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.

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 1H 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-beta-glucuronidase involved in the intracellular post-synthetic fragmentation of macromolecular heparin. Eight out of the 10 isolated oligosaccharides shared the trisaccharide sequence, -4IdceA alpha 1-4GlcNAc alpha 1-4GlcA beta 1-, and its reverse sequence, -4GlcA beta 1-4GlcNAc alpha 1-4IdceA alpha 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 delta HexA(2-sulfate)alpha 1-4GlcN(N-sulfate)alpha 1-4IdceA alpha 1-4GlcNAc alpha 1-4GlcA beta 1-4GlcN(N-sulfate) which contained the above trisaccharide sequence (delta HexA, IdceA, GlcN, and GlcA represent 4-deoxy-alpha-L-threo-hex-4-enepyranosyluronic acid, L-iduronic acid, D-glucosamine, and D-glucuronic acid, respectively) and additional sulfate groups. The specificity of the heparinase used for preparation of the oligosaccharides indicates the occurrence of the common pentasulfated octasaccharide core sequence, -4GlcN(N-sulfate)alpha 1-4HexA(2-sulfate)1-4GlcN(N-sulfate) alpha 1-4IdceA alpha 1-4GlcNAc alpha 1-4GlcA beta 1-4 GlcN(N-sulfate)alpha 1-4HexA(2-sulfate)1-, where the central hexasaccharide is flanked by GlcN(N-sulfate) and HexA(2-sulfate) on the nonreducing and reducing sides, respectively. The revealed common sequence constituted a low sulfated trisaccharide representing the irregular region sandwiched by highly sulfated regions and should reflect the control mechanism of heparin biosynthesis.