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• W2071472192 abstract "Ticks are blood-feeding parasites that secrete a number of immuno-modulatory factors to evade the host immune response. Saliva isolated from different species of ticks has recently been shown to contain chemokine neutralizing activity. To characterize this activity, we constructed a cDNA library from the salivary glands of the common brown dog tick, Rhipicephalus sanguineus. Pools of cDNA clones from the library were transfected into HEK293 cells, and the conditioned media from the transfected cells were tested for chemokine binding activity by chemical cross-linking to radiolabeled CCL3 followed by SDS-PAGE. By de-convolution of a single positive pool of 270 clones, we identified a full-length cDNA encoding a protein of 114 amino acids, which after signal peptide cleavage was predicted to yield a mature protein of 94 amino acids that we called Evasin-1. Recombinant Evasin-1 was produced in HEK293 cells and in insect cells. Using surface plasmon resonance we were able to show that Evasin-1 was exquisitely selective for 3 CC chemokines, CCL3 and CCL4 and the closely related chemokine CCL18, with KD values of 0.16, 0.81, and 3.21 nm, respectively. The affinities for CCL3 and CCL4 were confirmed in competition receptor binding assays. Analysis by size exclusion chromatography demonstrated that Evasin-1 was monomeric and formed a 1:1 complex with CCL3. Thus, unlike the other chemokine-binding proteins identified to date from viruses and from the parasitic worm Schistosoma mansoni, Evasin-1 is highly specific for a subgroup of CC chemokines, which may reflect a specific role for these chemokines in host defense against parasites. Ticks are blood-feeding parasites that secrete a number of immuno-modulatory factors to evade the host immune response. Saliva isolated from different species of ticks has recently been shown to contain chemokine neutralizing activity. To characterize this activity, we constructed a cDNA library from the salivary glands of the common brown dog tick, Rhipicephalus sanguineus. Pools of cDNA clones from the library were transfected into HEK293 cells, and the conditioned media from the transfected cells were tested for chemokine binding activity by chemical cross-linking to radiolabeled CCL3 followed by SDS-PAGE. By de-convolution of a single positive pool of 270 clones, we identified a full-length cDNA encoding a protein of 114 amino acids, which after signal peptide cleavage was predicted to yield a mature protein of 94 amino acids that we called Evasin-1. Recombinant Evasin-1 was produced in HEK293 cells and in insect cells. Using surface plasmon resonance we were able to show that Evasin-1 was exquisitely selective for 3 CC chemokines, CCL3 and CCL4 and the closely related chemokine CCL18, with KD values of 0.16, 0.81, and 3.21 nm, respectively. The affinities for CCL3 and CCL4 were confirmed in competition receptor binding assays. Analysis by size exclusion chromatography demonstrated that Evasin-1 was monomeric and formed a 1:1 complex with CCL3. Thus, unlike the other chemokine-binding proteins identified to date from viruses and from the parasitic worm Schistosoma mansoni, Evasin-1 is highly specific for a subgroup of CC chemokines, which may reflect a specific role for these chemokines in host defense against parasites. Ticks are blood-feeding external parasites that can infest a wide variety of mammals, including humans. The hard tick species, or Ixodidae, are characterized by the fact that they feed for extended periods of time on their hosts, ranging from a few days to 2–3 weeks depending on the stage in the life cycle and on the species. The normal mammalian response to parasites that penetrate the skin is to unleash an immunological response aimed at destroying or neutralizing the foreign agent. The tick has to block these responses to survive and feed successfully. To do this ticks have developed an armory of anti-inflammatory, anti-coagulant, and anti-pain molecules that they inject into their hosts, allowing them to remain essentially undetected while they feed (1Ribeiro J.M. Infect. Agents Dis. 1995; 4: 143-152PubMed Google Scholar, 2Ribeiro J.M. Makoul G.T. Levine J. Robinson D.R. Spielman A. J. Exp. Med. 1985; 161: 332-344Crossref PubMed Scopus (308) Google Scholar, 3Brossard M. Wikel S.K. Parasitology. 