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)

Cyclophilin B (CyPB) is a heparin-binding protein first identified as a receptor for cyclosporin A. In previous studies, we reported that CyPB triggers chemotaxis and integrin-mediated adhesion of T-lymphocytes by way of interaction with two types of binding sites. The first site corresponds to a signalling receptor; the second site has been identified as heparan sulphate (HS) and appears crucial to induce cell adhesion. Characterization of the HS-binding unit is critical to understand the requirement of HS in pro-adhesive activity of CyPB. By using a strategy based on gel mobility shift assays with fluorophore-labelled oligosaccharides, we demonstrated that the minimal heparin unit required for efficient binding of CyPB is an octasaccharide. The mutants CyPB(KKK-) [where KKK- refers to the substitutions K3A(Lys3-->Ala)/K4A/K5A] and CyPB(DeltaYFD) (where Tyr14-Phe-Asp16 has been deleted) failed to interact with octasaccharides, confirming that the Y14FD16 and K3KK5 clusters are required for CyPB binding. Molecular modelling revealed that both clusters are spatially arranged so that they may act synergistically to form a binding site for the octasaccharide. We then demonstrated that heparin-derived octasaccharides and higher degree of polymerization oligosaccharides inhibited the interaction between CyPB and fluorophore-labelled HS chains purified from T-lymphocytes, and strongly reduced the HS-dependent pro-adhesive activity of CyPB. However, oligosaccharides or heparin were unable to restore adhesion of heparinase-treated T-lymphocytes, indicating that HS has to be present on the cell membrane to support the pro-adhesive activity of CyPB. Altogether, these results demonstrate that the octasaccharide is likely to be the minimal length unit required for efficient binding of CyPB to cell surface HS and consequent HS-dependent cell responses.
Biochem J 2004 Sep 01
PMID:Octasaccharide is the minimal length unit required for efficient binding of cyclophilin B to heparin and cell surface heparan sulphate. 1510 1

C(18) and C(8) bonded stationary phases dynamically coated with cetyltrimethylammonium (CTA) and strong anion exchange (SAX) were developed to obtain separations of oligosaccharide mixtures resulting from chemical or enzymatic depolymerization of heparin. With this method, the retention of sulfated oligosaccharides is directly adjustable depending on the amount of CTA adsorbed into the column. Oligosaccharides containing up to 20 sulfates were separated with a resolving power superior to that of conventional SAX analysis. The stability of the column coating enables hundreds of injections. Using ammonium methane sulfonate aqueous solutions as ultraviolet transparent mobile phases, it was possible to set up double detection, including selective detection of acetylated oligosaccharides. Analytical gel permeation chromatography was directly coupled to CTA-SAX, obtaining a two-dimensional profile of analyzed oligosaccharidic mixtures. A sequencing method of heparin oligosaccharides using partial depolymerization by heparinases according to their size and sulfation pattern and digest analysis by CTA-SAX was developed. A direct application of this method to the analysis of oligosaccharide mixtures obtained by complete digestion of heparins by heparinases I, II, and III was done. It allowed a reliable quantification of heparin building blocks. We also focused our attention on di- and tetrasaccharidic species containing the 3-O-sulfated glucosamines taken as markers of the active sites for antithrombin III. The method was also applied to more complex mixtures resulting from porcine heparin partially depolymerized with heparinase I. The specificity of the reaction was studied up to decasaccharidic fractions.
Anal Biochem 2004 Sep 15
PMID:Chromatographic analysis and sequencing approach of heparin oligosaccharides using cetyltrimethylammonium dynamically coated stationary phases. 1532 99

