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Per Venge - One of the best experts on this subject based on the ideXlab platform.
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asparagine linked glycans determine the cytotoxic capacity of Eosinophil Cationic Protein ecp
Molecular Immunology, 2013Co-Authors: Jenny Rubin, Per VengeAbstract:Eosinophil Cationic Protein (ECP) is a toxic, granule-stored Protein of the Eosinophil granulocyte. It is a heterogeneous Protein; molecular weights can differ from 15 to 22 kDa, due to glycosylations. We purified high molecular weight ECP from blood donors with the ECP434GG (rs2073342) genotype, with the aim of examining whether removal of carbohydrates could enhance the cytotoxic capacity. The cytotoxic activity of the ECP pools was tested against the NCI-H69 cell line, before and after enzymatic deglycosylation. ECP was also analysed by SELDI-TOF MS to monitor the changes in molecular mass after deglycosylation. Five high molecular weight pools of ECP (HMW-ECP I-V) with decreasing degrees of glycosylation were tested at concentrations ranging from 0.02 to 0.6 mu M. The activity ranged from EC50 of >0.6 mu M to 0.04 mu M; HMW-ECP II had the lowest activity and HMW-ECP V the highest. After deglycosylation with N-glycosidase F, pools HMW-ECP I-III were reduced to the same molecular weight of 15.78 kDa and acquired potent cytotoxic activities. HMW-ECP IV and V with molecular species at 163 and 16.1 kDa were highly cytotoxic as such and were only partially deglycosylated, with slight enhancement of the toxic properties. The results suggest the presence of several HMW-ECP molecular species with differences in their post-translational modifications and cytotoxic properties. We conclude that a fraction of native ECP is stored in a non-cytotoxic form, which can be converted into a cytotoxic form by N-deglycosylation, whereas another fraction is stored as a highly cytotoxic form carrying different post-translational modifications.
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effect of Eosinophil Cationic Protein ecp on hodgkin lymphoma cell lines
Experimental Hematology, 2011Co-Authors: Ingrid Glimelius, Per Venge, Jenny Rubin, Marie Fischer, Daniel Molin, Rosemarie Amini, Gunilla EnbladAbstract:Objective In Hodgkin lymphoma (HL), tumor Eosinophilia indicates poor prognosis, probably caused by Eosinophil-induced stimulation of tumor cells. Our aim was to investigate the effects of Eosinophil Cationic Protein (ECP) on HL tumor cells in vitro. Materials and Methods A fluorometric microculture cytotoxicity assay was used to measure the survival index of cells from the HL cell lines: HDLM-2 (T-cell origin, nodular sclerosis histology), KMH2 (B-cell origin, mixed cellularity), and L428 (B-cell origin, nodular sclerosis) after incubation with ECP97arg variants with different glycosylations and with ECP97thr. Flow cytometry monitored the effects of ECP on markers of cell death. Results For KMH2 and L428, ECP was cytotoxic with a dose−response relationship similar to a previously investigated small-cell lung cancer cell line. HDLM-2 was more sensitive to ECP at low concentrations, but reached a plateau (survival index of 70%) at 0.018 μM. The IC 50 for KMH2 and L428 were 0.2 and 0.15 μM, respectively. The IC 50 was never reached for HDLM-2. All tested ECP variants displayed similar activity in HDLM-2, in contrast to KMH2 and L428, which were more sensitive to less glycosylated ECP. Positive DNA staining (propidium iodide) of HDLM-2 cells treated with ECP indicated cell death by necrosis. Conclusions ECP is cytotoxic for HL tumor cells even at low concentrations, but heterogeneity between cell lines exists and not all tumor cells are eradicated. Two cell lines of B-cell origin, KMH2 and L428, were sensitive to ECP in a dose−response manner, but for HDLM-2, which is of T-cell origin, the cytotoxicity reached a plateau.