2004; 129: S161-S176Crossref PubMed Scopus (205) Google Scholar). Many tick-borne pathogens such as the lyme disease-causing bacterium Borrelia burgdorferi and viruses (Rocky Mountain spotted fever, Colorado fever, tick-borne encephalitis virus) are also thought to exploit these activities to facilitate their own transmission and replication (4Nuttall P.A. Parasitology. 1998; 116: S65-S72Crossref PubMed Google Scholar, 5Gillespie R.D. Mbow M.L. Titus R.G. Parasite Immunol. (Oxf.). 2000; 22: 319-331Crossref PubMed Scopus (144) Google Scholar, 6Schoeler G.B. Wikel S.K. Ann. Trop. Med. Parasitol. 2001; 95: 755-771Crossref PubMed Scopus (95) Google Scholar, 7Jones L.D. Hodgson E. Nuttall P.A. J. Gen. Virol. 1989; 70: 1895-1898Crossref PubMed Scopus (102) Google Scholar). Chemokines are small chemoattractant cytokines that are key mediators of the inflammatory response against parasites. They alert the immune system to the assault and play an important role in recruiting specific leukocyte populations to the site of infection to destroy the invader. Many pathogenic microorganisms including viruses and protozoa have developed immune evasion strategies based on the interference of cytokine- and chemokine-mediated inflammatory signals. Over the last few years a number of distinct chemokine-binding proteins (CKBPs) 4The abbreviations used are:CKBPchemokine-binding proteinPBSphosphate-buffered proteinRANTESregulated on activation normal T cell expressed and secretedBS3bis(sulfosuccinimidyl) suberateNi-NTAnickel-nitrilotriacetic acidMALDI-TOFmatrix-assisted laser desorption ionization time-of-flightSPAscintillation proximity assayCHOChinese hamster ovaryHEKhuman embryonic kidneySPRsurface plasmon resonance have been isolated from members of the herpes and pox family of viruses, which have been shown to inhibit chemokine activity both in vitro and in vivo and play an important role in immune evasion (for reviews, see Refs. 8Alcami A. Nat. Rev. Immunol. 2003; 3: 36-50Crossref PubMed Scopus (452) Google Scholar and 9Lalani A.S. McFadden G. Cytokine Growth Factor Rev. 1999; 10: 219-233Crossref PubMed Scopus (50) Google Scholar). For example, deletion of the gene encoding the broad spectrum chemokine-binding protein, M-T1, from myxoma virus resulted in an extensive leukocyte infiltration in the dermis of the infected rabbits that could be substantially inhibited when animals were infected by the wild type virus, demonstrating that the virus had evolved a mechanism that prevented the recruitment of leukocytes to the site of infection in the form of a chemokine-binding protein (10Graham K.A. Lalani A.S. Macen J.L. Ness T.L. Barry M. Liu L.Y. Lucas A. Clark-Lewis I. Moyer R.W. McFadden G. Virology. 1997; 229: 12-24Crossref PubMed Scopus (193) Google Scholar). It would, therefore, seem likely that parasites such as ticks have evolved similar immune evasion mechanisms to ensure their survival. chemokine-binding protein phosphate-buffered protein regulated on activation normal T cell expressed and secreted bis(sulfosuccinimidyl) suberate nickel-nitrilotriacetic acid matrix-assisted laser desorption ionization time-of-flight scintillation proximity assay Chinese hamster ovary human embryonic kidney surface plasmon resonance Hajnicka et al. (11Hajnicka V. Kocakova P. Slavikova M. Slovak M. Gasperik J. Fuchsberger N. Nuttall P.A. Parasite Immunol. (Oxf.). 2001; 23: 483-489Crossref PubMed Scopus (79) Google Scholar, 12Kocakova P. Slavikova M. Hajnicka V. Slovak M. Gasperik J. Vancova I. Fuchsberger N. Nuttall P.A. Folia Parasitol. (Praha). 2003; 50: 79-84Crossref PubMed Scopus (17) Google Scholar) demonstrated the presence of anti-CXCL8 (interleukin-8) activity in salivary gland extracts from several ixodid tick species and have recently shown that tick saliva contains a variety of inhibitory activities directed against pro-inflammatory cytokines such as interleukin-2 and the chemokines CCL2/MCP-1, CCL3/MIP-1α, CCL5/RANTES, and CCL11/eotaxin (13Hajnicka V. Vancova I. Kocakova P. Slovak M. Gasperik J. Slavikova M. Hails R.S. Labuda M. Nuttall P.A. Parasitology. 