FRINK Linked Data Fragments server

Matches in SemOpenAlex for { <https://semopenalex.org/work/W2017987939> ?p ?o ?g. }

• W2017987939 endingPage "25474" @default.
• W2017987939 startingPage "25468" @default.
• W2017987939 abstract "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. 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. Eosinophil cationic protein (ECP), 2The abbreviations used are: ECPeosinophil cationic proteinAMAC2-aminoacridoneEDNeosinophil-derived neurotoxinFACEfluorescence-assisted carbohydrate electrophoresisHSheparan sulfateHSPGheparan sulfate proteoglycanITFEintrinsic tryptophan fluorescence emissionLMWHlow molecular weight heparinMBPmaltose-binding proteinELISAenzyme-linked immunosorbent assayPBSphosphate-buffered salineMTT3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromideHRPhorseradish peroxidase. 2The abbreviations used are: ECPeosinophil cationic proteinAMAC2-aminoacridoneEDNeosinophil-derived neurotoxinFACEfluorescence-assisted carbohydrate electrophoresisHSheparan sulfateHSPGheparan sulfate proteoglycanITFEintrinsic tryptophan fluorescence emissionLMWHlow molecular weight heparinMBPmaltose-binding proteinELISAenzyme-linked immunosorbent assayPBSphosphate-buffered salineMTT3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromideHRPhorseradish peroxidase. a member of the ribonuclease A (RNase A) superfamily, is found in the specific granules of eosinophilic leukocytes. It is a single polypeptide with a molecular mass ranging from 16 to 21.4 kDa due to varying degrees of glycosylation. It shows a 67% amino acid sequence identity with eosinophil-derived neurotoxin (EDN), another eosinophil-secreted RNase. Although ECP shares the overall three-dimensional structure of RNase A, it has relatively lower RNase activity (1Boix E. Nikolovski Z. Moiseyev G.P. Rosenberg H.F. Cuchillo C.M. Nogues M.V. J. Biol. Chem. 1999; 274: 15605-15614Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar). ECP released by activated eosinophils contributes to the toxicity against helminth parasites, bacteria, and single strand RNA viruses (2Molina H.A. Kierszenbaum F. Hamann K.J. Gleich G.J. Am. J. Trop. Med. Hyg. 1988; 38: 327-334Crossref PubMed Scopus (63) Google Scholar, 3Lehrer R. Szklarek D. Barton A. Ganz T. Hamann K. Gleich G. J. Immunol. 1989; 142: 4428-4434PubMed Google Scholar, 4Domachowske J.B. Dyer K.D. Adams A.G. Leto T.L. Rosenberg H.F. Nucleic Acids Res. 1998; 26: 3358-3363Crossref PubMed Scopus (168) Google Scholar). Together with other proteins secreted from eosinophils, ECP is thought to cause damage to epithelial cells, a common feature of airway inflammation in asthma (5Gleich G.J. J. Allergy Clin. Immunol. 2000; 105: 651-663Abstract Full Text Full Text PDF PubMed Scopus (681) Google Scholar). eosinophil cationic protein 2-aminoacridone eosinophil-derived neurotoxin fluorescence-assisted carbohydrate electrophoresis heparan sulfate heparan sulfate proteoglycan intrinsic tryptophan fluorescence emission low molecular weight heparin maltose-binding protein enzyme-linked immunosorbent assay phosphate-buffered saline 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide horseradish peroxidase. eosinophil cationic protein 2-aminoacridone eosinophil-derived neurotoxin fluorescence-assisted carbohydrate electrophoresis heparan sulfate heparan sulfate proteoglycan intrinsic tryptophan fluorescence emission low molecular weight heparin maltose-binding protein enzyme-linked immunosorbent assay phosphate-buffered saline 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide horseradish peroxidase. The mechanism underlying the cytotoxic property of ECP is unclear. It has been hypothesized that ECP cytotoxicity is due to destabilization of lipid membranes of target cells (6Young J. Peterson C. Venge P. Cohn Z. Nature. 1986; 321: 613-616Crossref PubMed Scopus (358) Google Scholar), and the degree of cytotoxicity is dependent on the cellular concentration (7Carreras E. Boix E. Navarro S. Rosenberg H.F. Cuchillo C.M. Nogues M.V. Mol. Cell. Biochem. 2005; 272: 1-7Crossref PubMed Scopus (47) Google Scholar). The binding of ECP to target cells has been attributed to its high arginine content (estimated pI = 10.8), which facilitates the interaction between ECP and negatively charged molecules on the cell surface (7Carreras E. Boix E. Navarro S. Rosenberg H.F. Cuchillo C.M. Nogues M.V. Mol. Cell. Biochem. 2005; 272: 1-7Crossref PubMed Scopus (47) Google Scholar, 8Carreras E. Boix E. Rosenberg H.F. Cuchillo C.M. Nogues M.V. Biochemistry. 