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Eosinophil Cationic Protein (ECP) Is Processed during Secretion
Journal of immunology (Baltimore Md. : 1950), 2009Co-Authors: Charlotte Woschnagg, Jenny Rubin, Per VengeAbstract:The Eosinophil granulocyte is an inflammatory cell involved in allergic diseases such as asthma and rhinitis. Eosinophil Cationic Protein (ECP) is a basic and potentially cytotoxic granule Protein that is released from the Eosinophil upon activation. The aim was to study secretion of molecular variants of ECP from blood Eosinophils with the hypothesis that the stored noncytotoxic ECP is altered into cytotoxic species upon release from the cell. Eosinophil granulocytes were purified to >95% from venous blood from birch pollen allergic subjects, with symptoms of rhinitis, and from healthy control subjects during the birch pollen season. The cells were stimulated with IL-5, GM-CSF, or serum-opsonized Sephadex particles. Concentration of ECP in cells or supernatants was measured by means of a fluoroenzyme immunoassay, and ECP heterogeneity was studied using an affinity capture assay with the surface-enhanced laser desorption/ionization-time of flight mass spectrometry technique. Extracts of unstimulated Eosinophils contained 10 major ECP variants, with molecular masses ranging from 16.1 to 17.7 kDa. Stimulation with particles mainly induced the secretion of two molecular variants at 16.1 and 16.3 kDa, while cytokine stimulation gave rise to a different secretion profile. ECP variants in the pellet extracts remained unaffected by cell activation. The modifications of secreted ECP were partly explained by differences in N-linked glycosylations. Secretion of ECP from Eosinophils involves Protein modification. The molecular masses of released ECP have acquired the masses of the cytotoxic species.
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purification and characterization of Eosinophil Cationic Protein from normal human Eosinophils
European Journal of Haematology, 2009Co-Authors: Christer Peterson, Hans Jornvall, Per VengeAbstract:ECP (Eosinophil Cationic Protein) was purified in high yield from the granules of human buffy coat Eosinophils obtained from healthy individuals. The separation procedure included gel filtration on Sephadex G-75, ion-exchange chromatography on Bio Rex 70, and chelating chromatography on zinc-chelate Sepharose 6B. The normal ECP is a single-chain, highly Cationic glycoProtein which separates on SDS-polyacrylamide gel electrophoresis into at least 3 molecular weight forms, with molecular weights of 18.5, 20 and 22 kDa. A heterogeneity in charge was also observed, with the 18.5 kDa form being the most Cationic one. The various molecular species of ECP exhibited antigenic identity, identical amino acid compositions, and identical amino-terminal amino acid sequences. The molecular heterogeneity was shown to be caused by differences in glycosylation of the Protein.
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Eosinophil Cationic Protein stimulates migration of human lung fibroblasts in vitro
Scandinavian Journal of Immunology, 2009Co-Authors: Ulrika Zagai, Per Venge, Joachim Lundahl, J Klominek, C M SkoldAbstract:Asthma is characterized by Eosinophilic inflammation and remodelling of the airways. Eosinophil Cationic Protein (ECP) is a Protein released by activated Eosinophils and the hypothesis that ECP contributes to the development of structural changes in the airways of asthmatics has been posed. Fibroblast recruitment is an important step in the remodelling process, and we therefore put the question whether ECP stimulates migration of human lung fibroblasts. Human peripheral Eosinophils isolated from buffycoats from healthy individuals were cultured and conditioned media (CM) were collected. Native ECP was extracted from human peripheral Eosinophils by gel filtration, ion-exchange and chelating chromatography. The ability of Eosinophil CM and ECP to stimulate fibroblast migration was determined using the 48-well Boyden chamber. ECP concentrations in CM were assayed by ECP-CAP-FEIA. Both CM and ECP significantly stimulated fibroblast migration (48.4+/-cells/field versus 33+/-2 and 36+/-6 versus 25+/-4; P<0.001 and 0.05 respectively) in a time- and concentration-dependent manner. Adding neutralizing ECP antibodies attenuated fibroblast migration induced by both ECP as well as CM. ECP stimulates migration of human lung fibroblasts, suggesting a potential mechanism for Eosinophils in the fibrotic response. This may be an important mechanism by which ECP promotes remodelling of extracellular matrix leading to airway fibrosis in asthmatics.
Margaret Dah-tsyr Chang - One of the best experts on this subject based on the ideXlab platform.
