Structures of Five Sulfated Hexasaccharides Prepared from Porcine Intestinal Heparin Using Bacterial Heparinase

Abstract

Porcine intestinal heparin was extensively digested with Flavobacterium heparinase and size-fractionated by gel chromatography. Subfractionation of the hexasaccharide fraction by anion exchange high pressure liquid chromatography yielded 10 fractions. Six contained oligosaccharides derived from the repeating disaccharide region, whereas four contained glycoserines from the glycosaminoglycan-protein linkage region. The latter structures were reported recently (Sugahara, K., Tsuda, H., Yoshida, K., Yamada, S., de Beer, T., and Vliegenthart, J.F.G. (1995) J. Biol. Chem. 270, 22914-22923). In this study, the structures of one tetra- and five hexasaccharides from the repeat region were determined by chemical and enzymatic analyses as well as 500-MHz 1H NMR spectroscopy. The tetrasaccharide has the hexasulfated structure typical of heparin. The five hexa- or heptasulfated hexasaccharides share the common core pentasulfated structure delta HexA(2S) alpha 1-4GlcN-(NS, 6S) alpha 1-4IdoA alpha/GlcA beta 1-4GlcN(6S) alpha 1-4GlcA beta 1-4GlcN (NS) with one or two additional sulfate groups (delta HexA, GlcN, IdoA, and GlcA represent 4-deoxy-alpha-L-threo-hex-4-enepyranosyluronic acid, D-glucosamine, L-iduronic acid, and D-glucuronic acid, whereas 2S, 6S and NS stand for 2-O-, 6-O-, and 2-N-sulfate, respectively). Three components have the following hitherto unreported structures: delta HexA(2S) alpha 1-4GlcN(NS, 6S) alpha 1-4GlcA beta 1-4GlcN(NS, 6S) alpha 1-4GlcA beta 1-4GlcN(NS,6S), delta HexA(2S) alpha 1-4GlcN(NS, 6S) alpha 1-4IdoA alpha 1-4GlcNAc(6S)-alpha 1-4GlcA beta 1-4GlcN(NS, 3S), and delta HexA(2S) alpha 1-4GlcN-(NS,6S) alpha 1-4IdoA (2S) alpha 1-4GlcNAc(6S) alpha 1-4GlcA beta 1-4GlcN(NS, 6S). Two of the five hexasaccharides are structural variants derived from the antithrombin III-binding sites containing 3-O-sulfated GlcN at the reducing termini with or without a 6-O-sulfate group on the reducing N,3-disulfated GlcN residue. Another contains the structure identical to that of the above heptasulfated antithrombin III-binding site fragment but lacks the 3-O-sulfate group and therefore is a pro-form for the binding site. Another has an extra sulfate group on the internal IdoA residue of this pro-form and therefore can be considered to have diverged from the binding site in the biosynthetic pathway. Thus, the isolated hexasacharides in this study include the three overlapping pairs of structural variants with an apparent biosynthetic precursor-product relationship, which may reflect biosynthetic regulatory mechanisms of the binding site.

Enzymatic Preparation of Heparin Oligosaccharides Containing Antithrombin III Binding Sites

“…Chemical synthesis of a pentasaccharide containing this unique 3,6-di-O-sulfated sequence residue substantiated these findings (12). A number of oligosaccharide structures having sequence motifs similar to the ATIII binding sequence have been reported by our group (13) and others (14,15). Detailed structure-activity relationship studies have also been performed using degradative methods (16,17) and chemical synthesis (18) and have elucidated the features of the pentasaccharide that are critical for ATIII binding.…”

“…A number of oligosaccharides containing a portion of the pentasaccharide sequence required for ATIII binding have been isolated and characterized by our group (13,36) and others (14,15) (Table IV). These were prepared by exhaustive treatment of heparin with heparin lyases.…”

Structural Recognition by Recombinant Human Heparanase That Plays Critical Roles in Tumor Metastasis

“…Fluorescein isothiocyanate (FITC) was from Sigma-Aldrich. Structurally defined oligosaccharides were isolated from porcine intestinal Hep or bovine kidney HS, and their homogeneity was judged by capillary electrophoresis as described previously (35)(36)(37)(38)(39)(40)(41).…”

“…Their fine structures have been established by 500-MHz NMR analysis (35)(36)(37)(38)(39)(40)(41). Eighteen contained GlcUA residue(s) at the internal position of each oligosaccharide sequence, whereas the other two contained only ⌬HexUA and IdoUA but not GlcUA residues as uronic acid components.…”

“…However, the susceptibility of the oligosaccharides lacking the GlcN(3S) structure demonstrated in the present study suggests that 3-O-sulfation is not obligatory. Hexa-1, Hexa-8, or Tetra-10 comprises a portion of the antithrombin III-binding pentasaccharide sequence containing the unique 3-O-sulfate group (35,39,40). The structures in Hexa-4, Hexa-15, and Tetra-25 lack this particular 3-O-sulfate residing on the reducing terminal GlcN residue found in the corresponding structures of Hexa-1, Hexa-8, and Tetra-10, respectively.…”