2003; 42: 6636-6644Crossref PubMed Scopus (86) Google Scholar). Recently, we found that binding and endocytosis of ECP into bronchial epithelial cells were greatly dependent on the cell surface glycosaminoglycan, specifically heparan sulfate proteoglycans (HSPG) (9Fan T.C. Chang H.T. Chen I.W. Wang H.Y. Chang M.D. Traffic. 2007; 8: 1778-1795Crossref PubMed Scopus (50) Google Scholar). The cytotoxicity of ECP was severely reduced toward cell lines with heparan sulfate (HS) deficiency. Heparin and HS are complex polysaccharides composed of alternating units of hexuronic acid and glucosamine. The uronic acid residues of heparin typically consist of 90% l-idopyranosyluronic acid and 10% d-glucopyranosyluronic acid (10Capila I. Linhardt R.J. Angew. Chem. Int. Ed. Engl. 2002; 41: 391-412Crossref PubMed Scopus (1543) Google Scholar). The N position of glucosamine may be substituted with an acetyl or sulfate group. The 3-O and 6-O positions of glucosamine and the 2-O of uronic acid may be sulfated. Through the combination of different negatively charged moieties, heparin and HS have been demonstrated to bind a variety of proteins with diverse functions, including growth factors, thrombin, chemokines, and viral proteins. The HS chains contain domains with a high level of sulfation and epimerization (S-domains), regions with mixed N-acetylation and N-sulfation (NA/S-domains), and unmodified domains that are mostly N-acetylated and contain little sulfate (11Tumova S. Woods A. Couchman J.R. Int. J. Biochem. Cell Biol. 2000; 32: 269-288Crossref PubMed Scopus (310) Google Scholar). Because HS chains contain heparin regions, heparin and its mimetics can be used to study interactions between proteins and polysaccharides. The structure of ECP has been determined and refined to a resolution up to 1.75 Å, displaying a folding topology that involves three α helices and five β strands (12Mallorqui-Fernandez G. Pous J. Peracaula R. Aymami J. Maeda T. Tada H. Yamada H. Seno M. Llorens R.d. Gomis-Ruth F.X. Coll M. J. Mol. Biol. 2000; 300: 1297-1307Crossref PubMed Scopus (57) Google Scholar). The most interesting feature is as many as 19 surface-oriented arginine residues, conferring a strong basic character to ECP. However, the heparin binding site in ECP has not been identified. Heparin binding domains within proteins usually contain a high proportion of positively charged residues, which bind to the acidic groups of heparin through electrostatic interactions. It has been proposed that the three-dimensional structure of the HS chain is critical for protein binding (13Hileman R.E. Fromm J.R. Weiler J.M. Linhardt R.J. BioEssays. 1998; 20: 156-167Crossref PubMed Scopus (515) Google Scholar). However, not much is known about the three-dimensional structure of HS. After examining a series of heparin-binding protein sequences, Cardin and Weintraub (14Cardin A.D. Weintraub H.J. Arteriosclerosis. 1989; 9: 21-32Crossref PubMed Google Scholar) proposed that the pattern XBBBXXBX or XBBXBX (where X represents hydrophobic or uncharged amino acids, and B represents basic amino acids) is responsible for HS binding to other proteins. In the present work, a linear heparin binding site on ECP and the shortest ECP-binding heparin oligosaccharide unit have been identified. Loop L3 of ECP may mediate the interaction between ECP and cell surface HSPG, contributing to ECP cytotoxicity. Furthermore, the dissociation constants between oligosaccharides and ECP were determined by tryptophan emission titration. Antibodies and Reagents—Mouse anti-maltose-binding protein (MBP) was obtained from Santa Cruz Biotechnology (Santa Cruz, CA). RNase A was purchased from Promega (Madison, WI). Chemicals were purchased from Sigma-Aldric unless otherwise specified. Recombinant Protein Purification—Recombinant human ECP containing a C-terminal His6 tag was expressed in Escherichia coli BL21 codon plus (DE3) (Novagen, Madison, WI), purified by chromatography, and refolded as previously described (1Boix E. Nikolovski Z. Moiseyev G.P. Rosenberg H.F. Cuchillo C.M. Nogues M.V. J. Biol. Chem. 1999; 274: 15605-15614Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar). Amino acid residues Arg-34, Trp-35, Arg-36, and Lys-38 of ECP were simultaneously substituted to alanine using QuikChange site-directed mutagenesis (Stratagene, La Jolla, CA), and the resultant mutant was named ECP mt1. The primers used were as follows: mt1 forward, 5′-TATGCAGCGGCTTGCGCAAACCAAAAT-3′; and mt1 reverse, 5′-TTTGCGCAAGCCGCTGCATAATTGTTA-3′. E. coli BL21 codon plus (DE3) cells were used to transform various plasmids. MBP-ECP was purified using amylose affinity chromatography (1Boix E. Nikolovski Z. Moiseyev