Abstract
Three discrete tetrasaccharide structures which are resistant to Flavobacterium heparinase and heparitinases I and II were isolated from porcine intestinal heparin after exhaustive digestion with a mixture of all the above enzymes, and the tri-, tetra-, and penta-sulfated structures were determined by negative ion mode fast atom bombardment mass spectrometry and 500-MHz 1H NMR analysis as delta 4,5GlcA beta 1-4GlcNAc (6-sulfate)alpha 1-4GlcA beta 1-4GlcN(N,3-disulfate), delta 4,5 GlcA beta 1-4GlcNAc(6-sulfate)alpha 1-4GlcA beta 1-4GlcN (N,3,6-trisulfate), and delta 4,5GlcA beta 1-4GlcN (N,6-disulfate)alpha 1-4GlcA beta 1-4GlcN(N,3,6-trisulfate). The three components share the 3-O-sulfated reducing GlcN and the 6-O-sulfated internal GlcN, indicating that they are structural variants derived from the nonreducing portion of the minimal pentasaccharide sequence required for binding to antithrombin III. Isolation of the pentasulfated component has never been reported. Their unexpected resistance to heparitinases I and II indicates that 3-O-sulfation of the reducing GlcN contributes to the resistant nature of these tetrasaccharides to the enzymes. The present study demonstrates that the nonreducing trisaccharide portion of the structural variants of the antithrombin III-binding pentasaccharide sequence can be isolated in tetrasaccharides resistant to heparinase/heparitinases I and II, while the rest of the repeating region is degraded into disaccharide units. The lyase treatment is applicable to evaluation of heparin/heparan sulfate preparations in terms of the presence or absence of the specific structure containing the 3-O-sulfated GlcN representing biosynthetic precursors, intermediates or final products of the binding site.
Highlights
Three discrete tetrasaccharidestructures which are Heparin exerts a variety of biological and biochemical acresistant to Flavobacterium heparinase and hepariti- tivities such as inhibitionof blood coagulation (l)m, odulation nases I and I1 were isolated from porcine intestinal of cellular proliferation [2, 3], potentiation of angiogenesis heparin after exhaustive digestion with a mixture of [4],and interactions with acidic and basic fibroblast growth all the above enzymes, and the tri, tetra, andpenta- factors [5,6,7]
Some of these activities seem to reside within sulfated structures were determined by negative ion mode fast atom bombardment mass spectrometry and 500-MHz ‘HNMR analysis as A4,6GlcA@1-4GlcNAc (6-sulfate)c~1-4GlcA@l-4GlcN(N,3-disulfate),A4,5
Inearlier work Stage 14 porcine intestinal heparin was digested with a mixture of heparinase/heparitinases 1 and I1 in order to isolate the carbohydrate-protein linkage region which is resistant to bacterial lyases
Summary
Three discrete tetrasaccharidestructures which are Heparin exerts a variety of biological and biochemical acresistant to Flavobacterium heparinase and hepariti- tivities such as inhibitionof blood coagulation (l)m, odulation nases I and I1 were isolated from porcine intestinal of cellular proliferation [2, 3], potentiation of angiogenesis heparin after exhaustive digestion with a mixture of [4],and interactions with acidic and basic fibroblast growth all the above enzymes, and the tri-, tetra-, andpenta- factors [5,6,7]. In the present study we isolated and characterized three after pyrolysis by ion chromatography where sulfate is well separated lyase-resistant tetrasaccharides, afterexhaustive digestion of from phosphate [29]. Inearlier work Stage 14 porcine intestinal heparin was digested with a mixture of heparinase/heparitinases 1 and I1 in order to isolate the carbohydrate-protein linkage region which is resistant to bacterial lyases.
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