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Cell Penetrating Peptide Derived from Human Eosinophil Cationic Protein Decreases Airway Allergic Inflammation
Nature Publishing Group, 2017Co-Authors: Shun-lung Fang, Jaw-ji Tsai, Heng-kuei Lin, Yee-jun Chen, Ting-yu Chen, Margaret Dah-tsyr ChangAbstract:Abstract Cell penetrating peptide derived from human Eosinophil Cationic Protein (CPPecp) is a 10-amino-acid peptide containing a core heparan sulfate (HS)-binding motif of human Eosinophil Cationic Protein (ECP). It binds and penetrates bronchial epithelial cells without cytotoxic effects. Here we investigated airway-protective effects of CPPecp in BEAS-2B cell line and mite-induced airway allergic inflammation in BALB/c mice. In BEAS-2B cell, CPPecp decreases ECP-induced eotaxin mRNA expression. CPPecp also decreases eotaxin secretion and p-STAT6 activation induced by ECP, as well as by IL-4. In vivo studies showed CPPecp decreased mite-induced airway inflammation in terms of Eosinophil and neutrophil count in broncho-alveolar lavage fluid, peri-bronchiolar and alveolar pathology scores, cytokine production in lung Protein extract including interleukin (IL)-5, IL-13, IL-17A/F, eotaxin; and pause enhancement from methacholine stimulation. CPPecp treated groups also showed lower serum mite-specific IgE level. In this study, we have demonstrated the in vitro and in vivo anti-asthma effects of CPPecp
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a novel cell penetrating peptide derived from human Eosinophil Cationic Protein
PLOS ONE, 2013Co-Authors: Shun-lung Fang, Tanchi Fan, Chishin Hwang, Chienjung Chen, Tajen Hung, Lihyuan Lin, Margaret Dah-tsyr ChangAbstract:Cell-penetrating peptides (CPPs) are short peptides which can carry various types of molecules into cells; however, although most CPPs rapidly penetrate cells in vitro, their in vivo tissue-targeting specificities are low. Herein, we describe cell-binding, internalization, and targeting characteristics of a newly identified 10-residue CPP, denoted ECP32–41, derived from the core heparin-binding motif of human Eosinophil Cationic Protein (ECP). Besides traditional emphasis on positively charged residues, the presence of cysteine and tryptophan residues was demonstrated to be essential for internalization. ECP32–41 entered Beas-2B and wild-type CHO-K1 cells, but not CHO cells lacking of cell-surface glycosaminoglycans (GAGs), indicating that binding of ECP32–41 to cell-surface GAGs was required for internalization. When cells were cultured with GAGs or pre-treated with GAG-digesting enzymes, significant decreases in ECP32–41 internalization were observed, suggesting that cell-surface GAGs, especially heparan sulfate proteoglycans were necessary for ECP32–41 attachment and penetration. Furthermore, treatment with pharmacological agents identified two forms of energy-dependent endocytosis, lipid-raft endocytosis and macropinocytosis, as the major ECP32–41 internalization routes. ECP32–41 was demonstrated to transport various cargoes including fluorescent chemical, fluorescent Protein, and peptidomimetic drug into cultured Beas-2B cells in vitro, and targeted broncho-epithelial and intestinal villi tissues in vivo. Hence this CPP has the potential to serve as a novel vehicle for intracellular delivery of biomolecules or medicines, especially for the treatment of pulmonary or gastrointestinal diseases.
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chemoattraction of macrophages by secretory molecules derived from cells expressing the signal peptide of Eosinophil Cationic Protein
BMC Systems Biology, 2012Co-Authors: Yu Shu Liu, Margaret Dah-tsyr Chang, Tanchi Fan, Pei Wen Tsai, Yong Wang, Chia Hung Hsieh, Tunwen Pai, Chien Fu Huang, Chungyu Lan, Haoteng ChangAbstract:Background: Eosinophil Cationic Protein is a clinical asthma biomarker that would be released into blood, especially gathered in bronchia. The signal peptide of Eosinophil Cationic Protein (ECPsp) plays an important role in translocating ECP to the extracellular space. We previously reported that ECPsp inhibits microbial growth and regulates the expression of mammalian genes encoding tumor growth factor-α (TGF-α) and epidermal growth factor receptor (EGFR). Results: In the present study, we first generated a DNA microarray dataset, which showed that ECPsp upregulated proinflammatory molecules, including chemokines, interferon-induced molecules, and Toll-like receptors. The levels of mRNAs encoding CCL5, CXCL10, CXCL11, CXCL16, STAT1, and STAT2 were increased in the presence of ECPsp by 2.07-, 4.21-, 7.52-, 2.6-, 3.58-, and 1.67-fold, respectively. We then constructed a functional linkage network by integrating the microarray dataset with the pathway database of Kyoto Encyclopedia of Genes and Genomes (KEGG). Follow-up analysis revealed that STAT1 and STAT2, important transcriptional factors that regulate cytokine expression and release, served as hubs to connect the pathways of cytokine stimulation (TGF-α and EGFR pathways) and inflammatory responses. Furthermore, integrating TGF-α and EGFR with the functional linkage network indicated that STAT1 and STAT2 served as hubs that connect two functional clusters, including (1) cell proliferation and survival, and (2) inflammation. Finally, we found that conditioned medium in which cells that express ECPsp had been cultured could chemoattract macrophages. Experimentally, we also demonstrated that the migration of macrophage could be inhibited by the individual treatment of siRNAs of STAT1 or STAT2. Therefore, we hypothesize that ECPsp may function as a regulator for enhancing the migration of macrophages through the upregualtion of the transcriptional factors STAT1 and STAT2. Conclusion: The increased expression and release of various cytokines triggered by ECPsp may attract macrophages to bronchia to purge damaged cells. Our approach, involving experimental and computational systems biology, predicts pathways and potential biological functions for further characterization of this novel function of ECPsp under inflammatory conditions.
