Heparan sulfate found in the cerebral plaques of Alzheimer's disease binds to ␤-amyloid (A␤) fibrils. This interaction has been proposed to enhance fibril deposition and mediate A␤-induced glia activation and neurotoxicity. On the other hand, heparan sulfate augments signaling of fibroblast growth factor-2 (FGF-2), a neuroprotective factor that antagonizes the neurotoxic effects of A␤. We defined structures in heparan sulfate from human cerebral cortex that bind A␤ fibrils. The minimal binding site is found in N-sulfated hexasaccharide domains and contains critical 2-O-sulfated iduronic acid residues. By contrast, binding of A␤ monomers requires, in addition, 6-O-sulfate groups on glucosamine residues. The binding specificity of fibrillar A␤ is shared by FGF-2, and we here show that cerebral heparan sulfate domains selected for binding to A␤-(1-40) fibrils bind also to FGF-2. These data suggest that neurotoxic and neuroprotective signals may converge by competing for the same binding sites on the heparan sulfate chain.

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...

Heparin lyases are valuable tools for generating oligosaccharide fragments and in sequence determination of heparan sulfate (HS). Heparin lyase III is known to cleave the linkages between N-acetylglucosamine (GlcNAc) or N-sulfated glucosamine (GlcNS) and glucuronic acid (GlcA) as the primary sites and the linkages between GlcNAc, GlcNAc(6S), or GlcNS and iduronic acid as secondary sites. N-Unsubstituted glucosamine (GlcN) occurs as a minor component in HS, and it has been associated with various bioactivities. Here we investigate the specificity of heparin lyase III toward the GlcN-GlcA linkage using a recombinant enzyme of high purity and as substrates the partially de-N-acetylated polysaccharide of Escherichia coli K5 strain and derived hexasaccharides. The specificity of lyase III toward the GlcN-GlcA linkage is deduced by sequencing of the oligosaccharide products using electrospray mass spectrometry with collision-induced dissociation and MS/MS scanning. The results demonstrate that under controlled conditions for partial digestion, lyase III does not act at the GlcN-GlcA linkage, whereas GlcNAc-GlcA is cleaved. Even under forced conditions for exhaustive digestion, the GlcN-GlcA linkage is only partly cleaved. It is this property of lyase III that has enabled the isolation of a unique, nonsulfated antigenic determinant DeltaUA-GlcN-UA-GlcNAc from HS and from partially de-N-acetylated K5 polysaccharide. It was unexpected that pentasaccharide fragments were also detected among the digestion products of the K5 polysaccharide used. It is possible that these are products of an additional glycosidase activity of lyase III, although other mechanisms cannot be completely ruled out.