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)

Heparin, in a concentration-dependent manner, inhibited the generation of conjugated dienes and thiobarbituric acid-positive substances when incubated with Fe2+ and gamma-linolenic acid. In the conjugated diene assay, other glycosaminoglycans, on a molar basis calculated with respect to their respective hydrated disaccharide repeat units, were less effective than heparin. Heparin which had been re-N-sulphonated after removal of both N-sulphonates and O-sulphates, and heparin in which iduronate residues had been reduced to idose residues, were largely unaffected in their activity. Removal of both N-sulphonates and O-sulphates greatly reduced the effectiveness of the heparin. Analysis of the effects of heparin fragments generated by heparinase I treatment of heparin showed that depolymerization decreased the effectiveness of the heparin. It is possible that heparins and related strongly acidic polysaccharides may function as endogenous antioxidants, and that sequestration by them, or harmless oxidation by them, of ions such as Fe2+, contributes to their effectiveness.
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PMID:Inhibition by heparin of Fe(II)-catalysed free-radical peroxidation of linolenic acid. 141 30

Two tetrasaccharides, two hexasaccharides, and a disaccharide have been purified from heparinase digests of porcine intestinal mucosal heparin in sufficient quantities to permit 13C-n.m.r. characterization of the species. The two tetrasaccharides are the sulfated iduronic acid-containing 4en-HexpA2SO3-(1----4)-alpha-D-GlcpNSO3;6SO3-(1- ---4)-alpha-L- IdopA2SO3-(1----4)-D-GlcpNSO3;6SO3 and the non-sulfated glucuronic acid-containing 4en-HexpA2SO3-(1----4)-alpha-D-GlcpNSO3;6SO3-(1- ---4)-beta-D-GlcpA-(1----4)-D- GlcpNSO3;6SO3. The two hexasaccharides are related to the two tetrasaccharides by the insertion of alpha-linked L-IdopA2SO3-(1----4)-D-GlcpNSO3;6SO3 after the non-reducing end sulfated glucosamine residue. The disaccharide is 4en-HexpA2SO3-(1----4)-alpha-D-GlcpNSO3;6SO3. The disaccharide, together with each of the iduronate-containing oligosaccharides, form one series of related di-, tetra-, and hexa-saccharides, while the disaccharide together with the glucuronate-containing oligosaccharides form a second series. Using inverse detection as a means of increasing sensitivity, two-dimensional n.m.r. 13C-1H heterocorrelation spectra have been obtained for all five oligosaccharides. The use of two-dimensional heterocorrelation n.m.r. spectroscopy offers a much less ambiguous means of making 13C resonance assignments than do traditional one-dimensional methods, while the use of inverse detection gives both greater sensitivity than direct detection, as well as values for the one-bond 13C-1H coupling constants. From a knowledge of the assignments of resonances in the 1H spectra of these species, it has been possible to assign almost all of the 13C resonances of these five oligosaccharides. Some corrections to previously published assignments for the tetrasaccharides have been made. In addition, one-bond 13C-1H coupling constant data have been obtained for all of the anomeric protons.
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PMID:One- and two-dimensional 13C-n.m.r. characterization of two series of oligosaccharides derived from porcine intestinal mucosal heparin by degradation with heparinase. 159 34

The molecular structure of human skin fibroblast heparan sulphate was examined by specific chemical or enzymic depolymerization and high-resolution separation of the resulting oligosaccharides and disaccharides. Important features of the molecular organization, disaccharide composition and O-sulphate disposition of this heparan sulphate were identified. Analysis of the products of HNO2 hydrolysis revealed a polymer in which 53% of disaccharide units were N-acetylated and 47% N-sulphated, with an N-/O-sulphate ratio of 1.8:1. These two types of disaccharide unit were mainly located in separate domains. Heparitinase and heparinase scission indicated that the iduronate residues (37% of total hexuronate) were largely present in contiguous disaccharide sequences of variable size that also contained the majority of the N-sulphate groups. Most of the iduronate residues (approx. 70%) were non-sulphated. About 8-10% of disaccharide units were cleaved by heparinase, but only a minority of these originated from contiguous sequences in the intact polymer. Trisulphated disaccharide units [alpha-N-sulpho-6-sulphoglucosaminyl-(1----4)-iduronate 2-sulphate], which are the major structural units in heparin, made up only 3% of the disaccharide units in heparan sulphate. O-Sulphate groups (approx. 26 per 100 disaccharide units) were distributed almost evenly among C-6 of N-acetylglucosamine, C-2 of iduronate and C-6 of N-sulphated glucosamine residues. The results indicate that the sulphated regions of heparan sulphate have distinctive and potentially variable structural characteristics. The high content of non-sulphated iduronate in this heparan sulphate species suggests a conformational versatility that could have important implications for the biological properties of the polymer.
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PMID:Molecular organization of heparan sulphate from human skin fibroblasts. 213 90

