Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:4.2.2.7 (heparinase)
1,270 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Four hexasaccharides representing major structural sequences of heparin were isolated and characterized after degradation of heparin by heparinase. The structures were determined from two-dimensional 1H NMR spectroscopy including TOCSY (total correlated spectroscopy), COSY (correlated spectroscopy), and ROESY (rotating frame nuclear Overhauser enhancement spectroscopy) methods, providing new data on hexasaccharides. One of the hexasaccharides, the last eluting component from anion exchange chromatography, was derived from the tri-sulfated repeating disaccharide, alpha-L-idopyranosyluronic acid 2-sulfate-(1-->4)-2-amino-2-deoxy-D-glucopyranose 6,N-disulfate, and having the structure delta UAp2S-(1)-->4)-alpha-D-GlcNp2S6S-(1-->4)-alpha-L- IdoAp2S-(1-->4)-alpha-D-GlcNp2S6S-(1-->4)-alpha-L- IdoAp2S-(1-->4)-alpha-D-GlcNp2S6S. The second hexasaccharide contained a nonsulfated D-glucuronic acid unit instead of the L-iduronic acid adjacent to the reducing end, and having the structure delta UAp2S-(1-->4)-alpha-D-GlcNp2S6S-(1-->4)-alpha-L- IdoAp2S-(1-->4)-alpha-D-GlcNp2S6S-(1-->4)-beta-D- GlcAp-(1-->4)-alpha-D-GlcNp2S6S. The last two hexasaccharides were obtained in lower yield and they have not been isolated and characterized before. The structure of the third saccharide corresponded to a trimer of the repeating disaccharide except for the lack of a 6-O-sulfate group at the reducing end glucosamine residue; deltaUAp2S-(1-->4)-alpha-D-Glcnp2S6S-(1-->4)-alpha-L- IdoAp2S-(1-->4)-alpha-D-GlcNp2S6S-(1-->4)-alpha-L-IdoAp2S -(1-->4)-alpha- D-GlcNp2S. The fourth and last hexasaccharide were less sulfated and the following structure was established delta UAp2S-(1-->4)-alpha-D-GlcNp2S6S-(1-->4)-alpha-L- Idop2S-(1-->4)-alpha-D-GlcNp2S6S-(1-->4)-alpha-L- IdoAp-(1-->4)-alpha-D-GlcNpAc6S. Analysis of the ROESY spectra revealed conformational difference of the glucosidic linkage alpha-L-IdoAp-(1-->4)-alpha-D-GlcNp between the hexasaccharides and longer heparin chains.
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PMID:Isolation and characterization of hexasaccharides derived from heparin. Analysis by HPLC and elucidation of structure by 1H NMR. 769 49

Eleven tetrasaccharides were isolated from the repeating disaccharide region of porcine intestinal heparin after strong digestion with Flavobacterium heparinase. Their structures were determined by composition analysis, enzymatic analysis, and 1H NMR spectroscopy. Nine of them have the common tetrasaccharide backbone, delta HexA alpha 1-4GlcN alpha 1-4IdoA alpha 1-4GlcN, where delta HexA and IdoA represent 4,5-unsaturated hexuronic acid and L-iduronic acid, respectively, and their structural variations are based upon the positions of sulfate groups. The nine compounds include one hexasulfated, three pentasulfated and five tetrasulfated compounds, and four of them have not been isolated previously as discrete structures. The other two of the 11 tetrasaccharides have the following hitherto unreported structures with novel glucuronate 2-O-sulfate at the internal position: delta HexA(2-sulfate) alpha 1- 4GlcN(N,6-disulfate) alpha 1-4GlcA(2-sulfate) beta 1-4GlcN(N-sulfate) and delta HexA(2-sulfate) alpha 1-4GlcN(N,6-disulfate) alpha 1-4GlcA(2-sulfate) beta 1-4GlcN(N,6-disulfate). Thus, 2-O-sulfated glucuronate in the highly sulfated tetrasaccharide structures typical of heparin has been demonstrated. The former and the latter tetrasaccharides account for 0.31 and 0.32% (w/w) of the starting heparin, respectively. Their yield, however, is an underestimation, since these tetrasaccharide structures in longer sequences will be degraded by heparinase. Although the latter tetrasaccharide described above was unexpectedly cleaved by heparinase into two disaccharide units, the former was not degraded by the enzyme most likely due to the lack of the 6-O-sulfate group on the GlcN residue at the reducing terminus. The results indicate its capability of catalyzing both anti and syn elimination, a property shared by heparitinases I and II and chondroitinase ABC. Both tetrasaccharides were degraded into disaccharides by heparitinase II. Therefore, it is necessary to reevaluate the disaccharide composition of heparin/heparan sulfate or oligosaccharide structures, which were previously determined after heparinase or heparitinase II digestion. It is no longer possible to conclude that the 2-O-sulfated unsaturated uronic acid residues obtained from heparin/heparan sulfate by lyase digestions are always derived from iduronate 2-O-sulfate residues in the original polymer. It is quite possible that the novel glucuronate 2-O-sulfate structure in the highly sulfated region of heparin is involved in some of the biological activities of heparin.
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PMID:Isolation of the porcine heparin tetrasaccharides with glucuronate 2-O-sulfate. Heparinase cleaves glucuronate 2-O-sulfate-containing disaccharides in highly sulfated blocks in heparin. 772 74