G.P. Rosenberg H.F. Cuchillo C.M. Nogues M.V. J. Biol. Chem. 1999; 274: 15605-15614Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar). Cells and Cell Culture—Beas-2B, a human bronchial epithelial cell line, was cultured in RPMI 1640 medium (Sigma-Aldrich) supplemented with heat-inactivated 10% fetal bovine serum (Invitrogen). Fluorescence-assisted Carbohydrate Electrophoresis—Synthetic heparin oligosaccharide from the degree of polymerization (dp) 3–9 (Fig. 1, Mr 926–2642) (16Lee C.J. Lu X.A. Kulkarni S.S. Wen Y.S. Hung S.C. J. Am. Chem. Soc. 2004; 126: 476-477Crossref PubMed Scopus (139) Google Scholar), low molecular weight heparin (LMWH, Sigma-Aldrich, average Mr 3,000), and PI-88 (kindly provided by Progen Inc., Australia) were labeled with 2-aminoacridone (AMAC) as described previously (17Calabro A. Benavides M. Tammi M. Hascall V.C. Midura R.J. Glycobiology. 2000; 10: 273-281Crossref PubMed Scopus (195) Google Scholar). AMAC-oligosaccharide and proteins were incubated at room temperature for 15 min, loaded onto 1% gels, and electrophoresed as described (18Holmes O. Pillozzi S. Deakin J.A. Carafoli F. Kemp L. Butler P.J. Lyon M. Gherardi E. J. Mol. Biol. 2007; 367: 395-408Crossref PubMed Scopus (74) Google Scholar). Cell ELISA—The ability of MBP-ECP to bind cells in the presence of serial dilutions of oligosaccharides or peptides was determined as described (9Fan T.C. Chang H.T. Chen I.W. Wang H.Y. Chang M.D. Traffic. 2007; 8: 1778-1795Crossref PubMed Scopus (50) Google Scholar). Briefly, confluent monolayers of Beas-2B cells in 96-well plates were pretreated with various concentrations of oligosaccharides or peptides in serum-free RPMI 1640 medium at 4 °C for 30 min before incubation with 5 μg/ml MBP-ECP at 4 °C for 1 h. The cells were then washed with ice-cold PBS and fixed with 2% paraformaldehyde at room temperature for 15 min prior to blocking with 2% bovine serum albumin/PBS at room temperature for 90 min. The level of bound MBP-ECP was quantified by ELISA analysis. MBP-ECP was detected using mouse monoclonal anti-MBP and goat anti-mouse HRP-conjugated secondary antibody, followed by the enhanced chemiluminescence detection system. The amount of MBP-ECP bound to cells without oligosaccharide or peptide treatment was set to 100%. Intrinsic Tryptophan Fluorescence Emission—Binding of ECP to heparin was monitored by changes in ITFE. ECP (200 nm) in PBS at 25 °C was titrated with small aliquots of a high concentration of heparin oligosaccharides with minimal dilution (<2%). Protein fluorescence measurements were recorded 2 min after each addition on a Hitachi 8000 spectrofluorometer at emission wavelength of 340 nm using an excitation wavelength of 280 nm. ΔF, the relative fluorescence change, equals F0 - Fobs, where F0 and Fobs represent the initial and observed fluorescence values, respectively. Binding constants were estimated from the titration data using a nonlinear least-squares computer fit to the equation based on 1:1 binding stoichiometry (19Venge P. Bystrom J. Int. J. Biochem. Cell Biol. 1998; 30: 433-437Crossref PubMed Scopus (49) Google Scholar), ΔF=ΔFmax×([P]+[H]+Kd-(([P]+[H]+Kd)2-4×[P]×[H])1/2)/(2×[P])(Eq. 1) where ΔFmax is the maximum relative fluorescence change, P is the total concentration of ECP, H is the concentration of heparin, and Kd is the dissociation constant for the ECP-heparin interaction. CD Spectroscopy—CD spectra were recorded using an Aviv model 202 CD Spectrometer equipped with a 450-watt xenon arc lamp. Far-UV spectra were recorded at 25 °C from 200 to 250 nm using a 0.1-cm cuvette containing 10 μm protein in PBS. The CD spectra were recorded at 1.5-min intervals with a bandwidth of 1 nm. Each spectrum is an average of three consecutive scans and was corrected by subtracting the buffer spectrum. Cell Growth Assay—The effect of ECP on the cell growth was determined by a colorimetric assay using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) (US Biological, Marblehead, MA). Cells were plated in a 96-well plate (5000 cells/well) and incubated at 37 °C overnight. Each sample was incubated with the indicated concentration (1–100 μm) of ECP. Forty-eight hours after treatment with ECP, MTT was added, and cell growth was monitored at A570 to measure the mitochondrial-dependent formation of a colored product. Synthetic Peptides—Human ECP peptide NYRWRCKNQNK-biotin (C1), EDN peptide NYQRRCKNQNK-biotin (D1), RNase1 peptide MTQGRCKPVNK-biotin (R1), and HIV-TAT peptide YGRKKRRQRRRK-biotin (Tat) were purchased from Genemed Synthesis (South San Francisco, CA). Immunohistochemical Staining—Adult