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101.
Identification and characterization of heparin/heparan sulfate binding domains of the endoglycosidase heparanase 总被引:6,自引:0,他引:6
Levy-Adam F Abboud-Jarrous G Guerrini M Beccati D Vlodavsky I Ilan N 《The Journal of biological chemistry》2005,280(21):20457-20466
The endo-beta-glucuronidase, heparanase, is an enzyme that cleaves heparan sulfate at specific intra-chain sites, yielding heparan sulfate fragments with appreciable size and biological activities. Heparanase activity has been traditionally correlated with cell invasion associated with cancer metastasis, angiogenesis, and inflammation. In addition, heparanase up-regulation has been documented in a variety of primary human tumors, correlating with increased vascular density and poor postoperative survival, suggesting that heparanase may be considered as a target for anticancer drugs. In an attempt to identify the protein motif that would serve as a target for the development of heparanase inhibitors, we looked for protein domains that mediate the interaction of heparanase with its heparan sulfate substrate. We have identified three potential heparin binding domains and provided evidence that one of these is mapped at the N terminus of the 50-kDa active heparanase subunit. A peptide corresponding to this region (Lys(158)-Asp(171)) physically associates with heparin and heparan sulfate. Moreover, the peptide inhibited heparanase enzymatic activity in a dose-responsive manner, presumably through competition with the heparan sulfate substrate. Furthermore, antibodies directed to this region inhibited heparanase activity, and a deletion construct lacking this domain exhibited no enzymatic activity. NMR titration experiments confirmed residues Lys(158)-Asn(162) as amino acids that firmly bound heparin. Deletion of a second heparin binding domain sequence (Gln(270)-Lys(280)) yielded an inactive enzyme that failed to interact with cell surface heparan sulfate and hence accumulated in the culture medium of transfected HEK 293 cells to exceptionally high levels. The two heparin/heparan sulfate recognition domains are potentially attractive targets for the development of heparanase inhibitors. 相似文献
102.
Cohen L Karbat I Gilles N Ilan N Benveniste M Gordon D Gurevitz M 《The Journal of biological chemistry》2005,280(6):5045-5053
Scorpion beta-toxins that affect the activation of mammalian voltage-gated sodium channels (Navs) have been studied extensively, but little is known about their functional surface and mode of interaction with the channel receptor. To enable a molecular approach to this question, we have established a successful expression system for the anti-mammalian scorpion beta-toxin, Css4, whose effects on rat brain Navs have been well characterized. A recombinant toxin, His-Css4, was obtained when fused to a His tag and a thrombin cleavage site and had similar binding affinity for and effect on Na currents of rat brain sodium channels as those of the native toxin isolated from the scorpion venom. Molecular dissection of His-Css4 elucidated a functional surface of 1245 A2 composed of the following: 1) a cluster of residues associated with the alpha-helix, which includes a putative "hot spot" (this cluster is conserved among scorpion beta-toxins and contains their "pharmacophore"); 2) a hydrophobic cluster associated mainly with the beta2 and beta3 strands, which is likely to confer the specificity for mammalian Navs; 3) a single bioactive residue (Trp-58) in the C-tail; and 4) a negatively charged residue (Glu-15) involved in voltage sensor trapping as inferred from our ability to uncouple toxin binding from activity upon its substitution. This study expands our understanding about the mode of action of scorpion beta-toxins and illuminates differences in the functional surfaces that may dictate their specificities for mammalian versus insect sodium channels. 相似文献
103.
Strugatsky D Zilberberg N Stankiewicz M Ilan N Turkov M Cohen L Pelhate M Gilles N Gordon D Gurevitz M 《Biochemistry》2005,44(25):9179-9187
We isolated from the venom of the scorpion Leiurus quinquestriatus hebraeus an extremely active anti-insect selective depressant toxin, Lqh-dprIT(3). Cloning of Lqh-dprIT(3) revealed a gene family encoding eight putative polypeptide variants (a-h) differing at three positions (37A/G, 50D/E, and 58N/D). All eight toxin variants were expressed in a functional form, and their toxicity to blowfly larvae, binding affinity for cockroach neuronal membranes, and CD spectra were compared. This analysis links Asn-58, which appears in variants a-d, to a toxin conformation associated with high binding affinity for insect sodium channels. Variants e-h, bearing Asp-58, exhibit a different conformation and are less potent. The importance of Asn-58, which is conserved in other depressant toxins, was further validated by construction and analysis of an N58D mutant of the well-characterized depressant toxin, LqhIT(2). Current and voltage clamp assays using the cockroach giant axon have shown that despite the vast difference in potency, the two types of Lqh-dprIT(3) variants (represented by Lqh-dprIT(3)-a and Lqh-dprIT(3)-e) are capable of blocking the action potentials (manifested as flaccid paralysis in blowfly larvae) and shift the voltage dependence of activation to more negative values, which typify the action of beta-toxins. Moreover, the stronger and faster shift in voltage dependence of activation and lack of tail currents observed in the presence of Lqh-dprIT(3)-a suggest an extremely efficient trapping of the voltage sensor compared to that of Lqh-dprIT(3)-e. The current clamp assays revealed that repetitive firing of the axon, which is reflected in contraction paralysis of blowfly larvae, can be obtained with either the less potent Lqh-dprIT(3)-e or the highly potent Lqh-dprIT(3)-a at more negative membrane potentials. Thus, the contraction symptoms in flies are likely to be dominated by the resting potential of neuronal membranes. This study clarifies the electrophysiological basis of the complex symptoms induced by scorpion depressant toxins in insects, and highlights for the first time molecular features involved in their activity. 相似文献
104.
