycling of glypican-1 in vascular endothelial cells.

We show here that the endothelial cell-line ECV 304 expresses the heparan sulfate proteoglycan glypican-1. The predominant cellular glycoform carries truncated side-chains and is accompanied by heparan sulfate oligosaccharides. Treatment with brefeldin A results in accumulation of a glypican proteoglycan with full-size side-chains while the oligosaccharides disappear. During chase the glypican proteoglycan is converted to partially degraded heparan sulfate chains and chain-truncated proteoglycan, both of which can be captured by treatment with suramin. The heparan sulfate chains in the intact proteoglycan can be depolymerized by nitrite-dependent cleavage at internally located N-unsubstituted glucosamine moieties. Inhibition of NO-synthase or nitrite-deprivation prevents regeneration of intact proteoglycan from truncated precursors as well as formation of oligosaccharides. In nitrite-deprived cells, formation of glypican proteoglycan is restored when NO-donor is supplied. We propose that, in recycling glypican-1, heparan sulfate chains are cleaved at or near glucosamines with unsubstituted amino groups. NO-derived nitrite is then required for the removal of short, nonreducing terminal saccharides containing these N-unsubstituted glucosamine residues from the core protein stubs, facilitating re-synthesis of heparan sulfate chains.

Brefeldin A↗

PubMed1999

Protective role for proteoglycans against cationic lipid cytotoxicity allowing optimal transfection efficiency in vitro.

A dependence on proteoglycans for cationic lipid-mediated gene transfer has been suggested in previous studies [Mislick and Baldeschwieler (1996) Proc. Natl. Acad. Sci. U.S.A. 93, 12349-12354; Mounkes, Zhong, Cipres-Palacin, Heath and Debs (1998) J. Biol. Chem. 273, 26164-26170]. We have evaluated the mechanism of proteoglycan involvement in cationic lipid-mediated gene transfer. DNA plasmid uptake and gene expression were studied in wild-type Chinese hamster ovary (CHO) cells (CHO-K1), heparan sulphate-deficient CHO cells (pgsD-677) and proteoglycan-deficient CHO cells (pgsB-618). At an optimal ratio of cationic lipid to DNA, a substantial decrease in reporter gene expression was observed in proteoglycan-deficient cells compared with that in heparan sulphate-deficient and wild-type cells. However, there were no differences in reporter gene expression between the cell lines when transfected by electroporation. Moreover, all cell lines exhibited equal cationic-lipid-DNA complex uptake activities, as assessed by the measurement of intracellular (32)P-labelled and rhodamine-labelled DNA plasmid. An analysis of reflected-light images of wild-type and proteoglycan-deficient cells suggested that cationic lipids were preferentially toxic to proteoglycan-deficient cells. Cell-growth assays confirmed this, showing that cationic lipids exhibited a greater anti-proliferative activity in proteoglycan-deficient cells and in chlorate-treated wild-type cells than in the other cell lines. The growth-inhibitory effect of cationic lipids was abrogated by the addition of exogenous sulphated glycosaminoglycans. We conclude that the glycosaminoglycan part of proteoglycans serves a protective role against cationic lipid cytotoxicity, allowing optimal transfection efficiency in vitro.

Animals↗

PubMed1999

Intracellular accumulation of secreted proteoglycans inhibits cationic lipid-mediated gene transfer. Co-transfer of glycosaminoglycans to the nucleus.

Molecules secreted by potential target cells may interfere with cationic lipid-mediated gene transfer. This has been studied using human lung fibroblasts and human epidermoid lung cancer cells. Secreted cell medium components caused a substantial decrease both in the uptake of cationic lipid-DNA complexes (2-4-fold) and in reporter gene expression (100-1000-fold). Metabolic labeling of the cell medium showed that especially [35S]sulfate-labeled macromolecules competed with DNA for binding to the cationic lipid. Release of DNA from the cationic lipid by cell medium components was demonstrated by an ethidium bromide intercalation assay. In the presence of the cationic lipid, the secreted macromolecules were internalized by the cells. By enzymatic digestions, it was shown that the competing macromolecules consist of chondroitin/dermatan sulfate and heparan sulfate proteoglycans and that the effects on transfection were mediated by the polyanionic glycosaminoglycan portion of the proteoglycan. Accordingly, pretreatment of cell medium with the polycationic peptide protamine sulfate abrogated the inhibitory effects on gene transfer. Fluorescence microscopy studies revealed that heparan sulfate, internalized as a complex with cationic lipids, accumulated in the cell nuclei. These results support the view that the lack of specificity of this type of gene transfer vehicle is a major hindrance to efficient and safe in vivo administration.