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tnf α mediates Eosinophil Cationic Protein induced apoptosis in beas 2b cells
BMC Cell Biology, 2010Co-Authors: Kunche Chang, Margaret Dah-tsyr Chang, Shun-lung Fang, Jaw-ji Tsai, Tanchi Fan, Chihwen Shu, Chuanhsin Chang, Chengta Chung, Chihchung Chao, Yiukay LaiAbstract:Eosinophilic granulocytes are important for the human immune system. Many Cationic Proteins with cytotoxic activities, such as Eosinophil Cationic Protein (ECP) and Eosinophil-derived neurotoxin (EDN), are released from activated Eosinophils. ECP, with low RNase activity, is widely used as a biomarker for asthma. ECP inhibits cell viability and induces apoptosis to cells. However, the specific pathway underlying the mechanisms of ECP-induced cytotoxicity remains unclear. This study investigated ECP-induced apoptosis in bronchial epithelial BEAS-2B cells and elucidated the specific pathway during apoptosis. To address the mechanisms involved in ECP-induced apoptosis in human BEAS-2B cells, investigation was carried out using chromatin condensation, cleavage of poly (ADP-ribose) polymerase (PARP), sub-G1 distribution in cell cycle, annexin V labeling, and general or specific caspase inhibitors. Caspase-8-dependent apoptosis was demonstrated by cleavage of caspase-8 after recombinant ECP treatment, accompanied with elevated level of tumor necrosis factor alpha (TNF-α). Moreover, ECP-induced apoptosis was effectively inhibited in the presence of neutralizing anti-TNF-α antibody. In conclusion, our results have demonstrated that ECP increased TNF-α production in BEAS-2B cells and triggered apoptosis by caspase-8 activation through mitochondria-independent pathway.
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characterization of molecular interactions between Eosinophil Cationic Protein and heparin
Journal of Biological Chemistry, 2008Co-Authors: Tanchi Fan, Shun-lung Fang, Chishin Hwang, Chihyen Hsu, Shangcheng Hung, Shuchuan Lin, Margaret Dah-tsyr ChangAbstract:Eosinophil Cationic Protein (ECP) is currently used as a biomarker for airway inflammation. It is a heparin-binding ribonuclease released by activated Eosinophils. Its cytotoxicity toward cancer cell lines is blocked by heparin. The objective of this study was to locate the heparin binding site of ECP by site-directed mutagenesis and construction of a synthetic peptide derived from this region. Synthetic heparin with ≥5 monosaccharide units showed strong inhibition of ECP binding to the cell surface. Analysis of ECP mt1 (R34A/W35A/R36A/K38A) showed that these charged and aromatic residues were involved in ECP binding to heparin and the cell surface. A potential binding motif is located in the loop L3 region between helix α2 and strand β1, outside the RNA binding domain. The synthetic peptide derived from the loop L3 region displayed strong pentasaccharide binding affinity and blocked ECP binding to cells. In addition, ECP mt1 showed reduced cytotoxicity. Thus, the tight interaction between ECP and heparin acts as the primary step for ECP endocytosis. These results provide new insights into the structure and function of ECP for anti-asthma therapy.
Alexander Kapp - One of the best experts on this subject based on the ideXlab platform.