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

The distribution and structure of heparan sulphate (HS) synthesised by bovine aortic endothelial cells (BAEC) has been studied. Confluent cultures were harvested and analysed as three separate compartments: (a) the culture medium, (b) the detergent-soluble cell-associated material and (c) the detergent-insoluble matrix material extracted with 6 M urea. HS was present in all three of the culture compartments, but the molecular size of the HS proteoglycans (PG) and the free polysaccharide chains varied according to compartment origin. The matrix pool accounted for almost 50% of the total HS which was present as a large HSPG possessing polysaccharide chains of 79 kDa. When studied in more detail, these large HS chains displayed an N-sulphate content and distribution (determined by low pH nitrous acid treatment) similar to that seen in the majority of other mammalian heparan sulphates. Extended iduronate sequences were also identified (i.e., heparitinase-resistant sequences); however, apart from these regions, the degree of O-sulphation was relatively low. In addition, the presence of heparin-like sequences (GlcNSO3(+/- 6S)-IdoA(2S)), characterised by heparinase sensitivity, accounted for only 5% of the disaccharides and such sequences appeared to be located with an ordered distribution, mainly in relatively short sulphated domains within the intact molecule. Given the strategic location of the large matrix-associated HSPG within the BAEC system studied, it is conceivable that the HS structure may be important in a number of functions such as cell attachment processes and/or the binding of growth factors.
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PMID:Molecular structure of heparan sulphate synthesised by bovine aortic endothelial cells. 776 44

Heparan sulphate (HS) is an abundant polysaccharide component of the pericellular domain and is found in most soft tissues and all adherent cells in culture. It interacts with a wide spectrum of proteins including polypeptide growth factors and glycoproteins of the extracellular matrix. These interactions might influence fundamental cellular activities such as adhesion, growth and migration. HS might therefore represent a highly adaptive mechanism by which cells respond to their environment. The present study shows that the interaction between fibroblast HS, metabolically labelled with [3H]glucosamine, and the C-terminal heparin-binding domain of human plasma fibronectin (HEPII), is determined by distinct regions of the polysaccharide chain. By using a very sensitive affinity-chromatography method and specific polysaccharide scission it was shown that the HEPII-binding regions of HS reside within sulphated domains that are resistant to degradation by heparinase III. In addition, optimal binding was achieved with specific heparinase III-resistant fragments of 14-16 monosaccharides in length. The affinity of HS for HEPII was significantly decreased when the polysaccharide was cleaved with heparinase I. Chondroitin sulphate and dermatan sulphate were poor competitive inhibitors of [3H]HS binding to HEPII whereas unlabelled HS and heparin gave a strong inhibitory activity, with heparin being the most potent inhibitor. These findings suggest that the interaction between HEPII and HS is specific and requires extended sequences of seven to eight N-sulphated disaccharides in which a proportion of the iduronate residues are sulphated at C-2. The results have important implications for the functions of HS in cell adhesion and migration.
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PMID:Structural domains of heparan sulphate for specific recognition of the C-terminal heparin-binding domain of human plasma fibronectin (HEPII). 876 Mar 76

Skin fibroblasts treated with brefeldin A produce a recycling variant of glypican (a glycosylphosphatidylinositolanchored heparan-sulfate proteoglycan) that is resistant to inositol-specific phospholipase C and incorporates sulfate and glucosamine into heparan sulfate chains (Fransson, L.-A. et al., Glycobiology, 5, 407-415, 1995). We have now investigated structural modifications of recycling glypican, such as fatty acylation from [3H]palmitate, and degradation and assembly of heparan sulfate side chains. Most of the 3H-radioactivity was recovered as lipid-like material after de-esterification. To distinguish between formation of heparan sulfate at vacant sites, elongation of existing chains or degradation followed by re-elongation of chain remnants, cells were pulse-labeled with [3H]glucosamine and then chase-labeled with [14C]glucosamine. Material isolated from the cells during the chase consisted of proteoglycan and mostly [3H]-labeled heparan-sulfate degradation products (molecular mass, 20-80 kDa) showing that the side chains were degraded during recycling. The degradation products were initially glucuronate-rich, but became more iduronate-rich with time. The glypican proteoglycan formed during the chase was degraded either with alkali to release intact side chains or with heparinase to generate distally located chain fragments that were separated from the core protein, containing the proximally located, covalently attached chain remnants. All of the [14C]-radioactivity incorporated during the pulse was found in peripheral chain fragments, and the chains formed were not significantly longer than the original ones. We therefore conclude that newly made heparan-sulfate chains were neither made on vacant sites, nor by extension of existing chains but rather by re-elongation of degraded chain remnants. The remodeled chains made during recycling appeared to be more extensively modified than the original ones.
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PMID:Glypican (heparan sulfate proteoglycan) is palmitoylated, deglycanated and reglycanated during recycling in skin fibroblasts. 906 69