specific-pathogen-free Sprague-Dawley rats (Narl:SD), with body weight of 200–300 g, were purchased and maintained at the National Laboratory Animal Center in Taiwan and heparin (FRAGMIN®, average Mr 6,000 and was 5,000 IU/0.2 ml). The rats were separated into three groups. In group 1, each rat was injected with 5 nmol of ECP through the tail vein. In group 2, each rat was co-injected with heparin (20 IU/100 g of body weight) and 5 nmol of ECP. In group 3, each rat was injected with 5 nmol of MBP as the negative control. All animals were sacrificed using CO2 narcosis 1 h after the injection of these agents. The lung and trachea of these rats were taken and immediately fixed with 10% neutral-buffered formaldehyde. The tissue samples were processed by routine methods to prepare paraffin wax-embedded block. These blocks were then sectioned into 6-μm slices. All tissue sections were examined using the Super Sensitive Non-Biotin HRP Detection System (BioGenex Laboratories, San Ramon, CA) as described (20Liang C.T. Chueh L.L. Pang V.F. Zhuo Y.X. Liang S.C. Yu C.K. Chiang H. Lee C.C. Liu C.H. J. Comp. Pathol. 2007; 136: 57-64Crossref PubMed Scopus (14) Google Scholar). Briefly, the mouse anti-ECP or anti-MBP monoclonal antibody was used as the primary antibody. Antigen unmasking was performed by immersion of sections in 5% Trilogy (Cell Marque, Rocklin, CA) antigen unmasking solution in Milli-Q water and boiled at 121 °C. Endogenous peroxidase activity was quenched with hydrogen peroxide (3%) in methanol. These sections were then incubated in Power Block solution, and mouse anti-ECP or anti-MBP at 1/200 dilution was applied and left for 24 h. The sections were incubated with Super Enhancer reagent, followed by Polymer-HRP reagent, and then incubated with 3-amino-9-ethylcarbazole chromogen solution. The sections were finally counter-stained with Mayer's hematoxylin and mounted with Super Mount permanent aqueous mounting media prior to examination with a light microscope (Axioplan, Zeiss, Germany). Oligosaccharide Length Dependence of ECP-Heparin Interaction—Previously, we demonstrated that binding and endocytosis of ECP are HSPG-dependent (9Fan T.C. Chang H.T. Chen I.W. Wang H.Y. Chang M.D. Traffic. 2007; 8: 1778-1795Crossref PubMed Scopus (50) Google Scholar). An early study demonstrated ECP purification using heparin-Sepharose chromatography (21Gleich G.J. Loegering D.A. Bell M.P. Checkel J.L. Ackerman S.J. McKean D.J. Proc. Natl. Acad. Sci. U. S. A. 1986; 83: 3146-3150Crossref PubMed Scopus (277) Google Scholar). Thus, the minimal heparin length (or disaccharide unit) required to interact with ECP was investigated by FACE. Initially, ECP was co-incubated with AMAC-labeled LMWH at various molar ratios, and the binding products were analyzed by gel electrophoresis. The decrease in the free LMWH signal was monitored as ECP concentration was increased (Fig. 2A). Subsequently, the dependence of ECP binding on heparin size was examined in the presence of synthetic heparin oligosaccharides dp3 to dp9 using LMWH as a positive control. It was apparent that dp5 served as the shortest heparin fragment that retained the ability to bind ECP (Fig. 2B). Furthermore, we tested a potent heparanase inhibitor (PI-88), undergoing clinical trials for its anti-angiogenic and anti-metastatic effects (22Joyce J.A. Freeman C. Meyer-Morse N. Parish C.R. Hanahan D. Oncogene. 2005; 24: 4037-4051Crossref PubMed Scopus (145) Google Scholar, 23Parish C.R. Freeman C. Brown K.J. Francis D.J. Cowden W.B. Cancer Res. 1999; 59: 3433-3441PubMed Google Scholar), that contains a mixture of highly sulfated mannose-containing di- to hexasaccharides (24Yu G. Gunay N.S. Linhardt R.J. Toida T. Fareed J. Hoppensteadt D.A. Shadid H. Ferro V. Li C. Fewings K. Palermo M.C. Podger D. Eur. J. Med. Chem. 2002; 37: 783-791Crossref PubMed Scopus (73) Google Scholar, 25Ferro V. Li C. Fewings K. Palermo M.C. Linhardt R.J. Toida T. Carbohydr. Res. 2002; 337: 139-146Crossref PubMed Scopus (43) Google Scholar). Interestingly, this heparin mimetic could also bind ECP (Fig. 2B). Competitive Inhibition of ECP Binding to Cells by Synthetic Oligosaccharides—Using cell ELISA, synthetic heparins (dp3–dp9) were tested for their ability to interfere with ECP binding to Beas-2B cells. Beas-2B cells were preincubated with oligosaccharides, and bound ECP was detected essentially as described (9Fan T.C. Chang H.T. Chen I.W. Wang H.Y. Chang M.D. Traffic. 2007; 8: 1778-1795Crossref PubMed Scopus (50) Google Scholar). The degree of inhibition increased with increasing oligosaccharide length (Fig. 3). 