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 in testinal heparin was investigated by characterizing the hexasaccharide fraction prepared by extensive digestion of the highly sulfated regio n with Flavobacterium heparinase and subsequent size fractionation by gel chromatography, Structures of a tetrasaccharide, a pentasaccharide , and eight hexasaccharide components in this fraction, which accounte d for approximately 19% (w/w) of the starting heparin representing the major oligosaccharide fraction derived from the irregular region, wer e determined by chemical and enzymatic analyses as well as H-1 NMR spe ctroscopy, Five compounds including one penta-and four hexasaccharides had hitherto unreported structures, The structure of the pentasacchar ide with a glucuronic acid at the reducing terminus was assumed to be derived from the reducing terminus of a heparin glycosaminoglycan chai n and may represent the reducing terminus exposed by a tissue endo-a-g lucuronidase involved in the intracellular post-synthetic fragmentatio n of macromolecular heparin, Eight out of the 10 isolated oligosacchar ides shared the trisaccharide sequence, -4IdceA alpha 1-4Glc-NAc alpha 1-4GlcAp beta 1-, and its reverse sequence, 4GlcA beta 1-4GlcNAc alph a 1-4IdceA alpha 1-, was not found, The latter has not been reported t o date for heparin/heparan sulfate, indicating the substrate specifici ty of the D-glucuronyl C-5 epimerase, Furthermore, seven hexasaccharid es shared the common trisulfated hexasaccharide core sequence Delta He xA(2-sulfate) alpha 1-4GlcN(N-sulfate) alpha 1-4IdceA alpha 1-4G1cNA a lpha 1-4GlcA beta 1-4GlcN(N-sulfate) which contained the above trisacc haride sequence (Delta HexA, IdceA, GlcN, and GlcA represent 4-deoxy-a lpha-L-threo-hex-4-enepyranosyluronic acid, L-iduronic acid, D-glucosa mine, and D-glucuronic acid, respectively) and additional sulfate grou ps, The specificity of the heparinase used for preparation of the olig osaccharides indicates the occurrence of the common pentasulfated octa saccharide core sequence, -4GlcN(N-sulfate)alpha 1-4HexA(B-sulfate)1-4 GlcN(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 hexasacch aride is flanked by GlcN(N-sulfate) and HexA(2-sulfate) on the nonredu cing and reducing sides, respectively, The revealed common sequence co nstituted a low sulfated trisaccharide representing the irregular regi on sandwiched by highly sulfated regions and should reflect the contro l mechanism of heparin biosynthesis.