Seven pharmaceutical heparins were investigated by oligosaccharide mapping by digestion with heparin lyase 1, 2 or 3, followed by high performance liquid chromatography analysis. The structure of one of the prepared mapping standards, ΔUA-Gal-Gal-Xyl-O-CH 2 CONHCH 2 COOH, (where ΔUA is 4-deoxy-α-L-threo-hex-4-eno-pyranosyluronic acid, Gal is β-D-galactpyranose, and Xyl is β-D-xylopyranose) released from the linkage region using either heparin lyase 2 or heparin lyase 3 digestion, is reported for the first time. A size-dependent susceptibility of site cleaved by heparin lyase 3 was also observed. Heparin lyase 3 acts on the under sulfated domains of the heparin chain and does not cleave the linkages within heparin's antithrombin III binding site. Thus, a novel low molecular weight heparin (LMWH) was afforded on heparin lyase 3 digestion of heparin due to this unique substrate specificity, which has anticoagulant activity comparable to that of currently available LMWH.
Heparin and heparan sulphate (HS) exert their wide range of biological activities by interacting with extracellular protein ligands. Among these important protein ligands are various angiogenic growth factors and cytokines. HS-binding to vascular endothelial growth factor (VEGF) regulates multiple aspects of vascular development and function through its specific interaction with HS. Many studies have focused on HS-derived or HS-mimicking structures for the characterization of VEGF165 interaction with HS. Using a heparinase 1-prepared small library of heparin-derived oligosaccharides ranging from hexasaccharide to octadecasaccharide, we systematically investigated the heparin-specific structural features required for VEGF binding. We report the apparent affinities for the association between the heparin-derived oligosaccharides with both VEGF165 and VEGF55, a peptide construct encompassing exclusively the heparin-binding domain of VEGF165. An octasaccharide was the minimum size of oligosaccharide within the library to efficiently bind to both forms of VEGF and that a tetradecasaccharide displayed an effective binding affinity to VEGF165 comparable to unfractionated heparin. The range of relative apparent binding affinities among VEGF and the panel of heparin-derived oligosaccharides demonstrate that VEGF binding affinity likely depends on the specific structural features of these oligosaccharides including their degree of sulphation and sugar ring stereochemistry and conformation. Notably, the unique 3-O-sulpho group found within the specific antithrombin binding site of heparin is not required for VEGF165 binding. These findings afford new insight into the inherent kinetics and affinities for VEGF association with heparin and heparin-derived oligosaccharides with key residue specific modifications and may potentially benefit the future design of oligosaccharide-based anti-angiogenesis drugs.
Two unique 8,9,30-phragmalin ortho esters, xyloccensins O (1) and P (2), were isolated from the mangrove plant Xylocarpus granatum. They are a new type of ortho ester of phragmalin. The structures were determined by spectroscopic and single-crystal X-ray diffraction techniques. The biogenetic pathway to these new phragmalins was also proposed. [structure: see text]
Heparin lyase I (heparinase I) specifically depolymerizes heparin, cleaving the glycosidic linkage next to iduronic acid. Here, we show the crystal structures of heparinase I from Bacteroides thetaiotaomicron at various stages of the reaction with heparin oligosaccharides before and just after cleavage and product disaccharide. The heparinase I structure is comprised of a -jellyroll domain harboring a long and deep substrate binding groove and an unusual thumb-resembling extension. This thumb, decorated with many basic residues, is of particular importance in activity especially on short heparin oligosaccharides. Unexpected structural similarity of the active site to that of heparinase II with an (␣/␣) 6 fold is observed. Mutational studies and kinetic analysis of this enzyme provide insights into the catalytic mechanism, the substrate recognition, and processivity.Heparin and heparan sulfate are linear, negatively charged polymers consisting of repeating units of 134-linked uronic acid (L-iduronic acid (IdoA) 4 and D-glucuronic acid (GlcA)) and glucosamine (1). Heparin consists of a high proportion of IdoA (ϳ90%) and is highly sulfated. It is widely used as an anticoagulant based on its binding to antithrombin, leading to the accelerated inhibition of the blood coagulation cascade (2). Heparin interacts with a variety of proteins, such as growth factors and chemokines, suggesting its relevance in various physiological and pathological processes (2, 3).Glycosaminoglycans in general, and heparin in particular, can be degraded by two mechanisms: hydrolysis and lytic elimination (4). Glycosaminoglycan hydrolases, present in eukaryotes and prokaryotes, break the glycosidic bond to the nonreducing end of the glucosamine, whereas glycosaminoglycan lyases, found only in prokaryotes, break the glycosidic linkage to the nonreducing end of uronic acid (5). The lyases that cleave chondroitin sulfate and hyaluronan have been extensively studied, structurally and biochemically. All of these lyases share a common fold, (␣/␣) 5 barrel, and antiparallel -sheet, and have similar catalytic mechanisms (6 -8). In contrast, dermatan sulfate (chondroitin B) lyase has a completely different fold as a parallel -helix, similar to pectate lyases, and employs very different catalytic machinery (9).The eliminative depolymerization of heparin/heparan sulfate affording unsaturated oligosaccharide products is carried out by three families of enzymes (10). Their primary sequences show no recognizable similarity, and they have distinct specificities (11). Thus, heparinase I is specific for heparin cleaving the glycosidic linkage to the nonreducing end of IdoA, heparin lyase III (heparinase III) cleaves the heparan sulfate next to glucuronic acid, and heparin lyase II (heparinase II) can depolymerize both of these substrates (see Fig. 1A). Structural information on the heparin degrading enzymes is limited to the Pedobacter heparinus (formerly Flavobacterium heparinum) heparinase II, which adopts an overall fold similar to chondroitin and hy...