Heparin, NAcHep, DS, and CS were labeled with deuterium by N-reacetylating, with the deuterated acetic anhydride (CD3CO)2O, GAGs previously N-deacetylated (by hydrazinolysis) to the desired extent. Degrees of deuteration of the present preparations, as determined by 2H- and 1H-NMR were 15%, 51%, 49%, and 79% for heparin, NAcHep, DS, and CS, respectively. The NMR analysis (including the 13C spectra) of the labeled products indicated that deuterium labeling did not involve any substantial modification of the GAG structures. Also NMR signals associated with specific sequences of heparin for antithrombin and of DS for heparin cofactor II were essentially the same in the unlabeled and in the deuterated GAGs. The substantial retention of the original structure was confirmed by data on the degree of sulfation (by conductimetry) and on the electrophoretic mobility in acid buffer. On the other hand, HPLC/SEC data indicated some depolymerization of heparin and DS in the N-deacetylation step of the labeling reactions. HPLC/MS spectrometry permitted a clear identification of disaccharide and tetrasaccharide fragments obtained from deuterated GAGs by enzymic (heparinase, chondroitinase ABC) or chemical depolymerization (deaminative cleavage, Smith degradation), opening new prospects for studies of human pharmacokinetics, with differentiation of exogenous from endogenous GAGs.
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PMID:Preparation and characterization of deuterium-labeled glycosaminoglycans. 799 88

We prepared a series of oligosaccharides from porcine intestinal heparin after extensive digestion with a mixture of Flavobacterium heparinase as well as heparitinases I and II. Previously, we reported the structures of the two glycoserines derived from the carbohydrate-protein linkage region [Sugahara et al., J. Biol. Chem., 267, 1528-1533 (1992)] and three tetrasaccharides derived from the antithrombin III-binding site [Yamada et al., J. Biol. Chem., 268, 4780-4787 (1993)]. In this study, we determined the structures of 10 other tetrasaccharides and a trisaccharide by enzymatic digestion, fast atom bombardment mass spectrometry and 500-MHz 1H NMR spectroscopy. These tetrasaccharides share the common disulphated structure, delta HexA alpha 1-4GlcN(N-sulphate)alpha 1-4IdoA(2-sulphate)alpha 1-4GlcN (where HexA is hexuronic acid and IdoA is L-iduronic acid), and their structural variations are based upon the positions of additional sulphate groups. Eight among the 10 have never been isolated as discrete structures. The structure of the trisaccharide is GlcN(N-sulphate)alpha 1-4IdoA(2-sulphate) alpha 1-4GlcN(N,6-disulphate) and is derived from the non-reducing terminus of heparin chains. This structure may represent the terminus of a biosynthetically formed native heparin chain or a newly formed non-reducing terminus exposed by a tissue endo-beta-glucuronidase which may be involved in the intracellular post-synthetic fragmentation of macromolecular heparin. The 11 structures characterized in the present study and 6 additional tetrasaccharides were used to investigate the substrate specificities of heparinase, as well as heparitinases I and II. The results indicate that modification of the adjacent glucosamine on the reducing side of the disaccharide cleavage site influences the enzymatic action of the lyases, whereas the adjacent uronic acid on the non-reducing side is not recognized by these enzymes.
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PMID:Structural studies on the tri- and tetrasaccharides isolated from porcine intestinal heparin and characterization of heparinase/heparitinases using them as substrates. 818 52

