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.-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+).
Highlights
Cationic polyamines are natural constituents of all cells and essential for growth and differentiation
High or Low Content of N-Unsubstituted Glucosamine—We have previously demonstrated that Gpc-1 can contain heparan sulfate (HS) chains with clusters of GlcNH3ϩ residues, especially near the linkage region to the core protein (Ref. 18; see Scheme 1)
When S-nitroso groups (SNO)-containing Gpc-1 from polyamine synthesis-inhibited cells was subjected to ion exchange FPLC on MonoQ, it eluted in pool III (Fig. 1H), indicating that it contained HS chains that had already been processed by deaminative cleavage at the GlcNH3ϩ residues
Summary
Glypican; anMan, anhydromannose; BMCC, 1-biotinamido-4-[4Ј-(maleimidomethyl)cyclohexane-carboxamido]butane; BFA, brefeldin A; DFMO, ␣-difluoromethylornithine; GlcNAc, N-acetylglucosamine; GlcNH3ϩ, N-unsubstituted glucosamine; GlcN, glucosamine with unspecified N-substituent; HexUA, unspecified hexuronic acid; HS, heparan sulfate; NDST, N-deacetylase/sulfotransfamily with six known members to date. Position 2, Gpc-1 from cells treated with DFMO carrying HS chains with an increased number of GlcNH3ϩ residues and sulfated uronic acids, often juxtaposed. We have found that brefeldin A (BFA)-treated cells accumulate a large size Gpc-1 glycoform with long HS chains containing clusters of GlcNH3ϩ residues [17] These clusters are concentrated to the reducing side of heparanase cleavage sites, especially near the linkage region to the core protein [18]. The large Gpc-1 glycoform is degraded by pericellular and intracellular heparanase, generating HS oligosaccharides and a glycoform with truncated HS chains, still containing GlcNH3ϩ residues [17, 18] These stubs are further eroded by NO-dependent deaminative cleavage at the GlcNH3ϩ residues [17]. We have identified and isolated subpopulations containing S-nitroso groups (SNO) as well as Gpc-1 glycoforms carrying HS with a high content of GlcNH3ϩ and studied their processing
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