Seven commercial heparin active pharmaceutical ingredients and one commercial low molecular weight from different manufacturers were characterized with a view profiling their physico-chemical properties. All heparins had similar molecular weight properties as determined by polyacrylamide gel electrophoresis (MN 10–11 kDa, MW 13–14 kDa, polydispersity (PD) 1.3–1.4) and by size exclusion chromatography (MN 14–16 kDa, MW 21–25 kDa, PD 1.4–1.6). 1D 1H- and 13C-NMR evaluation of the heparin samples was performed and peaks were fully assigned using 2D NMR. The percentage of glucosamine residues with 3-O-sulfo groups and the percentage of N-sulfo groups and N-acetyl groups ranged from 5.8–7.9, 78–82 and 13–14 %, respectively. There was substantial variability observed in the disaccharide composition with, as determined by high performance liquid chromatography (HPLC)-mass spectral analysis of heparin lyase I–III digested heparins. Heparin oligosaccharide mapping was performed using HPLC following separate treatments with heparin lyase I, II and III. These maps were useful in qualitatively and quantitatively identifying structural differences between these heparins. The binding affinities of these heparins to antithrombin III and thrombin were evaluated by using a SPR competitive binding assay. This study provides the physico-chemical and activity characterization necessary for the appropriate design and synthesis of a generic bioengineered heparin.
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.
Heparinase II (HepII) is an 85-kDa dimeric enzyme that depolymerizes both heparin and heparan sulfate glycosaminoglycans through a -elimination mechanism. Recently, we determined the crystal structure of HepII from Pedobacter heparinus (previously known as Flavobacterium heparinum) in complex with a heparin disaccharide product, and identified the location of its active site. Here we present the structure of HepII complexed with a heparan sulfate disaccharide product, proving that the same binding/active site is responsible for the degradation of both uronic acid epimers containing substrates. The key enzymatic step involves removal of a proton from the C5 carbon (a chiral center) of the uronic acid, posing a topological challenge to abstract the proton from either side of the ring in a single active site. We have identified three potential active site residues equidistant from C5 and located on both sides of the uronate product and determined their role in catalysis using a set of defined tetrasaccharide substrates. HepII H202A/Y257A mutant lost activity for both substrates and we determined its crystal structure complexed with a heparan sulfate-derived tetrasaccharide. Based on kinetic characterization of various mutants and the structure of the enzyme-substrate complex we propose residues participating in catalysis and their specific roles. Heparin and heparan sulfate (HS)3 glycosaminoglycans (GAGs) are negatively charged, linear polysaccharides composed of repeating disaccharide units of uronic acid and glucosamine residues (GlcN, 2-amino-2-deoxy-␣-D-glucopyranose) (1). Heparin typically contains ϳ90% iduronic acid (IdoA, ␣-Lidopyranosyluronic acid) and 10% glucuronic acid (GlcA, -Dglucopyranosyluronic acid), with a high content of 2-O-sulfo groups on the IdoA residue. The glucosamine residue in heparin is predominantly substituted with N-sulfo groups (GlcNS, where S is sulfo) and 6-O-sulfo groups with a small number of N-acetyl groups and much less frequently with 3-O-sulfo groups. In contrast, HS is somewhat more diverse in its primary structure and characterized by a higher percentage of the GlcA epimer, N-acetyl-substituted GlcN (GlcNAc) and a lower percentage of 2-O-sulfo, 6-O-sulfo, and N-sulfo groups. The modifications in HS are not uniform; rather, they are concentrated within specific regions of the polysaccharide, giving rise to a short, sulfo group containing sequence motifs responsible for the interactions between HS and a diverse repertoire of proteins leading to its multiple biological roles. These complex polysaccharides provide docking sites for numerous protein ligands involved in diverse biological processes, ranging from cancer and angiogenesis, anticoagulation, inflammatory processes, viral and microbial pathogenesis to multiple aspects of development (2-9). Moreover, HS-GAGs are abundant at the cell surface as part of the proteoglycan cell surface receptors (3, 4).Specialized microorganisms express GAG-degrading lyases serving nutritional purposes of both themselves and their vertebrat...
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