Structures of five sulfated hexasaccharides prepared from porcine intestinal heparin using bacterial heparinase structural variants with apparent biosynthetic precursor-product relationships for the antithrombin III-binding site

Hiromi Tsuda, Shuhei Yamada, Yukari Yamane, Keiichi Yoshida, John J. Hopwood, Kazuyuki Sugahara

Research output: Contribution to journalArticlepeer-review

25 Citations (Scopus)


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 ΔHexA(2S)α1-4GlcN-(NS,6S)α1-4IdoAα/GlcAβ1-4GlcN(6S) α1-4GlcAβ1-4GlcN(NS) with one or two additional sulfate groups (ΔHexA, GlcN, IdoA, and GlcA represent 4-deoxy-α-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: ΔHexA(2S)α1-4GlcN(NS,6S)α1-4GlcAβ1-4GlcN(NS,6S)α1- 4GlcAβ1-4GlcN(NS,6S), ΔHexA(2S)α1-4GlcN(NS,6S)α1-4IdoAα1-4GlcNAc(6S)-α1- 4GlcAβ1-4GlcN(NS,3S), and ΔHexA(2S)α1-4GlcN-(NS,6S)α1-4IdoA(2S)α1-4GlcNAc(6S) α1-4GlcAβ1-4GlcN(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 hexasaccharides 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.

Original languageEnglish
Pages (from-to)10495-10502
Number of pages8
JournalJournal of Biological Chemistry
Issue number18
Publication statusPublished or Issued - 1996
Externally publishedYes

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

Cite this