Heparinase II from Pedobacter heparinus (formerly Flavobacterium heparinum), which acts on both heparin and heparan sulfate, is one of several glycosaminoglycan-degrading enzymes produced by this organism. This enzyme, with a molecular weight of 84 kDa, utilizes a lytic mechanism to cleave the alpha(1-4) glycosidic bond between hexosamine (D-glucosamine) and L-iduronic or D-glucuronic acid, resulting in a product with an unsaturated sugar ring at the non-reducing end. The enzyme was crystallized by the hanging-drop vapour-diffusion method. The crystals belong to orthorhombic space group P2(1)2(1)2(1) and diffract to 2 A resolution. There are two molecules in the asymmetric unit, consistent with the finding that recombinant heparinase II functions as a dimer in solution.
Acta Crystallogr D Biol Crystallogr 2004 Sep
PMID:Crystallization and preliminary X-ray analysis of heparinase II from Pedobacter heparinus. 1533 43

Cell surface heparan sulfate functions as a co-receptor in HSV-1 entry. In order to study its significance in context with specific gD receptors (nectin-1, HVEM, and 3-O-sulfated heparan sulfate) a low speed centrifugation based virus inoculation (spinoculation) method was used. The experiments were performed at 1200 x g using glycosylaminoglycan positive (GAG+) or deficient (GAG-) cells expressing gD receptors. Clearly, spinoculation of GAG- nectin-1 or HVEM cells enhanced significantly viral entry compared to similar but unspun cells. The enhanced entry was due to increased virus deposition at the cell surface and not due to pelleting of the virus. Among the gD receptors, spinoculated GAG- HVEM cells showed restoration of HSV-1 entry compared to unspinoculated GAG+ HVEM cells. In contrast, spinoculated GAG- nectin-1 cells showed less entry than unspinoculated GAG+ nectin-1 cells. GAG- 3-O-sulfotransferase-expressing cells or heparinase treated GAG+ 3-O-sulfated heparan sulfate cells, in contrast, remained resistant to entry even after spinoculation. To investigate further, any potential effects of centrifugation on membrane fusion, a virus-free cell fusion assay was performed. Clearly, spinning had no effects on cell fusion, nor could it replace the need for all four essential glycoproteins. Taken together these results suggest that heparan sulfate plays a role of an attachment receptor, which could be substituted by spinoculation. This effect, however, varies with the gD receptor used, which in turn, could be used as a means for identifying gD receptor usage for entry into a cell type.
J Virol Methods 2005 Sep
PMID:Spinoculation of heparan sulfate deficient cells enhances HSV-1 entry, but does not abolish the need for essential glycoproteins in viral fusion. 1590 19

Lipoprotein lipase (LPL) is a key enzyme in the hydrolysis of triglyceride-rich lipoproteins. In vascular diseases, such as atherosclerosis, inflammation plays an important role in the pathogenesis of the disease. We examined the role of LPL in modulating tumor necrosis factor-alpha (TNF-alpha)- and interferon-gamma (IFN-gamma)-mediated inflammatory cytokine signal transduction pathways in human aortic endothelial cells (HAECs). LPL significantly suppressed TNF-alpha-induced gene expression, and this suppression was reversed by tetrahydrolipstatin and heparinase. In contrast, LPL synergistically enhanced IFN-gamma-induced gene expression in HAECs. To elucidate the molecular mechanisms of LPL action, we investigated the role of transcription factors nuclear factor kappa B (NF-kappaB) and signal transducer and activator of transcription factor 1 (Stat1). The anti-inflammatory response of LPL in suppressing TNF-alpha-induced gene expression was a result of its inhibition of NF-kappaB activity by the abrogation of IkappaB-alpha degradation and phosphorylation of the p65 subunit. Although LPL alone had no effect on Stat1 activation, LPL enhanced IFN-gamma-induced phosphorylation of Stat1 on tyrosine 701 and serine 727, as well as Stat1-mediated transactivation. The synergistic effect of LPL on IFN-gamma-induced Stat1 activation was mediated by enhanced activation of the tyrosine kinase JAK2 and was abrogated by LY294002, a specific inhibitor of the phosphatidylinositol 3'-kinase pathway. Our studies indicate that LPL has differential effects on several inflammatory pathways known to be important in atherosclerosis.
J Biol Chem 2005 Sep 02
PMID:Differential effects of lipoprotein lipase on tumor necrosis factor-alpha and interferon-gamma-mediated gene expression in human endothelial cells. 1599 21

