An unusual antithrombin-binding heparin octasaccharide with an additional 3-O-sulfated glucosamine in the active pentasaccharide sequence.
The 3-O-sulfation of N-sulfated glucosamine is the last event in the biosynthesis of heparin/heparan sulfate, giving rise to the antithrombin-binding pentasaccharide sequence AGA*IA, which is largely associated with the antithrombotic activity of these molecules. The aim of the present study was the structural and biochemical characterization of a previously unreported AGA*IA*-containing octasaccharide isolated from the very-low-molecular-mass heparin semuloparin, in which both glucosamine residues of the pentasaccharide moiety located at the non-reducing end bear 3-O-sulfate groups. Two-dimensional and STD (saturation transfer difference) NMR experiments clearly confirmed its structure and identified its ligand epitope binding to antithrombin. The molecular conformation of the octasaccharide-antithrombin complex has been determined by NMR experiments and docking/energy minimization. The presence of the second 3-O-sulfated glucosamine in the octasaccharide induced more than one order of magnitude increase in affinity to antithrombin compared to the pentasaccharide AGA*IA.
G. Ronzoni Institute for Chemical and Biochemical Research, via G. Colombo 81, 20133 Milan, Italy. guerrini@ronzoni.it
Marco
M
Guerrini
Stefano
S
Elli
Pierre
P
Mourier
Timothy R
TR
Rudd
Davide
D
Gaudesi
Benito
B
Casu
Christian
C
Boudier
Giangiacomo
G
Torri
Christian
C
Viskov
D000991:Antithrombins / Q000737:chemistry* / Q000378:metabolism; D002240:Carbohydrate Sequence; D005944:Glucosamine / Q000737:chemistry* / Q000378:metabolism; D006493:Heparin / Q000737:chemistry* / Q000378:metabolism; D007700:Kinetics; D009682:Magnetic Resonance Spectroscopy; D008958:Models, Molecular; D008968:Molecular Conformation; D008969:Molecular Sequence Data; D015394:Molecular Structure; D009844:Oligosaccharides / Q000737:chemistry* / Q000378:metabolism; D011485:Protein Binding; D011487:Protein Conformation; D013431:Sulfates / Q000737:chemistry / Q000378:metabolism; D013696:Temperature
D000991:Antithrombins; D009844:Oligosaccharides; D013431:Sulfates; D006493:Heparin; D005944:Glucosamine