50 μg/ml heparin dp5 inhibited 50% of ECP binding to cells, and the same amount of dp7 and dp9 was capable of inhibiting over 70 and 80% of ECP binding, respectively. These data revealed that pentasaccharide was the minimal length sufficient to interfere with ECP binding to Beas-2B cells. In addition, the concentration dependence of PI-88 against cellular binding of ECP was similar to that of dp7 (Fig. 3). Identification of Heparin-binding Sequence in ECP—A consensus sequence of the heparin binding site (e.g. XBBXBX or XBBBXXBX) has been found in many glycosaminoglycan-binding proteins or peptides (3Lehrer R. Szklarek D. Barton A. Ganz T. Hamann K. Gleich G. J. Immunol. 1989; 142: 4428-4434PubMed Google Scholar, 14Cardin A.D. Weintraub H.J. Arteriosclerosis. 1989; 9: 21-32Crossref PubMed Google Scholar). Inspection of the sequences of human RNase A family members revealed a surface loop L3 region, 34QRRCKN39, in EDN that exactly matched the XBBXBX motif (Fig. 4A), but no consensus heparin-binding motif was found in ECP. Therefore, we speculated that residues 34–39 (34RWRCKN39) in ECP that correspond to the consensus motif in EDN might also bind heparin. To determine whether this region contributes to cellular binding, cell ELISA analysis was conducted using MBP-ECP and MBP-ECP mt1 containing the mutations R34A/W35A/R36A/K38A. This mutant had only 50% of the cell-binding activity, indicating the importance of the 34RWRCKN39 motif in ECP for cellular HS binding (Fig. 4B). Characterization of Association between ECP and Heparin Oligosaccharides—The binding affinities of wild-type and mutant ECP for heparin oligosaccharides were subsequently monitored by intrinsic tryptophan fluorescence titration (27Lau E.K. Paavola C.D. Johnson Z. Gaudry J.P. Geretti E. Borlat F. Kungl A.J. Proudfoot A.E. Handel T.M. J. Biol. Chem. 2004; 279: 22294-22305Abstract Full Text Full Text PDF PubMed Scopus (170) Google Scholar). The change in tryptophan fluorescence revealed that wild-type ECP bound to dp5 with high affinity, and the corresponding Kd was 139.6 nm (Fig. 5). As expected, a 4- to 5-fold increase in Kd to 568.1 nm was observed for ECP mt1 (R34A/W35A/R36A/K38A) (Table 1), indicating decreased heparin-binding activity for mutant ECP lacking the key heparin-binding motif in the L3 region.TABLE 1Dissociation constant (Kd) determination for wild-type ECP and ECP mt1 with heparin oligosaccharides Kd was measured in PBS at 25 °C. The change in intrinsic tryptophan fluorescence was fit by the equation 1 to obtain Kd.OligosaccharideKdECP wtECP mt1nmdp962.6400.3dp5139.6568.1 Open table in a new tab CD spectroscopy to compare the conformations of wild-type ECP and ECP mt1 showed that the secondary structure of ECP mt1 was very similar to that of wild-type ECP. These results strongly indicated that the decrease in heparin binding affinity resulted from a loss of the specific recognition sequence motif, but not from a conformational change in ECP mt1. Competitive Inhibition of ECP Binding to Cells by Synthetic Peptides—To investigate whether the RWRCK motif was directly responsible for heparin binding, cell ELISA was conducted using a synthetic peptide, C1 (NYRWRCKNQNK), along with a positive control peptide derived from HIV-TAT, YGRKKRRQRRRK (28Vives E. Brodin P. Lebleu B. J. Biol. Chem. 1997; 272: 16010-16017Abstract Full Text Full Text PDF PubMed Scopus (2054) Google Scholar, 29Ziegler A. Seelig J. Biophys. J. 2004; 86: 254-263Abstract Full Text Full Text PDF PubMed Scopus (174) Google Scholar). The dose-dependent competition of these peptides is shown in Fig. 6A. Binding of MBP-ECP to cell-surface HS was significantly reduced in the presence of both C1 and TAT peptides. Therefore, synthetic peptides with heparin-binding activity may compete with ECP for cellular binding. Furthermore, the cell surface-binding ability of peptides, including D1 (NYQRRCKNQNK) derived from EDN and R1 (MTQGRCKPVNK) derived from RNase1 was tested using cell ELISA assay. As expected, similar to the TAT peptide, C1 and D1 peptides bound to the cell surface, whereas R1 peptide was devoid of such function (Fig. 6B). Interaction of Synthetic Peptides with LMWH—The ability of C1 to directly interact with LMWH was further investigated by FACE analysis. As expected, a decreased amount of free AMAC-LMWH signal was observed with increasing concentration of C1 peptide or TAT peptide (Fig. 7). In addition, D1 bound heparin as tightly as C1. Interestingly, although the peptide segment in the corresponding location of human RNase 1 also contains several positively charged residues, it did not bind heparin. Taken together, these