Biological Transport↗

PubMed1999

Proteoglycan involvement in polyamine uptake.

We have evaluated the possible role of proteoglycans in the uptake of spermine by human lung fibroblasts. Exogenous glycosaminoglycans behaved as competitive inhibitors of spermine uptake, the most efficient being heparan sulphate (Ki=0.16+/-0.04 microM). Treatment of fibroblasts with either heparan sulphate lyase, p-nitrophenyl-O-beta-D-xylopyranoside or chlorate reduced spermine uptake considerably, whereas chondroitin sulphate lyase had a limited effect. Inhibition of polyamine biosynthesis with alpha-difluoromethylornithine resulted in an increase of cell-associated heparan sulphate proteoglycans exhibiting higher affinity for spermine. The data indicate a specific role for heparan sulphate proteoglycans in the uptake of spermine by fibroblasts. Spermine uptake by pgsD-677, a mutant Chinese hamster ovary cell defective in heparan sulphate biosynthesis, was only moderately reduced (20%) compared with wild-type cells. Treatment of mutant cells with the above-mentioned xyloside resulted in a greater reduction of endogenous proteoglycan production as well as a higher inhibition of spermine uptake than in wild-type cells. Moreover, treatment with chondroitin sulphate lyase resulted in a selective inhibition of uptake in mutant cells, indicating a role for chondroitin/dermatan sulphate proteoglycans in the uptake of spermine by these cells. Fibroblasts, made growth-dependent on exogenous spermine by alpha-difluoromethylornithine treatment, were growth-inhibited by heparan sulphate or beta-D-xyloside, which might have future therapeutical implications.

Animals↗

PubMed1996

Heparan sulphate/heparin glycosaminoglycans with strong affinity for the growth-promoter spermine have high antiproliferative activity.

Depletion of intracellular polyamine pools inhibits cell proliferation. Polyamine pools are maintained by intracellular synthesis and by uptake from the extracellular environment. It may be expected that cationic polyamines are sequestered by the polyanionic glycosaminoglycan substituents of extracellular proteoglycans. Moreover, highly sulphated heparin-related glycans inhibit growth of human embryonic lung fibroblasts. We have therefore investigated interactions between polyamines and heparin-related glycosaminoglycans. Affinity chromatography of various polyamines on heparin-agarose indicated that spermine was the only polyamine that bound efficiently to this type of glycan. By using equilibrium dialysis we found that spermine binds to a highly sulphated heparan sulphate/heparin preparation with a dissociation constant of 3.7 x 10(-5)M. Enzymatic degradation of heparan sulphate using three different heparan sulphate/heparin lyases, separately or in combination and in the absence or presence of spermine, was used to generate spermine-binding and degradation-protected oligosaccharides. As indicated by chromatographic and electrophoretic analysis a size- and chargeheterogeneous collection was obtained. However, protected oligosaccharides derived from antiproliferative heparan sulphates were inactive. Highly sulphated, antiproliferative heparan sulphates were subfractionated on spermine-agarose yielding high-affinity material with increased antiproliferative activity. A very potent material was obtained from pig skin. Although there was generally a clear correlation between high spermine-affinity and strong growth-inhibition, no correlation with sulphate content or oligosaccharide mapping patterns could be detected. Beef lung heparan sulphate comprised naturally occurring fragments of eicosasaccharide size with substantially increased specific activity. As these fragments were longer than oligosaccharides generated by enzymatic degradation in the presence of spermine (hexa- to tetradecasaccharide), multiple spermine-binding sites in tandem may be necessary to induce antiproliferative activity.

Animals↗

PubMed1993

The growth promoter spermine interacts specifically with dermatan sulfate regions that are rich in L-iduronic acid and possess antiproliferative activity.

We have investigated interactions between spermine, a member of the growth promoting polyamine family, and various glycosaminoglycans. By using gel chromatography and equilibrium dialysis experiments we found that spermine binds to L-iduronic acid-rich dermatan sulfate (Kd, approximately 3.9 x 10(-4) M) with an affinity similar to that between spermine and DNA. By digesting spermine-dermatan sulfate complexes with chondroitin ABC lyase, the formation of oligosaccharide fragments (tetra-to-decasaccharides) was demonstrated by polyacrylamide gel electrophoresis. Chondroitin sulfate, which is deficient in L-iduronic acid, generates no spermine-protected fragments. Analysis of protected dermatan sulfate oligosaccharides indicates that the majority of the L-iduronic acid residue is non-sulfated and in a periodate-resistant conformation. The oligosaccharides also possess antiproliferative activity.

Cell Division↗