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)

A human endometrial adenocarcinoma cell line (Ishikawa) has been shown to incorporate [3H]glucosamine and to secrete a radiolabeled high molecular weight compound which is excluded from a Sepharose CL-2B column. The excluded material was resistant to hyaluronidase, chondroitinase ABC, and heparinase. These findings rule out the possibility of this material being a proteoglycan. The susceptibility of this material to digestion with pronase, neuraminidase, and alkaline borohydride treatment strongly suggests that the excluded material is an O-glycosidic glycoprotein. The glycoprotein secreted by Ishikawa cells (ICGP) did not react immunologically with antibodies against either lactoferrin or fibronectin, but did react with an antibody made against tracheal mucin. Conversely, immunoblot analysis revealed that an antibody made against ICGP did not recognize hyaluronic acid, chondroitin, heparin, nasal turbinate mucin, bovine submaxillary gland mucin, lactoferrin, or fibronectin, but did recognize tracheal mucin. Analysis of ICGP amino acid and carbohydrate composition showed that it is rich in serine, threonine, glutamic acid, aspartic acid, and N-acetylneuraminic acid. In this respect, ICGP differs from other mucins, even though it is immunologically similar to respiratory mucin; hence we may consider ICGP to be a mucin-like glycoprotein. Secretion of ICGP can be modulated by Ca(2+)-ionophore and other mucus secretagogues, such as platelet activating factor, carbachol, and monocyte/macrophage mucus secretagogue, all mediators of lung inflammation. Ishikawa cells and anti-ICGP antibody may be used in studies on in vitro regulation of mucin-like glycoprotein synthesis and secretion in the respiratory tract as well as in the endometrium.
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PMID:Characterization of a unique mucin-like glycoprotein secreted by a human endometrial adenocarcinoma cell line (Ishikawa). 818 54

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

In a recent study (D. J. Culp, D. K. P. Lee, D. P. Penney, and M. G. Marin. Am. J. Physiol. 263: L264-275, 1992), we reported that primary cultures of cat tracheal gland cells expressed histological, ultrastructural, and immunological characteristics of mucous cells when cultured on floating gels of rat tail collagen (released-gel cultures) compared with cells cultured on glutaraldehyde-fixed collagen gels (fixed-gel cultures). We therefore collected culture medium from gland cells grown under both culture conditions for determination and comparison of glycoconjugates with characteristics of mucin glycoproteins. Cells were cultured in the presence of [3H]glucosamine, and material of high molecular weight and density (HMD material) was isolated. HMD material from both culture conditions were each resistant to heparitinase and heparinase, whereas 72 and 25% of the radiolabel in released-gel and fixed-gel HMD material, respectively, was resistant to chondroitinase ABC. Material resistant to chondroitinase ABC was analyzed further. Both samples contained a single broad glycoprotein band [relative molecular weight (M(r)) > 250,000] after sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and had amino acid profiles similar to airway mucin. The sample from fixed-gel cultures had nearly equal amounts of carbohydrate and protein, was highly enriched in N-acetylglucosamine, contained mannose, displayed little blood group A immunoreactivity, and had few O-linked oligosaccharides. Conversely, the sample from released-gel cultures contained 80% carbohydrate, was composed of monosaccharides characteristic of airway mucins, displayed blood group A immunoreactivity, and contained oligosaccharides O-linked via N-acetylgalactosamine.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Mucinlike glycoproteins from cat tracheal gland cells in primary culture. 821 86

The application of capillary electrophoresis to total compositional analysis of heparin and low-molecular-weight heparin samples has been studied. Optimum resolution of 17 defined oligosaccharides was obtained with the buffer system composed of 10 mM sodium borate and 50 mM sodium dodecyl sulfate at pH 8.81 and at a constant voltage of 20 kV. The ratio of oligosaccharide charge to the number of saccharide residues correlated with the migration time. For oligosaccharides having the same charge to saccharide ratio, the larger of the oligosaccharides eluted earlier. A hexasaccharide having a 3-O-sulfated glucosamine residue at the reducing end and arising from heparin's antithrombin III binding site, migrated in an unusual fashion. The limit of oligosaccharide detection was from 600 fmol to 1 pmol. Quantitative analysis could conveniently be performed on 10 pmol of an oligosaccharide sample. Oligosaccharide composition using capillary electrophoresis was obtained by nearly complete depolymerization of heparins with a mixture of heparin lyase I, II, and III. The analysis resulted in 95% mass balance for both heparin and low-molecular-weight heparin. Capillary electropherograms of heparin and different low-molecular-weight heparins depolymerized with heparin lyase I alone showed a high level of structural heterogeneity in the products formed. The oligosaccharide maps thus obtained might find use in fingerprinting the heparin and low-molecular-weight samples.
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PMID:Oligosaccharide composition of heparin and low-molecular-weight heparins by capillary electrophoresis. 823 64