results indicate that the RWRCK motif within loop L3 of ECP serves as a specific heparin binding site. Characterization of Association between C1 and Heparin Oligosaccharides—The definitive heparin-binding activity of the C1 peptide was determined by ITFE, which clearly demonstrated that C1 has high affinity for heparin (Fig. 8). The Kd values obtained for LMWH, dp9, and dp5 binding to the C1 peptide were 192.0, 229.1, and 274.8 nm, respectively (Table 2), indicating that C1 bound tighter to longer heparin oligosaccharides.TABLE 2Determination of dissociation constants (Kd) for C1 peptide with heparin oligosaccharidesOligosaccharideKdnmLMWH192.0dp9229.1dp5274.8 Open table in a new tab Growth Inhibitory Effect of ECP—The inhibition of lymphocyte and mammalian cell growth by ECP has been reported (9Fan T.C. Chang H.T. Chen I.W. Wang H.Y. Chang M.D. Traffic. 2007; 8: 1778-1795Crossref PubMed Scopus (50) Google Scholar, 30Peterson C.G. Skoog V. Venge P. Immunobiology. 1986; 171: 1-13Crossref PubMed Scopus (80) Google Scholar, 31Kimata H. Yoshida A. Ishioka C. Jiang Y. Mikawa H. Cell. Immunol. 1992; 141: 422-432Crossref PubMed Scopus (19) Google Scholar, 32Maeda T. Kitazoe M. Tada H. Llorens R.d. Salomon D.S. Ueda M. Yamada H. Seno M. Eur. J. Biochem. 2002; 269: 307-316Crossref PubMed Scopus (56) Google Scholar). As a physiological test, the cytotoxicity of ECP mt1 was monitored by MTT assay. As compared with wild-type ECP, ECP mt1 (R34A/W35A/R36A/K38A) exhibited a 2- to 3-fold increased IC50 value toward the Beas-2B cell line (Fig. 9 and Table 3). Thus, ECP mt1 without the key heparin binding sequence motif appears to be less cytotoxic than wild-type ECP, presumably due to weaker interaction with the cell surface, which in turn leads to decreased endocytosis.TABLE 3IC50 values for cytotoxic ECPProteinIC50μmECP wtaData taken from Ref. 916.05ECP mt138.52a Data taken from Ref. 9Fan T.C. Chang H.T. Chen I.W. Wang H.Y. Chang M.D. Traffic. 2007; 8: 1778-1795Crossref PubMed Scopus (50) Google Scholar Open table in a new tab Immunohistochemical Localization of ECP—To better understand the possible cellular targets of ECP, recombinant mature ECP was injected into the rat circulation through the tail vein. Immunohistochemical localization showed strong internalization of ECP in the tracheo- and bronchoepithelial cells 1 h post-injection (Fig. 10, A and B). ECP internalization was reduced when co-injection with heparin was carried out (Fig. 10C). For the negative control, no MBP signal was detected in tracheo- and bronchoepithelial cells (Fig. 10D), despite the MBP signal that could be detected in circulating blood (Fig. 10D). Here we provide the first direct evidence that a linear heparin binding site within ECP contributes to its cellular binding and cytotoxicity. The peptide derived from this domain exhibits a substantial binding affinity to heparin. The specific interaction with heparin mediated by the loop L3 region of ECP was analyzed by site-directed mutagenesis and synthetic peptides to determine the dissociation constant. We have shown that heparin dp5 binds to ECP with a Kd of 139.6 nm. Increased ECP binding affinity with increasing heparin oligosaccharide chain length was observed, indicating that longer heparin chains with an additional structural motif might strengthen polysaccharide-protein interactions. The mutagenesis study revealed that ECP mt1 (R34A/W35A/R36A/K38A), lacking the crucial heparin binding sequence motif, had reduced heparin-binding activity and was thus less cytotoxic than wild-type ECP. ITFE demonstrated that ECP mt1 had reduced heparin-binding activity. To further show direct heparin-binding activity of the loop L3 in ECP, the C1 and D1 peptides spanning residues 32–41 of ECP and EDN, respectively, were synthesized. Cell ELISA analysis revealed that C1 and D1 peptides interfered molecular interaction between ECP and cell surface HSPG. Studies of heparin binding by FACE showed that ECP C1 and EDN D1 bound as tightly to heparin as HIV-TAT peptide. On the other hand, the R1 peptide derived from RNase1, with positive charge density similar to C1, showed much lower affinity toward heparin, indicating that specific heparin-binding activity of C1 resulted not only from the positive charges but also from the amino acid sequence. Furthermore, in ITFE binding assays, C1 had high binding capacity for heparin, indicating that the heparin binding site in ECP is indeed located within the loop L3 region connecting helix α2 and strand β1. It has been shown that N-bromosuccinimide-modified ECP elutes from heparin chromatography at a slightly lower concentration of NaCl than unmodified ECP, indicating the contribution of tryptophan residues (Trp-10 and Trp-35) to heparin binding (32Maeda T. Kitazoe M. Tada H. Llorens R.d. Salomon D.S. Ueda M. Yamada H. Seno M. Eur. J. Biochem. 