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.
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PMID:Structural studies on the bacterial lyase-resistant tetrasaccharides derived from the antithrombin III-binding site of porcine intestinal heparin. 844 55

Heparin is a polydisperse sulphated copolymer consisting mostly of 1-->4 linked glucosamine and uronic acid residues, i.e. 2-deoxy-2-sulphamido-D-glucopyranose 6-sulphate and L-idopyranosyluronic acid 2-sulphate. 13C NMR has been used to study the interactions of heparinase-derived and purified heparin disaccharide with N- and C-terminally-blocked tripeptides GRG and GKG. Titration of the disaccharide with peptide indicates that GRG binds the disaccharide more strongly than does GKG, with interactions in either case being stronger at uronate ring positions. In the presence of GRG, a carboxylate pKa depression suggests electrostatic interactions between the arginine guanidinium group and the uronate carboxylate group. 13C relaxation data have been acquired for all disaccharide and peptide carbons in the presence and absence of GRG and GKG. 13C relaxation rates for the disaccharide are significantly faster in the presence of peptide, especially with GRG. Analysis of these relaxation data has been done in terms of molecular diffusion constants, D [symbol: see text] and D parallel, and an angle alpha between D parallel and a molecular frame defined by the moment of inertia tensor calculated for an internally rigid disaccharide. Disaccharide conformational space in these calculations has been sampled for both uronate half-chair forms (2H1 and 1H2) and over a range of glycosidic bond angles defined by motional order parameters and inter-residue nuclear Overhauser effects (+/- 30 degree from the average). In the absence of peptide, the ratio D [symbol: see text] /D parallel falls between 0.4 and 0.7; therefore molecular diffusion occurs preferentially about D parallel, which runs through both disaccharide rings. In the presence of peptide, D [symbol: see text] /D parallel is decreased, indicating that GRG is oriented along D parallel and proximal to the uronic acid ring. A model for this is shown.
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PMID:13C-NMR relation study of heparin-disaccharide interactions with tripeptides GRG and GKG. 861 13

Porcine intestinal heparin was extensively digested with Flavobacterium heparinase and size-fractionated by gel chromatography. Subfractionation of the hexasaccharide fraction by anion exchange high pressure liquid chromatography yielded 10 fractions. Six contained oligosaccharides derived from the repeating disaccharide region, whereas four contained glycoserines from the glycosaminoglycan-protein linkage region. The latter structures were reported recently (Sugahara, K., Tsuda, H., Yoshida, K., Yamada, S., de Beer, T., and Vliegenthart, J.F.G. (1995) J. Biol. Chem. 270, 22914-22923). In this study, the structures of one tetra- and five hexasaccharides from the repeat region were determined by chemical and enzymatic analyses as well as 500-MHz 1H NMR spectroscopy. The tetrasaccharide has the hexasulfated structure typical of heparin. The five hexa- or heptasulfated hexasaccharides share the common core pentasulfated structure delta HexA(2S) alpha 1-4GlcN-(NS, 6S) alpha 1-4IdoA alpha/GlcA beta 1-4GlcN(6S) alpha 1-4GlcA beta 1-4GlcN (NS) with one or two additional sulfate groups (delta HexA, GlcN, IdoA, and GlcA represent 4-deoxy-alpha-L-threo-hex-4-enepyranosyluronic acid, D-glucosamine, L-iduronic acid, and D-glucuronic acid, whereas 2S, 6S and NS stand for 2-O-, 6-O-, and 2-N-sulfate, respectively). Three components have the following hitherto unreported structures: delta HexA(2S) alpha 1-4GlcN(NS, 6S) alpha 1-4GlcA beta 1-4GlcN(NS, 6S) alpha 1-4GlcA beta 1-4GlcN(NS,6S), delta HexA(2S) alpha 1-4GlcN(NS, 6S) alpha 1-4IdoA alpha 1-4GlcNAc(6S)-alpha 1-4GlcA beta 1-4GlcN(NS, 3S), and delta HexA(2S) alpha 1-4GlcN-(NS,6S) alpha 1-4IdoA (2S) alpha 1-4GlcNAc(6S) alpha 1-4GlcA beta 1-4GlcN(NS, 6S). Two of the five hexasaccharides are structural variants derived from the antithrombin III-binding sites containing 3-O-sulfated GlcN at the reducing termini with or without a 6-O-sulfate group on the reducing N,3-disulfated GlcN residue. Another contains the structure identical to that of the above heptasulfated antithrombin III-binding site fragment but lacks the 3-O-sulfate group and therefore is a pro-form for the binding site. Another has an extra sulfate group on the internal IdoA residue of this pro-form and therefore can be considered to have diverged from the binding site in the biosynthetic pathway. Thus, the isolated hexasacharides in this study include the three overlapping pairs of structural variants with an apparent biosynthetic precursor-product relationship, which may reflect biosynthetic regulatory mechanisms of the binding site.
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PMID:Structures of five sulfated hexasaccharides prepared from porcine intestinal heparin using bacterial heparinase. Structural variants with apparent biosynthetic precursor-product relationships for the antithrombin III-binding site. 863 46

