A Major Common Trisulfated Hexasaccharide Core Sequence, Hexuronic Acid(2-Sulfate)-Glucosamine(N-Sulfate)-Iduronic Acid-N-Acetylglucosamine-Glucuronic Acid-Glucosamine(N-Sulfate), Isolated from the Low Sulfated Irregular Region of Porcine Intestinal Heparin

Abstract

The major structure of the low sulfated irregular region of porcine intestinal heparin was investigated by characterizing the hexasaccharide fraction prepared by extensive digestion of the highly sulfated region with Flavobacterium heparinase and subsequent size fractionation by gel chromatography. Structures of a tetrasaccharide, a pentasaccharide, and eight hexasaccharide components in this fraction, which accounted for approximately 19% (w/w) of the starting heparin representing the major oligosaccharide fraction derived from the irregular region, were determined by chemical and enzymatic analyses as well as 1 H NMR spectroscopy. Five compounds including one penta-and four hexasaccharides had hitherto unreported structures. The structure of the pentasaccharide with a glucuronic acid at the reducing terminus was assumed to be derived from the reducing terminus of a heparin glycosaminoglycan chain and may represent the reducing terminus exposed by a tissue endo-␤-glucuronidase involved in the intracellular post-synthetic fragmentation of macromolecular heparin. Eight out of the 10 isolated oligosaccharides shared the trisaccharide sequence, -4IdceA␣1-4Glc-NAc␣1-4GlcA␤1-, and its reverse sequence, -4GlcA␤1-4GlcNAc␣1-4IdceA␣1-, was not found. The latter has not been reported to date for heparin/heparan sulfate, indicating the substrate specificity of the D-glucuronyl C-5 epimerase. Furthermore, seven hexasaccharides shared the common trisulfated hexasaccharide core sequence ⌬HexA(2-sulfate) ␣1-4GlcN(N-sulfate)␣1-4IdceA␣1-4Gl-cNAc␣1-4GlcA␤1-4GlcN(N-sulfate)

A strategy for rapid sequencing of heparan sulfate and heparin saccharides

“…Together, these data identify the nonreducing disaccharide structure as ⌬HexA(2S)-GlcNS(6S). The complete sequence of heparin hexasaccharide 6-27 identified by IGS was in full agreement with the known sequence established by NMR (16).…”

“…26), and heparin hexasaccharide 6-27 was a gift from Kazuyuki Sugahara (University of Kobe, Kobe, Japan; ref. 16). Recombinant exoenzymes were prepared as described below and are available from Oxford Glycosciences (Abingdon, U.K.), which also supplies kits for 2-aminobenzoic acid (2AA)-labeling and PAGE gel solutions.…”

Profiling glycol-split heparins by high-performance liquid chromatography/mass spectrometry analysis of their heparinase-generated oligosaccharides

“…Such a comparison is especially feasible for GlcA/IdoA-containing heparin oligosaccharides, whose gs derivatives survive digestion with heparinase I but are susceptible of cleavage with heparinase II and are amenable to further analysis through their glycol-split derivatives. The structure of the major GlcA/IdoA-containing tetra- and hexasaccharides identified in porcine mucosal heparin (4,49,52,54,55) is shown on row A of Table 3, while row B shows the structures expected for the corresponding glycol-split oligosaccharides. It should be noted that four of the major six heparin tetrasaccharide fragments (Δ4,4,1-A; Δ4,4,1-B; Δ4,4,0-A, and Δ4,5,0) obtained from unmodified heparin upon treatment with heparinase I (4, 54, 55), terminate with a GlcNS,6S reducing residue.…”

Microanalysis of Glycosaminoglycan-Derived Oligosaccharides Labeled with a Fluorophore 2-Aminobenzamide by High-Performance Liquid Chromatography: Application to Disaccharide Composition Analysis and Exosequencing of Oligosaccharides

“…The HS-derived tetrasaccharides Tetra-H1-H3 were isolated from bovine kidney HS after heparitinase digestion (18). The HP-derived tetrasaccharides Tetra-H4 -H8 (19,20) and hexasaccharides Hexa-H1-H5 (21,22) were isolated from porcine intestinal HP after heparinase digestion, whereas the tetrasaccharides Tetra-H9 and H10 (20) were isolated from the same HP preparation 4 A. Kinoshita, T. Inui, and K. …”