Hinderlich S Salama I Eisenberg I Potikha T Mantey LR Yarema KJ Horstkorte R Argov Z Sadeh M Reutter W Mitrani-Rosenbaum S 《FEBS letters》2004,568(1-3):105-109
In vivo growth of bacterial flagellar filaments by self-assembly of flagellin is promoted by a capping structure composed of a pentameric assembly of hook associated protein 2 (HAP2). Isolated native filaments with intact HAP2 cap exhibited higher melting temperature (deltaTm = 4 degrees C) and significantly increased resistance against heat-induced depolymerization than non-capped ones. Reconstituted filaments were also stabilized by HAP2 binding, but the obtained filament-HAP2 complexes were less stable than native assemblies. Their fast depolymerization at elevated temperatures and sensitivity to proteolysis indicated that native-like filament-HAP2 complexes are rarely obtained by in vitro reconstitution. A procedure was developed to isolate perfectly capped native filaments to facilitate high-resolution structural analysis. 相似文献
105.
106.
Barbier J Lamthanh H Le Gall F Favreau P Benoit E Chen H Gilles N Ilan N Heinemann SH Gordon D Ménez A Molgó J 《The Journal of biological chemistry》2004,279(6):4680-4685
We have isolated delta-conotoxin EVIA (delta-EVIA), a conopeptide in Conus ermineus venom that contains 32 amino acid residues and a six-cysteine/four-loop framework similar to that of previously described omega-, delta-, microO-, and kappa-conotoxins. However, it displays low sequence homology with the latter conotoxins. delta-EVIA inhibits Na+ channel inactivation with unique tissue specificity upon binding to receptor site 6 of neuronal Na+ channels. Using amphibian myelinated axons and spinal neurons, we showed that delta-EVIA increases the duration of action potentials by inhibiting Na+ channel inactivation. delta-EVIA considerably enhanced nerve terminal excitability and synaptic efficacy at the frog neuromuscular junction but did not affect directly elicited muscle action potentials. The neuronally selective property of delta-EVIA was confirmed by showing that a fluorescent derivative of delta-EVIA labeled motor nerve endings but not skeletal muscle fibers. In a heterologous expression system, delta-EVIA inhibited inactivation of rat neuronal Na+ channel subtypes (rNaV1.2a, rNaV1.3, and rNaV1.6) but did not affect rat skeletal (rNaV1.4) and human cardiac muscle (hNaV1.5) Na+ channel subtypes. delta-EVIA, in the range of concentrations used, is the first conotoxin found to affect neuronal Na+ channels without acting on Na+ channels of skeletal and cardiac muscle. Therefore, it is a unique tool for discriminating voltage-sensitive Na+ channel subtypes and for studying the distribution and modulation mechanisms of neuronal Na+ channels, and it may serve as a lead to design new drugs adapted to treat diseases characterized by defective nerve conduction. 相似文献
107.
Gingis-Velitski S Zetser A Kaplan V Ben-Zaken O Cohen E Levy-Adam F Bashenko Y Flugelman MY Vlodavsky I Ilan N 《The Journal of biological chemistry》2004,279(42):44084-44092
Heparanase is a mammalian endoglycosidase that degrades heparan sulfate (HS) at specific intrachain sites, an activity that is strongly implicated in cell dissemination associated with metastasis and inflammation. In addition to its structural role in extracellular matrix assembly and integrity, HS sequesters a multitude of polypeptides that reside in the extracellular matrix as a reservoir. A variety of growth factors, cytokines, chemokines, and enzymes can be released by heparanase activity and profoundly affect cell and tissue function. Thus, heparanase bioavailability, accessibility, and activity should be kept tightly regulated. We provide evidence that HS is not only a substrate for, but also a regulator of, heparanase. Addition of heparin or xylosides to cell cultures resulted in a pronounced accumulation of, heparanase in the culture medium, whereas sodium chlorate had no such effect. Moreover, cellular uptake of heparanase was markedly reduced in HS-deficient CHO-745 mutant cells, heparan sulfate proteoglycan-deficient HT-29 colon cancer cells, and heparinase-treated cells. We also studied the heparanase biosynthetic route and found that the half-life of the active enzyme is approximately 30 h. This and previous localization studies suggest that heparanase resides in the endosomal/lysosomal compartment for a relatively long period of time and is likely to play a role in the normal turnover of HS. Co-localization studies and cell fractionation following heparanase addition have identified syndecan family members as candidate molecules responsible for heparanase uptake, providing an efficient mechanism that limits extracellular accumulation and function of heparanase. 相似文献
108.