2002; 269: 307-316Crossref PubMed Scopus (56) Google Scholar). The cellular binding activity of ECP W10A was only slightly decreased (<10%) as compared with that of wild-type ECP, whereas ECP W35A exhibited 50% lower binding, strongly indicating the involvement of Trp-35 in heparin binding. 3T.-c. Fan, S.-l. Fang, C.-s. Hwang, C.-y. Hsu, X.-a. Lu, S.-c. Hung, S.-C. Lin, and M. D.-T. Chang, unpublished data. In this study, Trp-35 together with the positively charged residues in loop L3 of ECP are likely to constitute a novel heparin-binding motif and, thus, are responsible for the heparin-binding activity. The heparin-binding activity of ECP mt1 was reduced 5-fold as compared with that of wild-type ECP, suggesting that residues Arg-34, Trp-35, Arg-36, and Lys-38 contribute to the overall free energy of the heparin-ECP interaction. However, this mutant still retained some heparin-binding ability. Because there are 19 arginine residues on the surface of ECP, some of which may form an extended binding surface together with the loop L3, detailed analysis of the contribution of each positively charged residue remains to be determined. Interestingly, the putative binding site identified in this study shares common features with that of EDN, indicating that the loop L3 of eosinophil RNases may contribute to their shared functions. These results suggest that the heparin binding and uptake mechanism of EDN are likely to be similar to those of ECP. The accumulation of a high concentration of ECP on the cell surface through interaction with HSPGs likely results in membrane perturbation and subsequent cytotoxicity. Immunohistochemical staining showed that ECP had high affinity for tracheo- and bronchoepithelial cells. Although HSPGs are ubiquitously expressed on all kinds of cell surfaces and in the extracellular matrix, the HS disaccharide composition seems to be tissue-specific (33Maccarana M. Sakura Y. Tawada A. Yoshida K. Lindahl U. J. Biol. Chem. 1996; 271: 17804-17810Abstract Full Text Full Text PDF PubMed Scopus (241) Google Scholar, 34Ledin J. Staatz W. Li J.P. Gotte M. Selleck S. Kjellen L. Spillmann D. J. Biol. Chem. 2004; 279: 42732-42741Abstract Full Text Full Text PDF PubMed Scopus (208) Google Scholar). The HS disaccharide composition and the overall degree of sulfation of murine tissues have been analyzed. The HS fingerprint has shown that lung tissue contains relatively high overall 2-O-sulfation due to the presence of 2-O-sulfated glucuronic acid-containing disaccharides (34Ledin J. Staatz W. Li J.P. Gotte M. Selleck S. Kjellen L. Spillmann D. J. Biol. Chem. 2004; 279: 42732-42741Abstract Full Text Full Text PDF PubMed Scopus (208) Google Scholar). Moreover, HS is presented in block structures, consisting of highly sulfated and non-sulfated domains. It is possible that HSs of tracheo- and bronchoepithelial cells have unique characteristics that serve as the principle receptor or co-receptor for ECP. The most prominent example is the requirement of contiguous stretches of the disaccharide 2-O-sulfated iduronic acid N-sulfated glucosamine for efficient interaction with fibroblast growth factor-2, and sulfation at the 6-O position of heparin may be required for cell signaling (15Guimond S. Maccarana M. Olwin B.B. Lindahl U. Rapraeger A.C. J. Biol. Chem. 1993; 268: 23906-23914Abstract Full Text PDF PubMed Google Scholar, 26Raman R. Sasisekharan V. Sasisekharan R. Chem. Biol. 2005; 12: 267-277Abstract Full Text Full Text PDF PubMed Scopus (339) Google Scholar). In summary, we have determined that pentasaccharides are the minimal sugar motif that can bind to ECP. The specific heparin binding site located within the loop L3 of ECP is critical for its physiological function. Our results may assist in determining the physiological role of ECP in vivo and further rational design of heparin mimetics to interfere with the cellular interactions of cytotoxic ECP." @default.
• W2017987939 created "2016-06-24" @default.
• W2017987939 creator A5011622872 @default.
• W2017987939 creator A5012219010 @default.
• W2017987939 creator A5023852685 @default.
• W2017987939 creator A5051824902 @default.
• W2017987939 creator A5059689909 @default.
• W2017987939 creator A5078300335 @default.
• W2017987939 creator A5086666509 @default.
• W2017987939 creator A5088155237 @default.
• W2017987939 date "2008-09-01" @default.