The versatile biological activities of proteoglycans are mainly mediated by their glycosaminoglycan (GAG) components. Unlike proteins and nucleic acids, no satisfactory method for sequencing GAGs has been developed. This paper describes a strategy to sequence the GAG chains of heparin. Heparin, prepared from animal tissue, and processed by proteinases and endoglucuronidases, is 90% GAG heparin and 10% peptidoglycan heparin (containing small remnants of core protein). Raw porcine mucosal heparin was labelled on the amino termini of these core protein remnants with a hydrophobic, fluorescent tag [N-4-(6-dimethylamino-2-benzofuranyl) phenyl (NDBP)-isothiocyanate]. Enrichment of the NDBP-heparin using phenyl-Sepharose chromatography, followed by treatment with a mixture of heparin lyase I and III, resulted in a single NDBP-linkage region tetrasaccharide, which was characterized as deltaUAp(1-->3)-beta-D-Galp(1-->3)-beta-D-Galp(1-->4)-beta-Xylp -(1-->O-Ser-NDBP (deltaUAp is 4-deoxy-alpha-L-threo-hex-4-enopyranosyl uronic acid). Several NDBP-octasaccharides were isolated when NDBP-heparin was treated with only heparin lyase I. The structure of one of these NDBP-octasaccharides, deltaUAp2S(1-->4)-alpha-D-GlcNpAc(1-->4)-alpha-L-IdoAp (1-->4)-alpha-D-GlcNpAc6S(1-->4)-beta-D-GlcAp(1-->3)-beta-D- Galp(1-->3)-beta-D-Galp(1-->4)-beta-Xylp-(1-->O-Ser NDBP (S is sulphate, Ac is acetate), was determined by 1H-NMR and enzymatic methods. Enriched NDBP-heparin was treated with lithium hydroxide to release heparin, and the GAG chain was then labelled at xylose with 7-amino-1,3-naphthalene disulphonic acid (AGA). The resulting AGA-Xyl-heparin was sequenced on gradient PAGE using heparin lyase I and heparin lyase III. A predominant sequence in heparin at the protein core attachment site was deduced to be -D-GlcNp2S6S(or 6OH)(1-->4)-alpha-L-IdoAp2S-(1-->4)-alpha-D-GlcNp2S6S (or60H) (1-->4)-alpha-L-IdoAp2S(1-->4)-alpha-D-GlcNp2S6S( or 6OH)(1-->4)-alpha-L-IdoAp2S(1-->4)-alpha-D-GlcNpAc (1- ->4)-alpha-L-IdoAp(1-->4)-alpha-D-GlcNpAc6S(1-->4)-beta-D-++ +GlcAp(1-->3)-beta-D-Galp(1-->3)-beta-D-Galp(1-->4)-beta-Xyl-AGA.
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PMID:Strategy for the sequence analysis of heparin. 872 74

