Lars-Åke Fransson

Structure and function of cell-associated proteoglycans

Abstract Proteoglycans which consist of a central core protein decorated with complex polysaccharide side chains occur in the intercellular matrix, at the cell surface and in intracellular storage granules. The cell-associated proteoglycans are diverse in structure and function. Recent studies indicate a role for these macromolecules in cell adhesion, growth control, activation of proteinase inhibitors, and in the regulation of receptor functions and cytotoxic events.

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, α-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.

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.

TGF-β enhances the production of hyaluronan in human lung but not in skin fibroblasts

Transforming growth factor-beta (TGF-beta) enhances the production of extracellular matrix components, such as type I and type III collagen, fibronectin, proteoglycans, in various cell types. The effect on hyaluronan synthesis in relation to proteoglycan synthesis has not been investigated. Human lung or skin fibroblast cultures were treated with TGF-beta in serum-free medium for various periods of time. 35SO4 or [3H]glucosamine was then added to the cultures in the absence of TGF-beta for up to 48 h. Hyaluronan and proteoglycans were isolated by ion-exchange chromatography and quantitated. TGF-beta induced a three- to fourfold increase in hyaluronan production by lung cells but had no effect on skin fibroblasts. In contrast, proteoglycan synthesis was enhanced in both cell types, although skin fibroblasts responded at lower concentrations of TGF-beta. Increased accumulation of hyaluronan was noted only in the cell medium, whereas proteoglycan accumulation was observed both in the medium and in the cell layer. The ED50 for TGF-beta on hyaluronan accumulation in lung cells was the same as that for proteoglycan accumulation, i.e., 40 pM. In skin fibroblasts the ED50 was considerably lower (4 pM). The induction time needed to attain full effect of TGF-beta was 6 h for both hyaluronan and proteoglycan synthesis. These results indicate that TGF-beta has tissue-specific effects on matrix production which may be of importance for control of cell proliferation in various disease states.

Biosynthesis of L-iduronic acid in heparin: Epimerization of D-glucuronic acid on the polymer level

Summary Incubation of a microsomal fraction from mouse mastocytoma with UDP-14C-glucuronic acid and unlabelled UDP-N-acetylglucosamine resulted in the incorporation of 14C-glucuronic acid into endogenous polysaccharide. When 3′-phosphoadenosine 5′-phosphosulfate (PAPS) was included in such incubations the polysaccharide product contained 14C-iduronic acid in addition to 14C-glucuronic acid. Pulse-chase experiments showed that 14C-glucuronic acid units incorporated into the polymer during the pulse period (in the absence of PAPS), were subsequently converted to 14C-iduronic acid units during the chase period (in the presence of PAPS). It is concluded that the iduronic acid residues had been formed by epimerization on the polymer level at C-5 of glucuronic acid residues.

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.

Periodate oxidation and alkaline degradation of heparin-related glycans

Abstract Heparin, heparan sulphate, and various derivatives thereof have been oxidised with periodate at pH 3.0 and 4° and at pH 7.0 and 37°. Whereas oxidation under the latter conditions destroys all of the nonsulphated uronic acids, treatment with periodate at low pH and temperature causes selective oxidation of uronic acid residues. The reactivity of uronic acid residues depends on the nature of neighbouring 2-amino-2-deoxyglucose residues. d -Glucuronic acid residues are susceptible to oxidation when flanked by N-acetylated amino sugars, but resistant when adjacent residues are either unsubstituted or N-sulphated. L -Iduronic acid residues in their natural environment (2-deoxy-2-sulphoamino- d -glucose) are resistant to oxidation, whereas removal of N-sulphate groups renders a portion of these residues periodate-sensitive. Oxidised uronic acid residues in heparin-related glycans may be cleaved by alkali, producing a series of oligosaccharide fragments. Thus, periodate oxidation-alkaline elimination provides an additional method for the controlled degradation of heparin.

Periodate oxidation of the d-glucuronic acid residues in heparan sulphate and heparin

Abstract On oxidation with periodate at pH 7.0 and 37°, the uronic acid residues of heparan sulphate preparations were almost completely destroyed, whereas only 20% of those in heparin were susceptible to oxidation. At pH 3.0 and 4°, 30–40% of the uronic acid residues in heparan sulphates were destroyed, but very few in heparin. In all cases at pH 3.0 and 4°, the l -iduronic acid residues were resistant to oxidation, whereas a large proportion of the d -glucuronic acid residues were affected. However, a small but significant proportion of the d -glucuronic acid residues resisted oxidation. The initially periodate-resistant d -glucuronic acid residues were destroyed when alkali-treated oxyheparan sulphate was treated with periodate. When heparan sulphate and heparin derivatives containing mainly N -acetylated 2-amino-2-deoxy- d -glucose residues were treated with periodate at pH 3.0 and 4°, most of the d -glucuronic acid residues were destroyed, whereas the l -iduronic acid residues were resistant.

Copper-dependent autocleavage of glypican-1heparan sulfate by nitric oxide derived fromintrinsic 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.-Å., 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 chargedN-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.-Å. (2001)J. Biol. Chem. 276, 46779–46791). Here, using both biochemical and microscopic techniques, we have identified and isolatedS-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 Cu2+/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 Cu2+ 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 Cu2+.

Periodate oxidation of L-iduronic acid residues in dermatan sulphate.

Abstract The rate of periodate oxidation of dermatan sulphate and chondroitin sulphate was studied at various pH values, ionic strengths, and temperatures. The L -iduronic acid residues of dermatan sulphate were readily oxidized under a variety of conditions, whereas oxidation of D -glucuronic acid was essentially zero at pH 3 and 4°. Extensive depolymerization occurred at temperatures greater than 4°. Periodate oxidation, performed at relatively high ionic-strength, yielded hemiacetal-type cross-linkages between oxidized and unoxidized uronic acid residues in separate chains. After oxidation of a mixture of dermatan sulphate and -labelled dermatan sulphate octasaccharide, a large proportion of the radioactivity was eluted with the polysaccharide on gel chromatography. Reductive cleavage of these cross-linkages allowed further consumption of oxidant. At the correct oxidation-limit for dermatan sulphate, > 20% of the L -iduronic acid residues were resistant to periodate. Although some of these residues were undoubtedly sulphated at 2 or 3, the presence of a small proportion of L -iduronic acid residues in the 1C conformation cannot be excluded.