As the spherical diameter of pulmonary capillaries is smaller than that of neutrophils, increased neutrophil stiffness or conversely, decreased neutrophil deformability is a key step in the initial sequestration of neutrophils within the lungs during inflammatory processes. Antithrombin III (AT) is known to exert a therapeutic effect against disseminated intravascular coagulation, and accumulating evidence suggests that AT also has anti-inflammatory properties. The mechanisms of its anti-inflammatory effects remain unclear, but in a rat endotoxin model, AT apparently inhibited neutrophil sequestration in the lung. In the present in vitro study, therefore, we examined the effect of AT on the deformability of human neutrophils and correlated those findings with their F-actin content. Isolated human neutrophils were stimulated with formyl-Met-Leu-Phe (1 muM, 2 min) in the presence or absence of the alpha, beta, or low heparin-affinity isoforms of AT (1 IU/ml, 20 min), and deformability was evaluated using a filter assay system. Neutrophils were also stained with fluorescein isothiocyanate-phalloidin and subjected to a fluorescein-activated cell sorter scan to assess F-actin content. The results showed that pretreatment with any of the three AT isoforms similarly inhibited the decreased neutrophil deformability and increased F-actin content of stimulated cells. Notably, heparinase had no effect on deformability or F-actin content in the presence or absence of AT, which was somewhat unexpected, as heparin sulfate proteoglycans likely function as AT receptors. These findings suggested that AT inhibits the increase in neutrophil stiffness seen during inflammatory processes by inhibiting actin polymerization via a heparin-independent pathway.
J Leukoc Biol 2005 Sep
PMID:Effect of antithrombin III on neutrophil deformability. 1600 Mar 88

Vascular endothelial growth factor (VEGF) is a family of glycoproteins with potent angiogenic activity. We reported previously that heparin has an affinity for VEGF165, the major isoform of VEGF, whereas 2-O-desulfated heparin and 6-O-desulfated heparin have weak but significant affinity (Ashikari-Hada, S., Habuchi, H., Kariya, Y., Itoh, N., Reddi, A. H., and Kimata, K. (2004) J. Biol. Chem. 279, 12346-12354). In this study, we first examined the effect of heparin and modified heparins (completely desulfated N-sulfated heparin, 2-O-desulfated heparin, and 6-O-desulfated heparin) on VEGF165-dependent mitogenic activity and tube formation on type I collagen gels of human umbilical vein endothelial cells. Both were enhanced by heparin, but not by modified heparins, suggesting that both the 2-O-sulfate group of hexuronic acid and the 6-O-sulfation group of N-sulfoglucosamine in heparin/heparan sulfate are necessary for VEGF165 activity. We then examined the activation of VEGF receptor (VEGFR) to understand the mechanism. We have made several new findings; 1) heparin yielded a 1.7-fold enhancement of VEGF165-induced phosphorylation of VEGFR-2; 2) depletion of cell surface heparan sulfate by heparinase/heparitinase treatment and preferential reduction of trisulfated disaccharide units of cell surface HS by sodium chlorate treatment resulted in the reduction of such phosphorylation, suggesting the involvement of a heparin-like domain in the phosphorylation of VEGFR-2; and 3) VEGF121, an isoform without the exon 7-encoded region, which has no capacity to bind to heparin, did not show these effects. It is therefore likely that a heparin-like domain of heparan sulfate/heparin forms a complex with VEGF165 and VEGFR-2 via the exon 7-encoded region, thereby enhancing VEGF165-dependent signaling.
J Biol Chem 2005 Sep 09
PMID:Heparin regulates vascular endothelial growth factor165-dependent mitogenic activity, tube formation, and its receptor phosphorylation of human endothelial cells. Comparison of the effects of heparin and modified heparins. 1602 24