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childhood atopic dermatitis brain derived neurotrophic factor correlates with serum Eosinophil Cationic Protein and disease severity
Allergy, 2016Co-Authors: Regina Folsterholst, Alexander Kapp, Eleni Papakonstantinou, Urda Rudrich, Matthias Buchner, Helene Pite, Manuela Gehring, Stephan Weidinger, Ulrike RaapAbstract:Several studies have shown that neurotrophins including brain-derived neurotrophic factor (BDNF) play a role in chronic inflammatory skin diseases such as atopic dermatitis (AD). BDNF is increased in the serum samples of adults with AD. Interestingly, Eosinophils of these patients can release and produce BDNF. We analyzed BDNF serum levels with ELISA and their correlation with SCORAD score, Eosinophil Cationic Protein (ECP), total IgE, IL-4, IL-13 and IL-31 in children with AD (n = 56) compared to nonatopic healthy children (n = 25). In addition, we analyzed FLG loss-of-function mutations in 17 children with AD and their connection to BDNF. BDNF serum levels were significantly higher in children with AD. Further, BDNF correlated with disease activity, serum ECP, and total IgE serum levels in AD. There was no difference in BDNF levels of filaggrin-positive or filaggrin-negative children with AD, and there was no correlation of BDNF with IL-31 and Th2 cytokines including IL-4 and IL-13. Together, our data add new insights into the pathophysiology of AD, suggesting that serum BDNF which correlates with disease severity contributes to the regulation of inflammation in an Eosinophil-, but not Th2-dependent manner.
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serum Eosinophil Cationic Protein ecp is a sensitive measure for disease activity in atopic dermatitis
British Journal of Dermatology, 1992Co-Authors: Wolfgang Czech, Jean Krutmann, Erwin Schöpf, Alexander KappAbstract:Atopic dermatitis (AD) is characterized by alterations in cellular and humoral immunity including elevated serum levels of IgE, IL-2 receptor (IL-2R) and Eosinophil Cationic Protein (ECP). In order to evaluate the relevance of these serum parameters as indicators of disease activity, the concentrations of IgE, IL-2R and ECP were measured in serum samples of patients with an acute exacerbation of AD (n = 19) on admission to hospital and every 6 days up to discharge, and compared with those from normal non-atopic controls (n = 15). The severity of the disease in the AD patients was examined using an established clinical scoring system. On admission, AD patients showed significantly elevated serum levels of IgE, IL-2R and ECP compared with normal controls (P less than or equal to 0.0001). Clinical improvement was associated with a decrease of both the clinical score (P less than or equal to 0.001) and serum ECP levels (P less than or equal to 0.005). No significant changes in serum IgE and serum IL-2R were observed. In addition, there was a significant correlation between serum ECP and the clinical score (R = 0.67, P less than or equal to 0.001). These data indicate that serum ECP may be a helpful tool for monitoring disease activity in AD.
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Eosinophil Cationic Protein in sera of patients with atopic dermatitis.
Journal of the American Academy of Dermatology, 1991Co-Authors: Alexander Kapp, Wolfgang Czech, Jean Krutmann, Erwin SchöpfAbstract:Patients with atopic dermatitis frequently show elevated blood Eosinophil counts, and Eosinophil-derived major basic Protein has been demonstrated in the eczematous skin from patients with atopic dermatitis. To evaluate further the role of Eosinophils in the pathogenesis of atopic dermatitis, the concentration of Eosinophil Cationic Protein was measured in serum samples of 42 patients with moderate to severe disease. The results were compared with those obtained in 32 patients with psoriasis with ( n = 9) or without ( n = 23) a history of inhalant allergy, 12 patients with a history of Pseudoallergie reactions to acetylsalicylic acid, 14 patients with a history of inhalant allergy, and 31 nonatopic healthy control subjects. Eosinophil Cationic Protein levels were significantly increased in the serum of patients with atopic dermatitis ( p ≤ 0.005) and patients with a history of Pseudoallergie reactions to acetylsalicylic acid ( p ≤ 0.01). There was no significant difference between Eosinophil Cationic Protein levels in patients with psoriasis or a history of inhalant allergy and in control subjects. Moreover, Eosinophil Cationic Protein levels did not differ significantly in psoriasis patients with or without inhalant allergy. These studies support the concept of an active participation of Eosinophils in atopic dermatitis and point to a possible role for Eosinophils in pseudoallergy.
Ester Boix - One of the best experts on this subject based on the ideXlab platform.