Two new oligosaccharides were prepared from heparin by its partial depolymerization using heparin lyase I (EC 4.2.2.7) in an attempt to prepare oligosaccharides having intact antithrombin III binding sites. The oligosaccharides were purified by chromatography on the basis of both size and charge and demonstrated a high level of purity by capillary electrophoresis. One- and two-dimensional 1H NMR spectroscopy at 500 MHz revealed the structure of each oligosaccharide. The octasaccharide and decasaccharide are DeltaUAp2S(1-->4)-alpha-DGlcNpS6S(1-->4)-alpha-L-IdoAp (1-->4)-alpha-D -GlcNpAc6S(1-->4)-betaD-GlcAp(1-->4)-alpha-D-GlcNpS 3S6S(1-->4)-alpha- L-IdoAp2S(1-->4)alpha-D-GlcNpS6S (where DeltaUAp is 4-deoxy-alpha-L-threo-hex-enopyranosyluronic acid, GlcNp is 2-amino-2-deoxy-glucopyranose, GlcAp is glucopyranosyluronic acid, S is sulfate and Ac is acetate) and DeltaUAp2S(1-->4)-alpha-D-GlcNpS6S(1-->4)-alpha-L-IdoAp++ +(1-->4)-alpha- D-GlcNpAc6S (1-->4)-beta-D-GlcAp(1-->4)-alpha-D-GlcNpS3S6S(1-->4)-alpha- L-IdoAp2S (1-->4)-alpha-D-GlcNpS6S(1-->4)-alpha-L-IdoAp2S(1-->4)-alpha -D-GlcNpS 6S, respectively. A hexasaccharide containing a similar structural motif to that found in the antithrombin III binding site and having greatly reduced anticoagulant activity was also isolated. The structure of the hexasaccharide is DeltaUAp2S(1-->4)-alpha-D-GlcNpAc6S(1-->4)-beta-D-GlcAp++ +(1-->4)-alpha- D-GlcNpS3S6S(1-->4)-alpha-L-IdoAp(1-->4)-alpha-D-GlcNpS6S . The octasaccharide and decasaccharide correspond to the predominant structural motif found in porcine intestinal mucosal heparin. Sufficient quantities of the decasaccharide were obtained to examine its interaction with antithrombin III using microtitration calorimetry. This decasaccharide bound to antithrombin III with similar avidity as heparin and showed comparable anticoagulant activity, as determined using an antithrombin III dependent anti-factor Xa assay. Interestingly, while both decasaccharide and heparin bound to antithrombin with nanomolar affinity, very little heat of binding was observed.
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PMID:Enzymatic preparation of heparin oligosaccharides containing antithrombin III binding sites. 894 54

This study presents a comparison of heparan sulphate chains isolated from various porcine and bovine tissues. 1H-NMR spectroscopy (500 MHz) was applied for structural and compositional studies on intact heparan sulphate chains. After enzymic digestion of heparan sulphate using heparin lyase I (EC 4.2.2.7) II and III (EC 4.2.2.8), the compositions of unsaturated disaccharides obtained were determined by analytical capillary electrophoresis. Correlations between the N-sulphated glucosamine residues and O-sulphation and between iduronic acid content and total sulphation were discovered using the data obtained by NMR and disaccharide analysis. Heparan sulphate chains could be classified into two groups based on the sulphation degree and the iduronic acid content. Heparan sulphate chains with a high degree of sulphation possessed also a significant number of iduronic acid residues and were isolated exclusively from porcine brain, liver and kidney medulla. The presence and amount of N-unsubstituted glucosamine residues (GlcNp) was established in all of the heparan sulphates examined. The structural context in which this residue occurs was demonstrated to be: high sulphation domain --> 4)-beta-D-GlcAp-(1 --> 4)-alpha-D-GlcNp-(1 --> 4)-beta-D-GlcAp-(1 --> low sulphation domain (where GlcNp is 2-amino-2-deoxyglucopyranose, and GlcAp is glucopyranosyluronic acid), based on the isolation and characterization of a novel, heparin lyase III-derived, GlcNp containing tetrasaccharide and hexasaccharide. The results presented suggest that structural differences may play a role in important biological events controlled by heparan sulphate in different tissues.
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PMID:Structural differences and the presence of unsubstituted amino groups in heparan sulphates from different tissues and species. 906 69


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