• W2017987939 modified "2023-10-16" @default.
• W2017987939 title "Characterization of Molecular Interactions between Eosinophil Cationic Protein and Heparin" @default.
• W2017987939 cites W1503068946 @default.
• W2017987939 cites W1578995377 @default.
• W2017987939 cites W1662030037 @default.
• W2017987939 cites W1966944551 @default.
• W2017987939 cites W1969102762 @default.
• W2017987939 cites W1973920331 @default.
• W2017987939 cites W1982423863 @default.
• W2017987939 cites W1988687667 @default.
• W2017987939 cites W1991046721 @default.
• W2017987939 cites W1991710519 @default.
• W2017987939 cites W1991954944 @default.
• W2017987939 cites W1995889983 @default.
• W2017987939 cites W2000891730 @default.
• W2017987939 cites W2001058584 @default.
• W2017987939 cites W2002947104 @default.
• W2017987939 cites W2019428611 @default.
• W2017987939 cites W2032498806 @default.
• W2017987939 cites W2033818437 @default.
• W2017987939 cites W2042143995 @default.
• W2017987939 cites W2045045541 @default.
• W2017987939 cites W2045741895 @default.
• W2017987939 cites W2055203083 @default.
• W2017987939 cites W2064292429 @default.
• W2017987939 cites W2070010734 @default.
• W2017987939 cites W2074987771 @default.
• W2017987939 cites W2092751827 @default.
• W2017987939 cites W2093432710 @default.
• W2017987939 cites W2109065012 @default.
• W2017987939 cites W2113458633 @default.
• W2017987939 cites W2135044461 @default.
• W2017987939 cites W2155494269 @default.
• W2017987939 cites W2170387755 @default.
• W2017987939 cites W2418798292 @default.
• W2017987939 doi "https://doi.org/10.1074/jbc.m803516200" @default.
• W2017987939 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/18593710" @default.
• W2017987939 hasPublicationYear "2008" @default.
• W2017987939 type Work @default.
• W2017987939 sameAs 2017987939 @default.
• W2017987939 citedByCount "40" @default.
• W2017987939 countsByYear W20179879392012 @default.
• W2017987939 countsByYear W20179879392013 @default.
• W2017987939 countsByYear W20179879392014 @default.
• W2017987939 countsByYear W20179879392016 @default.
• W2017987939 countsByYear W20179879392017 @default.
• W2017987939 countsByYear W20179879392018 @default.
• W2017987939 crossrefType "journal-article" @default.
• W2017987939 hasAuthorship W2017987939A5011622872 @default.
• W2017987939 hasAuthorship W2017987939A5012219010 @default.
• W2017987939 hasAuthorship W2017987939A5023852685 @default.
• W2017987939 hasAuthorship W2017987939A5051824902 @default.
• W2017987939 hasAuthorship W2017987939A5059689909 @default.
• W2017987939 hasAuthorship W2017987939A5078300335 @default.
• W2017987939 hasAuthorship W2017987939A5086666509 @default.
• W2017987939 hasAuthorship W2017987939A5088155237 @default.
• W2017987939 hasBestOaLocation W20179879391 @default.
• W2017987939 hasConcept C171250308 @default.
• W2017987939 hasConcept C183882617 @default.
• W2017987939 hasConcept C185592680 @default.
• W2017987939 hasConcept C188027245 @default.
• W2017987939 hasConcept C192562407 @default.
• W2017987939 hasConcept C203014093 @default.
• W2017987939 hasConcept C2776042228 @default.
• W2017987939 hasConcept C2777037409 @default.
• W2017987939 hasConcept C2777557582 @default.
• W2017987939 hasConcept C2780841128 @default.
• W2017987939 hasConcept C55493867 @default.
• W2017987939 hasConcept C60483475 @default.
• W2017987939 hasConcept C86803240 @default.
• W2017987939 hasConcept C88675164 @default.
• W2017987939 hasConceptScore W2017987939C171250308 @default.
• W2017987939 hasConceptScore W2017987939C183882617 @default.
• W2017987939 hasConceptScore W2017987939C185592680 @default.
• W2017987939 hasConceptScore W2017987939C188027245 @default.
• W2017987939 hasConceptScore W2017987939C192562407 @default.
• W2017987939 hasConceptScore W2017987939C203014093 @default.
• W2017987939 hasConceptScore W2017987939C2776042228 @default.
• W2017987939 hasConceptScore W2017987939C2777037409 @default.
• W2017987939 hasConceptScore W2017987939C2777557582 @default.
• W2017987939 hasConceptScore W2017987939C2780841128 @default.
• W2017987939 hasConceptScore W2017987939C55493867 @default.
• W2017987939 hasConceptScore W2017987939C60483475 @default.
• W2017987939 hasConceptScore W2017987939C86803240 @default.
• W2017987939 hasConceptScore W2017987939C88675164 @default.
• W2017987939 hasIssue "37" @default.
• W2017987939 hasLocation W20179879391 @default.

• next