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structural basis for endotoxin neutralization by the Eosinophil Cationic Protein
FEBS Journal, 2016Co-Authors: David Pulido, Marc Torrent, Maria Flor Garciamayoral, Mohammed Moussaoui, Diego Velazquez, Marta Bruix, Ester BoixAbstract:Acute infection by Gram-negative pathogens can induce an exacerbated immune response that leads to lethal septic shock syndrome. Bacterial lipopolysaccharide (LPS) is a major pathogen-associated molecular pattern (PAMP) molecule that can initiate massive and lethal immune system stimulation. Therefore, the development of new and effective LPS-neutralizing agents is a top priority. The Eosinophil Cationic Protein (ECP) is an antimicrobial Protein secreted in response to infection, with a remarkable affinity for LPS. Here, we demonstrate that ECP is able to neutralize bacterial LPS and inhibit tumor necrosis factor-α (TNF-α) production in human macrophages. We also characterized ECP neutralizing activity using progressively truncated LPS mutants, and conclude that the polysaccharide moiety and lipid A portions are required for LPS-mediated neutralization. In addition, we mapped the structural determinants required for the ECP-LPS interaction by nuclear magnetic resonance (NMR). Our results show that ECP is able to neutralize LPS and therefore opens a new route to develop novel therapeutic agents based on the ECP structural scaffolding. This article is protected by copyright. All rights reserved.
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two human host defense ribonucleases against mycobacteria the Eosinophil Cationic Protein rnase 3 and rnase 7
Antimicrobial Agents and Chemotherapy, 2013Co-Authors: David Pulido, Marc Torrent, Victoria M Nogues, David Andreu, Ester BoixAbstract:There is an urgent need to develop new agents against mycobacterial infections, such as tuberculosis and other respiratory tract or skin affections. In this study, we have tested two human antimicrobial RNases against mycobacteria. RNase 3, also called the Eosinophil Cationic Protein, and RNase 7 are two small Cationic Proteins secreted by innate cells during host defense. Both Proteins are induced upon infection displaying a wide range of antipathogen activities. In particular, they are released by leukocytes and epithelial cells, contributing to tissue protection. Here, the two RNases have been proven effective against Mycobacterium vaccae at a low micromolar level. High bactericidal activity correlated with their bacterial membrane depolarization and permeabilization activities. Further analysis on both Protein-derived peptides identified for RNase 3 an N-terminus fragment that is even more active than the parental Protein. Also, a potent bacterial agglutinating activity was unique to RNase 3 and its derived peptide. The particular biophysical properties of the RNase 3 active peptide are envisaged as a suitable reference for the development of novel antimycobacterial drugs. The results support the contribution of secreted RNases to the host immune response against mycobacteria.
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insights into the glycosaminoglycan mediated cytotoxic mechanism of Eosinophil Cationic Protein revealed by nmr
ACS Chemical Biology, 2013Co-Authors: Flor M Garciamayoral, Mohammed Moussaoui, Angeles Canales, Dolores Diaz, Javier Lopezprados, Jose L De Paz, Jesus Angulo, Pedro M Nieto, Jesus Jimenezbarbero, Ester BoixAbstract:Protein-glycosaminoglycan interactions are essential in many biological processes and human diseases, yet how their recognition occurs is poorly understood. Eosinophil Cationic Protein (ECP) is a cytotoxic ribonuclease that interacts with glycosaminoglycans at the cell surface; this promotes the destabilization of the cellular membrane and triggers ECP’s toxic activity. To understand this membrane destabilization event and the differences in the toxicity of ECP and its homologues, the high resolution solution structure of the complex between full length folded ECP and a heparin-derived trisaccharide (O-iPr-α-d-GlcNS6S-α(1–4)-l-IdoA2S-α(1–4)-d-GlcNS6S) has been solved by NMR methods and molecular dynamics simulations. The bound Protein retains the tertiary structure of the free Protein. The 2S0 conformation of the IdoA ring is preferably recognized by the Protein. We have identified the precise location of the heparin binding site, dissected the specific interactions responsible for molecular recognition, ...