In a previous study, we showed that heparitinase releases a 14-saccharide sequence (Oligo-H) from heparan sulfate (HS) with the structure delta GlcUA beta 1,4GlcNSO3-alpha 1,4[IdceA(2S)alpha 1,4GlcNSO3]5 alpha 1,4IdceA alpha 1,4GlcNAc (where IdceA(2S) represents iduronic acid 2-sulfate), which binds to basic fibroblast growth factor (bFGF) with high affinity (Turnbull, J. E., Fernig, D., Ke, Y., Wilkinson, M. C. & Gallagher, J. T. (1992) J. Biol. Chem. 267, 10337-10341). This paper describes further work on the binding properties of HS saccharides and their capacity to mediate bFGF activity in a mitogenesis assay in which responsiveness is dependent on the addition of HS or heparin. Saccharides prepared by heparinase or nitrous acid digestion and heparitinase-resistant fragments five disaccharide units (degree of polymerization (dp) = 10) or less in size were unable to activate bFGF. However, heparitinase-resistant saccharides of dp12-16 were active in the assay; the dp14 and dp16 fractions were equivalent in activity to heparin and more active than the parent HS. Saccharides of the same size and basic structure as the active fractions (> or = dp12) bound to bFGF with high relative affinity. Active saccharides were composed mainly of N-sulfated disaccharides, the predominant unit being IdceA(2S)-GlcNSO3. This was enriched at least 5-fold in the active saccharides by comparison with the original HS. In addition, the dp12 and dp14 active fractions had a notably low content of trisulfated disaccharides (IdceA(2S)-GlcNSO3(6S)) (where GlcNSO3(6S) represents N-sulfated glucosamine 6-sulfate), which are the major repeat units of heparin. The data show that sequences similar in size and basic structure to Oligo-H can mediate the mitogenic activity of bFGF. Overall, the results provide further evidence that specific HS sequences are generated biosynthetically in order to fulfill particular biological functions such as activation of bFGF.
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PMID:Specific heparan sulfate saccharides mediate the activity of basic fibroblast growth factor. 828 46

Previous studies have suggested that mucin gene expression is tissue-specific; however, the relationship between unique mucin gene products and the biochemical properties of mucins is unknown. The purpose of this study was to determine the biochemical and molecular characteristics of mucin synthesized by adenocarcinoma cell lines derived from breast (ZR-75-1), stomach (MGC-803), pancreas (Capan-2), and lung (Chago K-1). Mucin was quantitated by [3H]glucosamine labeling and Sepharose CL-4B chromatography. The mucinous nature of the labeled high molecular weight glycoproteins (HMG) was verified by alkaline borohydride treatment, cesium chloride density gradient ultracentrifugation, and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Specific mucin gene expression was determined using cDNA probes for 2 distinct intestinal mucins (MUC-2 and MUC-3) and one breast cancer mucin (MUC-1). Specific core mucin proteins were confirmed by immunoblots using antibodies that recognize MUC-1, MUC-2, and MUC-3 core peptides. These experiments demonstrate that all cell lines contained HMG in the medium, cytosol, and membrane fractions. The HMG was mucinous in breast, pancreatic, and lung cell lines. In contrast, most of the HMG secreted by the gastric cell line was proteoglycan-like, due to its susceptibility to hyaluronidase, heparinase, and chondroitinase avidin-biotin complex. Ion-exchange (DEAE-Sephacel) chromatography of [3H]glucosamine-labeled HMG demonstrated that the acidic or basic nature of the mucin was different in all cancer cell lines tested. Despite these differences, mRNA and immunoblot analysis suggest that all cell lines predominantly express MUC-1 apomucin, small amounts of MUC-2 apomucin, and no MUC-3. Immunoprecipitation of MUC-1-type mucin using the 139H2 monoclonal antibody demonstrated that different sizes of mucin peptides were present in all cell lines, corresponding to the known length polymorphism of this mucin. The amount and nature of carbohydrate epitopes were analyzed by immunoblots using anti-T (peanut lectin), anti-Tn (91S8 monoclonal antibody), and anti-sialosyl Tn (JT10e monoclonal antibody). T and Tn antigens were significantly higher in breast and pancreatic cells as compared with lung and gastric cell lines. These findings correlated with increased activities of polypeptidyl N-acetylgalactosaminyl transferase and beta-1,3-galactosyltransferase.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:Mucin synthesis and secretion in various human epithelial cancer cell lines that express the MUC-1 mucin gene. 844 22

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

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

A rapid, sensitive and accurate high-performance capillary electrophoresis method is described for the determination of the sulfation pattern of heparin and heparan sulfate disaccharides. The analysis, performed after enzymic degradation of the polysaccharides with heparinase and heparinases II and III in combination, yields highly UV-absorbing delta-disaccharides. The separation is performed with reversed polarity using 15 mM phosphate buffer, pH 3.50. This method is superior to others since all known 12 disaccharides carrying N-acetylated, N-sulfated or unsubstituent glucosamine can be separated in a single run of 15 min. At the highest sensitivity the analysis consumes only a few femtograms of glycosaminoglycan and allows a determination of delta-disaccharides at the attomole level.
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PMID:High performance capillary electrophoresis method to characterize heparin and heparan sulfate disaccharides. 890 Sep 48

Heparinases I, II and III from F. heparinum cleave heparin-like molecules, with a high degree of substrate specificity, at the glucosamine-uronate linkage by elimination, leaving an unsaturated C4-C5 bond in the uronic acid. The primary sequences of these enzymes have been reported earlier. In this study we perform a comparative analysis of the properties and primary sequences of heparinase I, II and III. Alignment of the primary sequences revealed little sequence homology (15% residue identity in a LALIGN alignment) at both DNA and amino acid levels. There are three basic clusters in heparinase II satisfying the heparin binding consensus sequence with one of the sequences sharing homology with a consensus sequence in the heparin binding site of heparinase I and two basic clusters in heparinase III. Similar to heparinase I, there are two putative 'EF-hand' calcium coordinating motifs in heparinase III, while heparinase II does not contain any such motifs. Recombinant heparinases II and III's degradation of the substrate and the subsequent separation of the oligosaccharide products by POROS anion exchange chromatography were identical to those obtained from native heparinases II and III from F. heparinum.
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PMID:A comparative analysis of the primary sequences and characteristics of heparinases I, II, and III from Flavobacterium heparinum. 895 71


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