title: Structural studies on the bacterial lyase-resistant tetrasaccharides derived from the antithrombin III-binding site of porcine intestinal heparin.
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.
authors.affiliation: Department of Physiological Chemistry, Kobe Women's College of Pharmacy, Japan.
authors.forename: S
authors.initials: S
authors.lastname: Yamada
authors.forename: K
authors.initials: K
authors.lastname: Yoshida
authors.forename: M
authors.initials: M
authors.lastname: Sugiura
authors.forename: K
authors.initials: K
authors.lastname: Sugahara
authors.forename: K H
authors.initials: KH
authors.lastname: Khoo
authors.forename: H R
authors.initials: HR
authors.lastname: Morris
authors.forename: A
authors.initials: A
authors.lastname: Dell
mesh_terms: D000818:Animals; D000990:Antithrombin III / Q000378:metabolism*; D001665:Binding Sites; D002236:Carbohydrate Conformation; D002240:Carbohydrate Sequence; D005417:Flavobacterium / Q000201:enzymology; D006493:Heparin / Q000737:chemistry* / Q000378:metabolism; D007422:Intestines / Q000737:chemistry*; D008190:Lyases / Q000378:metabolism; D009682:Magnetic Resonance Spectroscopy; D008969:Molecular Sequence Data; D009844:Oligosaccharides / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism; D016339:Spectrometry, Mass, Fast Atom Bombardment; D013379:Substrate Specificity; D013552:Swine
chemical_list: D009844:Oligosaccharides; D000990:Antithrombin III; D006493:Heparin; D008190:Lyases