Location of N-Unsubstituted Glucosamine Residues in Heparan Sulfate

Abstract

Functional properties of heparan sulfate (HS) are generally ascribed to the sulfation pattern of the polysaccharide. However, recently reported functional implications of rare N-unsubstituted glucosamine (GlcNH 2 ) residues in native HS prompted our structural characterization of sequences around such residues. HS preparations were cleaved with nitrous acid at either Nsulfated or N-unsubstituted glucosamine units followed by reduction with NaB 3 H 4 . The labeled products were characterized following complementary deamination steps. The proportion of GlcNH 2 units varied from 0.7-4% of total glucosamine in different HS preparations. The GlcNH 2 units occurred largely clustered at the polysaccharide-protein linkage region in intestinal HS, also more peripherally in aortic HS. They were preferentially located within N-acetylated domains, or in transition sequences between N-acetylated and N-sulfated domains, only 20 -30% of the adjacent upstream and downstream disaccharide units being N-sulfated. The nearest downstream (toward the polysaccharideprotein linkage) hexuronic acid was invariably GlcUA, whereas the upstream neighbor could be either GlcUA or IdoUA. The highly sulfated but N-unsubstituted disaccharide unit, -IdoUA2S-GlcNH 2 6S-, was detected in human renal and porcine intestinal HS, but not in HS from human aorta. These results are interpreted in terms of a biosynthetic mechanism, whereby GlcNH 2 residues are formed through regulated, incomplete action of an N-deacetylase/N-sulfotransferase enzyme.Many biological processes depend on interactions between heparan sulfate proteoglycans (HSPGs) 1 and proteins, such as enzymes, cytokines, growth factors, extracellular matrix proteins, and proteins produced by microbial pathogens (1-6). HSPGs are widely expressed on cell surfaces and in the extracellular matrices of most tissues. Their biological functions generally involve the carbohydrate moieties, i.e. one or more HS chains. These linear, sulfate-substituted glycosaminoglycans associate with basic amino acid residues in target proteins, sometimes through highly specific sequence motifs in the HS chain (see also reviews in Refs. 7 and 8). The proteinase inhibitor antithrombin thus binds to a unique pentasaccharide sequence that contains a rare 3-O-sulfated D-glucosamine (GlcN) unit (9). Other such rare constituents are 2-O-sulfated D-glucuronic acid (GlcUA) and N-unsubstituted GlcN residues (2). 2HS and the structurally related heparin are both synthesized through a non-sulfated precursor structure composed of alternating GlcUA and N-acetylated GlcN (GlcNAc) units (2,4,10,11). This precursor is modified through a series of enzymatic reactions, initiated by N-deacetylation and N-sulfation of GlcNAc residues. The resultant N-sulfated GlcN (GlcNS) residues are prerequisite to subsequent modification, involving C5-epimerization of GlcUA to L-iduronic acid (IdoUA), O-sulfation at C2 of the hexuronic acid (HexUA, i.e. GlcUA or IdoUA) and O-sulfation at C6 of GlcNS or GlcNAc units, or, less common, at C3 of GlcNS. H...

Glypican-1 Is a Vehicle for Polyamine Uptake in Mammalian Cells

“…The polyzwitterionic character of HS is enhanced, as there is an increased number of both the NO-sensitive GlcNH 3 ϩ residues and the highly sulfated high affinity sites for polyamines. Normally, GlcNH 3 ϩ residues are scarce in HS (35) and are predominantly located close to the linkage region to protein (27,35). The GlcNH 3 ϩ residues formed upon polyamine depletion appear to be located inside or near the polyamine-binding sites (15).…”

“…The GlcNH 3 ϩ residues formed upon polyamine depletion appear to be located inside or near the polyamine-binding sites (15). N-Desubstitution could be catalyzed by endo-N-deacetylase or endo-N-sulfamidase or both (15,17,27,35). How polyamine depletion is sensed and conveyed to the HS-modulating enzymes remains unknown.…”

Characterizing the Non-reducing End Structure of Heparan Sulfate

“…1, available in the on-line version of this article), suggesting that it might have been sulfated at its 6-O position. Also, considering the rareness and positioning of 3-O-sulfate and free amino group (28,29), this dp2-2S* was likely to be GlcA-GlcNS6S. No m/z value was found to have solution with Equation 3, suggesting no HS chain terminated on GlcN.…”

Prion, Amyloid β-derived Cu(II) Ions, or Free Zn(II) Ions Support S-Nitroso-dependent Autocleavage of Glypican-1 Heparan Sulfate

“…The N-unsubstituted glucosamines in HS (GlcNH 3 ϩ ) are usually cleaved by using high concentrations of nitrite at pH 3.9 (see Ref. 50). Presumably, NO 2 ⅐ Ϫ is the reactive species in this case.…”

“…Also NO gas (53) and acidified NO species derived from SNO-glutathione (54) can cleave HS and heparin quite extensively at these units. If NO ϩ is the reactive species in HNO 2 at pH 1.5 (52), this may explain why GlcNH 3 ϩ residues are resistant under these conditions (50).…”