Heparin preparations from pig intestinal mucosa and from bovine lung were separated by chromatography on antithrombin-Sepharose into a high-affinity fraction (with high anticoagulant activity) and a low-affinity fraction (with low anticoagulant activity). Antithrombin-binding heparin fragments (12-16 monosaccharide units) were prepared, either by digesting a high-affinity heparin-antithrombin complex with bacterial heparinase or by partial deaminative cleavage of the unfractionated polysaccharide with nitrous acid followed by affinity chromatography on (7) claimed that a tetrasaccharide sequence with a N-sulfated glucosamine residue at its reducing end, a N-acetylated internal glucosamine unit, and one residue each of D-glucuronic and L-iduronic acid would represent a critical structural element required for anticoagulant activity.The present study involves the structural characterization of antithrombin-binding regions from two different types'of heparin, isolated from pig intestinal mucosa and from bovine lung, respectively.

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 Nunsubstituted 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.

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 Nunsubstituted glucosamines, colocalized extensively with caveolin-1 but not with Rab9. Suramin, which inhibits heparanase, induced the appearance of Snitrosylated 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. Growthquiescent 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.Mammalian glypican-1 (Gpc-1) 1 is a member of a glycosylphosphatidylinositol (GPI)-linked cell-surface proteoglycan (PG) family with six known members to date. These PG, like other cell surface PG, are selective regulators of ligand-receptor encounters and thereby control growth and development (1-4). Gpc proteins are post-translationally modified by the addition of the glycosaminoglycan heparan sulfate (HS) at sites located close to the C-terminal GPI-membrane anchor (see Scheme 1). The central part of the protein consists of a cysteine-rich domain containing information that ensures a high level of HS substitution (5). Many of the functions of Gpc are dependent on the HS side chains, which are capable of binding and/or activating and/or transporting a variety of growth factors, cytokines, enzymes, viral proteins, and polyamines (6 -11).GPI-anchored proteins are usually associated with sphingolipid-and cholesterol-rich plasma membrane domains. Such enriched domains may exist either as small phase-separated "rafts" or, when associated with caveolin-1 (Cav-1), form flaskshaped plasmalemmal invaginations called caveolae, which are involved in signal transduction and special forms of non-clathrindependent endocytosis mediated by Cav-1-containing endosomes, also called caveosomes (12)(13)(14)(15)(16)(17).Biochemical studies using radioactively labeled precursors have demonstrated recycling of newly made Gpc-1 in normal fibroblasts as well as in transformed cells (18,19). During recycling, the HS side chains are degraded both by heparanase and by NO-dependent deaminative cleavage at N-unsubstituted glucosamine residues (GlcNH 3 ϩ ) (20). New HS chains can then be synthesized on the stubs remaining on the core protein (see Scheme 1). Biosynthesis of HS takes place in the Golgi and involves many interacting enzymes (21,22). The stubs should first be extended with a GlcNAc-hexuronic acid (HexUA) repeat backbone. The GlcNH 3 ϩ residues are either a result of inadequate sulfation dur...

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.

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, ␣-difluoromethylornithine (DFMO). However, the presence of cellsurface 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.metastasis ͉ proteoglycans ͉ spermine ͉ chemotherapy ͉ xylosides