Heparinase 1 selectivity for the 3,6-di-O-sulfo-2-deoxy-2-sulfamido-alpha-D-glucopyranose (1,4) 2-O-sulfo-alpha-L-idopyranosyluronic acid (GlcNS3S6S-IdoA2S) linkages.
Porcine intestinal mucosa heparin was partially depolymerized by recombinant heparinase 1 (heparin lyase 1, originating from Flavobacterium heparinum and expressed in Escherichia coli) and then fractionated, leading to the isolation of 22 homogeneous oligosaccharides with sizes ranging from disaccharide to hexadecasaccharide. The purity of these oligosaccharides was determined by gel electrophoresis, strong anion exchange and reversed-phase ion-pairing high-performance liquid chromatography. The molecular mass of oligosaccharides was determined using electrospray ionization-mass spectrometry and their structures were elucidated using one- and two-dimensional nuclear magnetic resonance spectroscopy at 600 MHz. Five of the characterized oligosaccharides represent new compounds. The most prominent oligosaccharide comprises the common repeating unit of heparin, ΔUA2S-[-GlcNS6S-IdoA2S-](n)-GlcNS6S, where ΔUA is 4-deoxy-α-l-threo-hex-4-eno-pyranosyluronic acid, GlcN is 2-deoxy-2-amino-d-glucopyranose, IdoA is l-idopyranosyluronic acid, S is sulfate and n = 0-7. A second prominent heparin oligosaccharide motif corresponds to ΔUA2S-[GlcNS6S-IdoA2S](n)-GlcNS6S-IdoA-GlcNAc6S-GlcA-GlcNS3S6S (where n = 0-5 and GlcA is d-glucopyranosyluronic acid), a fragment of the antithrombin III binding site in heparin. The prominence of this second set of oligosaccharides and the absence of intact antithrombin III binding sites suggest that the -GlcNS3S6S-IdoA2S- linkage is particularly susceptible to heparinase 1.
Xiao
Zhongping
Z
Key Laboratory of Marine Drugs, Chinese Ministry of Education, Institute of Marine Drug and Food, Ocean University of China, Qingdao 266003, China.
Zhao
Wenjing
W
Yang
Bo
B
Zhang
Zhenqing
Z
Guan
Huashi
H
Linhardt
Robert J
RJ
D000818:Animals; D000990:Antithrombin III / Q000378:metabolism; D001665:Binding Sites; D002236:Carbohydrate Conformation; D002240:Carbohydrate Sequence; D006493:Heparin / Q000737:chemistry*; D019763:Heparin Lyase / Q000378:metabolism*; D009844:Oligosaccharides / Q000737:chemistry* / Q000378:metabolism; D014574:Uronic Acids / Q000737:chemistry*
D009844:Oligosaccharides; D014574:Uronic Acids; D000990:Antithrombin III; D006493:Heparin; D019763:Heparin Lyase