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.
Highlights
Porcine intestinal heparin was extensively digested with Flavobacterium heparinase and size-fractionated by gel chromatography
The principal structure of the repeating disaccharide region, known as the regular region (Casu, 1985), is composed of the major trisulfated disaccharide unit, 34IdoA(2sulfate)␣134GlcN(N,6-disulfate)␣13, undersulfation and substantial structural variability are observed in the irregular region, which is distributed along the chain flanked by the regular region and accounts for approximately one quarter of the heparin polysaccharide chain
It has been demonstrated that the binding domain to basic fibroblast growth factor requires a 2-O-sulfated IdoA residue and Nsulfated GlcN residue(s) for its specific interaction with the
Summary
Materials—Stage 14 heparin was purchased from American Diagnostica (New York, NY) and purified by DEAE-cellulose chromatography as reported previously (Sugahara et al, 1992). The reaction was terminated by boiling for 1 min, and the digest was fractionated by HPLC on an amine-bound silica column as described below. Nitrous Acid Degradation—Each heparin hexasaccharide (5.0 nmol) was treated at room temperature with HNO2 at pH 1.5 for 30 min (Shively and Conrad, 1976a), and the resultant di- and/or tetrasaccharides were reduced under alkaline conditions with [3H]sodium borohydride (0.50 mCi) as reported previously (Yamada et al, 1995). Di- and tetrasaccharide fractions were separately pooled, lyophilized at least three consecutive times to ensure complete removal of the ammonium bicarbonate, and reconstituted in water These fractions were separated, respectively, by HPLC on an amine-bound silica column at a flow rate of 1 ml/min using a stepwise gradient of NaH2PO4. 4)GlcN(N,6-disulfate); NS, 2-N-sulfate; 2S, 2-O-sulfate; 3S, 3-O-sulfate; 6S, 6-O-sulfate; ATIII, antithrombin III, Fr., fraction(s)
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