Capillary liquid chromatography-mass spectrometry using graphitised carbon stationary phase and ion trap mass spectrometry was shown to be a powerful technique for analysing glycosaminoglycans digested with endoglycosidases. Commonly found disaccharides from heparin/heparan sulphate digests at sub nanomole levels were found to be separated by mass and/or retention time and detected by negative ion electrospray mass spectrometry predominantly as [M-H]- ions using a standard electrospray interface and flow rate between 6-10 microL/min. Graphitised carbon liquid chromatography-fragmentation mass spectrometry provided sequence data of disaccharides and oligosaccharides. Sequence information was obtained from either collision of the [M-H]- ions (low sulphated disaccharides) or of the [M+Na-2H]- ions (highly sulphated disaccharides). This separation and identification method of endoglycosidase digestion and sample preparation using a combination of cation exchange and graphitised carbon, was used to successfully analyse digests of keratan sulphate (keratanase) and heparin (heparinase) standards, and hyaluronic acid (hyaluronidase) from synovial fluid samples.
J Chromatogr B Analyt Technol Biomed Life Sci 2005 Sep 25
PMID:Use of graphitised carbon negative ion LC-MS to analyse enzymatically digested glycosaminoglycans. 1605 81

Glial cell line-derived neurotrophic factor (GDNF), a known survival factor for neurons, has recently been shown to stimulate the migration of Schwann cells (SCs) and to enhance myelination. GDNF exerts its biological effects by activating the Ret tyrosine kinase in the presence of glycosylphosphatidylinositol-linked receptor, GDNF family receptor (GFR) alpha1. In Ret-negative cells, the alternative transmembrane coreceptor is the 140-kDa isoform of neural cell adhesion molecule (NCAM) associated with a non-receptor tyrosine kinase Fyn. We confirmed that GDNF, GFRalpha1 and NCAM are expressed in neonatal rat SCs. We found that GDNF induces an increase in the partitioning of NCAM and heparan sulfate proteoglycan agrin into lipid rafts and that heparinase inhibits GDNF-signaling in SCs. In addition to activation of extracellular signal-regulated kinases, and phosphorylation of cAMP response element binding protein, we found that cAMP-dependent protein kinase A and protein kinase C are involved in GDNF-mediated signaling in SCs. Although GDNF did not promote the differentiation of purified SCs into the myelinating phenotype, it enhanced myelination in neuron-SC cocultures. We conclude that GDNF utilizes NCAM signaling pathways to regulate SC function prior to myelination and at early stages of myelin formation.
J Neurochem 2005 Sep
PMID:Glial cell line-derived neurotrophic factor-induced signaling in Schwann cells. 1608 1

A capillary electrophoresis (CE) method was developed for the separation of heparin oligosaccharides compatible to study the interactions between the oligosaccharides and granulocyte-colony stimulating factor (G-CSF). Unfractionated heparin was eliminitively degraded to heparin oligosaccharides by an endolytic heparinase. The degraded smaller oligosaccharides (M(r) < 1000) were baseline-separated by CE under a 50 mM phosphate buffer (pH 9.0) in 10 min. Standard heparin disaccharides and larger oligosaccharides (1000 < M(r) < 8000) were all separated under optimized separation conditions. Compared with standard heparin disaccharides, smaller oligosaccharides contained one nonsulfated, two monosulfated, and two disulfated disaccharides, but trisulfated disaccharides were not found. The smaller oligosaccharides were also identified and molecular mass was deduced by electrospray ionization-mass spectrometry (ESI-MS). Furthermore, interactions between G-CSF and the oligosaccharides were studied by using capillary zone electrophoresis (CZE) under the above separation conditions. It was found that larger oligosaccharides could interact with G-CSF while smaller oligosaccharides were not observed to bind to G-CSF under the experimental conditions. In conclusion, the purified heparinase could selectively degrade heparin into oligosaccharides and the interaction between G-CSF and heparin was correlated with the chain length of heparin.
Electrophoresis 2005 Sep
PMID:Separation, identification, and interaction of heparin oligosaccharides with granulocyte-colony stimulating factor using capillary electrophoresis and mass spectrometry. 1610 Jul 43


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