Platelet factor 4 is a tetrameric heparin binding chemokine released from the alpha-granules of activated platelets. In this study we show that platelet factor 4 binds with high affinity and specificity to an approximately 9-kDa sequence in heparan sulfate, which it protects from degradation by heparinase enzymes. This protected fragment is enriched in N-sulfated disaccharides and iduronate 2-O-sulfate residues, the latter being important for binding to platelet factor 4. The major structural motif of the fragment appears to consist of a pair of sulfated domains positioned at both ends separated by a central mainly N-acetylated region. On the basis of these findings, we propose a model in which the heparan sulfate fragment wraps around the ring of positive charges on platelet factor 4 with the iduronate 2-O-sulfates within the sulfated domains binding strongly to lysine clusters on opposite faces of the tetramer.
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PMID:Specific binding of the chemokine platelet factor 4 to heparan sulfate. 925 63

Heparin-like glycosaminoglycans, acidic complex polysaccharides present on cell surfaces and in the extracellular matrix, regulate important physiological processes such as anticoagulation and angiogenesis. Heparin-like glycosaminoglycan degrading enzymes or heparinases are powerful tools that have enabled the elucidation of important biological properties of heparin-like glycosaminoglycans in vitro and in vivo. With an overall goal of developing an approach to sequence heparin-like glycosaminoglycans using the heparinases, we recently have elaborated a mass spectrometry methodology to elucidate the mechanism of depolymerization of heparin-like glycosaminoglycans by heparinase I. In this study, we investigate the mechanism of depolymerization of heparin-like glycosaminoglycans by heparinase II, which possesses the broadest known substrate specificity of the heparinases. We show here that heparinase II cleaves heparin-like glycosaminoglycans endolytically in a nonrandom manner. In addition, we show that heparinase II has two distinct active sites and provide evidence that one of the active sites is heparinase I-like, cleaving at hexosamine-sulfated iduronate linkages, whereas the other is presumably heparinase III-like, cleaving at hexosamine-glucuronate linkages. Elucidation of the mechanism of depolymerization of heparin-like glycosaminoglycans by the heparinases and mutant heparinases could pave the way to the development of much needed methods to sequence heparin-like glycosaminoglycans.
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PMID:Mass spectrometric evidence for the enzymatic mechanism of the depolymerization of heparin-like glycosaminoglycans by heparinase II. 977 Apr 69

The capsular polysaccharide from E. Coli, strain K5 composed of ...-->4)beta-D-GlcA(1-->4)alpha-D-GlcNAc(1-->4)beta-D-GlcA (1-->..., chemically modified K5 polysaccharides, bearing sulfates at C-2 and C-6 of the hexosamine moiety and at the C-2 of the glucuronic acid residues as well as 2-O desulfated heparin were used as substrates to study the specificity of heparitinases I and II and heparinase from Flavobacterium heparinum. The natural K5 polysaccharide was susceptible only to heparitinase I forming deltaU-GlcNAc. N-deacetylated, N-sulfated K5 became susceptible to both heparitinases I and II producing deltaU-GlcNS. The K5 polysaccharides containing sulfate at the C-2 and C-6 positions of the hexosamine moiety and C-2 position of the glucuronic acid residues were susceptible only to heparitinase II producing deltaU-GlcNS,6S and deltaU,2S-GlcNS,6S respectively. These combined results led to the conclusion that the sulfate at C-6 position of the glucosamine is impeditive for the action of heparitinase I and that heparitinase II requires at least a C-2 or a C-6 sulfate in the glucosamine residues of the substrate for its activity. Iduronic acid-2-O-desulfated heparin was susceptible only to heparitinase II producing deltaU-GlcNS,6S. All the modified K5 polysaccharides as well as the desulfated heparin were not substrates for heparinase. This led to the conclusion that heparitinase II acts upon linkages containing non-sulfated iduronic acid residues and that heparinase requires C-2 sulfated iduronic acid residues for its activity.
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PMID:New insights on the specificity of heparin and heparan sulfate lyases from Flavobacterium heparinum revealed by the use of synthetic derivatives of K5 polysaccharide from E. coli and 2-O-desulfated heparin. 1057 95


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