Khateeb S Flusser H Ofir R Shelef I Narkis G Vardi G Shorer Z Levy R Galil A Elbedour K Birk OS 《American journal of human genetics》2006,79(5):942-948
Infantile neuroaxonal dystrophy (INAD) is an autosomal recessive progressive neurodegenerative disease that presents within the first 2 years of life and culminates in death by age 10 years. Affected individuals from two unrelated Bedouin Israeli kindreds were studied. Brain imaging demonstrated diffuse cerebellar atrophy and abnormal iron deposition in the medial and lateral globus pallidum. Progressive white-matter disease and reduction of the N-acetyl aspartate : chromium ratio were evident on magnetic resonance spectroscopy, suggesting loss of myelination. The clinical and radiological diagnosis of INAD was verified by sural nerve biopsy. The disease gene was mapped to a 1.17-Mb locus on chromosome 22q13.1 (LOD score 4.7 at recombination fraction 0 for SNP rs139897), and an underlying mutation common to both affected families was identified in PLA2G6, the gene encoding phospholipase A2 group VI (cytosolic, calcium-independent). These findings highlight a role of phospholipase in neurodegenerative disorders. 相似文献
109.
Groups of common carp (Cyprinus carpio) were trained for 33 days to discriminate between two acoustic signals differing in frequency and temporal pattern. One signal (positive stimulus) was reinforced by food, while the other (negative stimulus) was not rewarded. When exposed to 3 positive and 3 negative stimuli per day (training Paradigm 1) the fish responded similarly to the two signals. When daily negative stimuli outnumbered positive stimuli (3 positive and 33 negative, training Paradigm 2) the fish responded similarly to the two signals for the first 2 weeks and then began to discriminate between them. Discriminatory response was statistically significant from Day 21 of training. After 33 days, training was switched from Paradigm 2 to Paradigm 1 and the fish maintained their discriminatory capability. The results are interpreted in terms of anticipatory frustration and risk management. 相似文献
110.
Klotz S Hay I Dickstein ML Yi GH Wang J Maurer MS Kass DA Burkhoff D 《American journal of physiology. Heart and circulatory physiology》2006,291(1):H403-H412
Whereas end-systolic and end-diastolic pressure-volume relations (ESPVR, EDPVR) characterize left ventricular (LV) pump properties, clinical utility of these relations has been hampered by the need for invasive measurements over a range of pressure and volumes. We propose a single-beat approach to estimate the whole EDPVR from one measured volume-pressure (Vm and Pm) point. Ex vivo EDPVRs were measured from 80 human hearts of different etiologies (normal, congestive heart failure, left ventricular assist device support). Independent of etiology, when EDPVRs were normalized (EDPVRn) by appropriate scaling of LV volumes, EDPVRns were nearly identical and were optimally described by the relation EDP = An.EDV (Bn), with An = 28.2 mmHg and Bn = 2.79. V0 (the volume at the pressure of approximately 0 mmHg) was predicted by using the relation V0 = Vm.(0.6 - 0.006.Pm) and V30 by V30 = V0 + (Vm,n - V0)/(Pm/An) (1/Bn). The entire EDPVR of an individual heart was then predicted by forcing the curve through Vm, Pm, and the predicted V0 and V30. This technique was applied prospectively to the ex vivo human EDPVRs not used in determining optimal An and Bn values and to 36 in vivo human, 12 acute and 14 chronic canine, and 80 in vivo and ex vivo rat studies. The root-mean-square error (RMSE) in pressure between measured and predicted EDPVRs over the range of 0-40 mmHg was < 3 mmHg of measured EDPVR in all settings, indicating a good predictive value of this approach. Volume-normalized EDPVRs have a common shape, despite different etiology and species. This allows the entire curve to be predicted by a new method with a potential for noninvasive application. The results are most accurate when applied to groups of hearts rather than to individual hearts. 相似文献