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structural determinants of the Eosinophil Cationic Protein antimicrobial activity
Biological Chemistry, 2012Co-Authors: Ester Boix, M. Victòria Nogués, Marc Torrent, David Pulido, Vivian A Salazar, Mohamed MoussaouiAbstract:Antimicrobial RNases are small Cationic Proteins belonging to the vertebrate RNase A superfamily and endowed with a wide range of antipathogen activities. Vertebrate RNases, while sharing the active site architecture, are found to display a variety of noncatalytical biological properties, providing an excellent example of multitask Proteins. The antibacterial activity of distant related RNases suggested that the family evolved from an ancestral host-defence function. The review provides a structural insight into antimicrobial RNases, taking as a reference the human RNase 3, also named Eosinophil Cationic Protein (ECP). A particular high binding affi nity against bacterial wall structures mediates the Protein action. In particular, the interaction with the lipopolysaccharides at the Gram-negative outer membrane correlates with the Protein antimicrobial and specifi c cell agglutinating activity. Although a direct mechanical action at the bacteria wall seems to be suffi cient to trigger bacterial death, a potential intracellular target cannot be discarded. Indeed, the Cationic clusters at the Protein surface may serve both to interact with nucleic acids and cell surface heterosaccharides. Sequence determinants for ECP activity were screened by prediction tools, proteolysis and peptide synthesis. Docking results are complementing the structural analysis to delineate the Protein anchoring sites for anionic targets of biological signifi cance.
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refining the Eosinophil Cationic Protein antibacterial pharmacophore by rational structure minimization
Journal of Medicinal Chemistry, 2011Co-Authors: Marc Torrent, David Pulido, Marta Bruix, Beatriz G De La Torre, Flor M Garciamayoral, Victoria M Nogues, David Andreu, Ester BoixAbstract:Sequence analysis of Eosinophil Cationic Protein (ECP), a ribonuclease of broad antimicrobial activity, allowed identification of residues 1–45 as the antimicrobial domain. We have further dissected ECP(1–45) with a view to defining the minimal requirements for antimicrobial activity. Structure-based downsizing has focused on both α-helices of ECP(1–45) and yielded analogues with substantial potency against Gram-negative and -positive strains. Analogues ECP(8–36) and ECP(6–17)-Ahx-(23–36) (Ahx, 6-aminohexanoic acid) involve 36% and 40% size reduction relative to (1–45), respectively, and display a remarkably ECP-like antimicrobial profile. Both retain segments required for self-aggregation and lipolysaccharide binding, as well as the bacterial agglutination ability of parent ECP. Analogue (6–17)-Ahx-(23–36), in particular, is shown by NMR to preserve the helical traits of the native 8–16 (α1) and 33–36 (α2) regions and can be proposed as the minimal structure capable of reproducing the activity of the ent...
Hiroshi Fujishima - One of the best experts on this subject based on the ideXlab platform.
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IgE and Eosinophil Cationic Protein (ECP) as markers of severity in the diagnosis of atopic keratoconjunctivitis
The British journal of ophthalmology, 2012Co-Authors: Tais Hitomi Wakamatsu, Kazumi Fukagawa, Yoshiyuki Satake, Ayako Igarashi, Osama M. A. Ibrahim, Jun Shimazaki, Naoko Okada, Hiroshi FujishimaAbstract:Aims To evaluate tear and serum IgE and Eosinophil Cationic Protein (ECP) as severity markers for atopic keratoconjunctivitis (AKC). Methods Thirty eyes of 30 patients with AKC and 10 eyes of 10 healthy control subjects were examined in this prospective study. All subjects underwent fluorescein staining, conjunctival injection, conjunctival oedema and papillary formation grading. Tear and serum IgE and ECP levels were measured, and correlations between them investigated with reference to the ocular surface clinical parameters. Results The mean fluorescein scores, conjunctival injection, oedema scores and papillary formation were significantly higher in AKC patients compared to controls (p Conclusion This study suggests the presence of an Eosinophilic response in AKC disease independent of IgE sensitisation. Tear ECP was a useful marker delineating the severity of ocular surface disease in AKC.
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Eosinophil Cationic Protein as a marker for assessing the efficacy of tacrolimus ophthalmic solution in the treatment of atopic keratoconjunctivitis
Molecular vision, 2011Co-Authors: Tais Hitomi Wakamatsu, Kazumi Fukagawa, Mari Tanaka, Yoshiyuki Satake, Ayako Igarashi, Hiroshi FujishimaAbstract:Purpose To examine the clinical efficacy and anti–inflammatory effects of tacrolimus eye drops; we studied the changes in clinical ocular findings and measured tear Eosinophil Cationic Protein (ECP) levels of atopic keratoconjunctivitis (AKC) patients before and after the treatment.