2005; 130: 333-342Crossref PubMed Scopus (66) Google Scholar). We have, therefore, used an expression cloning approach to identify chemokine-binding proteins secreted in tick saliva. A cDNA library was prepared from salivary glands, resected from the ixodid tick, Rhipicephalus sanguineus (common brown dog tick), during feeding. A chemokine cross-linking assay was used to detect chemokine binding activity in conditioned medium harvested from HEK293 cells which had been transfected with the tick salivary gland cDNA library. Using this method we identified a family of chemokine-binding proteins that we have termed Evasins. In this paper we describe the cloning and characterization of Evasin-1, a CC chemokine-binding protein highly specific for CCL3, CCL4, and CCL18 (Pulmonary and activation-regulated chemokine (PARC)). Reagents and Recombinant Chemokines—Unless otherwise stated all reagents and chemicals were purchased from Sigma. Enzymes were obtained from New England Biolabs (Beverly, MA). 125I-Radiolabeled chemokines and chromatographic materials were obtained from GE Healthcare. Chemokines were purified as described previously (14Proudfoot A.E. Borlat F. Methods Mol. Biol. 2000; 138: 75-87PubMed Google Scholar). The cDNA encoding the ectromelia virus chemokine-binding protein (GenBank™/EBI Data Bank accession number AJ277111) was kindly provided by Dr. A. Alcami (University of Cambridge). The cowpox virus CKBP p35 protein was a gift from Dr. D. B. Wigley (Howard Hughes Medical Institute, Boston, MA). Preparation of Tick Saliva—R. sanguineus ticks were laboratory-reared as previously described (15Ferreira B.R. Machado R.Z. Bechara G.H. Vet. Parasitol. 1996; 62: 161-174Crossref PubMed Scopus (9) Google Scholar). All ticks used for infestations were 1–3-month-old adults. To obtain engorged ticks for saliva collection, 20 dogs were infested with 70 pairs of adult R. sanguineus ticks contained in plastic feeding chambers fixed to their backs. The saliva-collection procedure was performed using engorged female ticks (after 3–5 days of feeding) by inoculation of 10–15 μl of a 0.2% (v/v) solution of dopamine in phosphate-buffered saline (PBS), pH 7.4, using a 12.7 × 0.33-mm gauge needle (BD Biosciences). Saliva was harvested using a micropipette and placed on ice. Pooled saliva samples were centrifuged through a 0.22-μm pore filter (Costar-Corning, Inc., Cambridge, MA) and stored at -20 °C until further use. Each saliva pool consisted of material harvested from more than 200 female ticks. The saliva protein concentration, determined using a bicinchoninic acid solution (Sigma), ranged from 1000 to 2000 μg/ml. Chemical Cross-linking Assay—Lyophilized, iodinated chemokines were resuspended at 0.23 nm in 50 mm HEPES buffer, pH 7.5, containing 1 mm CaCl2, 5 mm MgCl2, and 0.1% bovine serum albumin and incubated with 10 μl of conditioned media from transfected HEK293 cells or with 10 μl of tick saliva in the presence or absence of 25 mm bis(sulfosuccinimidyl) suberate (BS3) in a final volume of 50 μl for 2 h at room temperature with shaking. The cross-linking reaction was quenched by the addition of 5 μl of 10× SDS-PAGE sample buffer. The samples were analyzed by SDS-PAGE and scanned using a Personal FX phosphorimaging system (Bio-Rad) at a resolution of 100 μm. Construction of a Tick Salivary Gland cDNA Library—Tick salivary glands were dissected, rinsed with ice-cold PBS, and stored in RNAlater™ solution (50 mg of tissue/ml) (Ambion, Inc., Canada) at -70 °C until use. Total RNA was extracted from ∼50 mg of salivary glands using TRIzol™ (Invitrogen) according to the manufacturer's directions. A tick salivary gland cDNA library was constructed using a SMART® cDNA library construction kit (Clontech, Palo Alto, CA) according to the manufacturer's directions. An aliquot of the resultant λTriplEx2 phage cDNA library containing 2 × 106 plaque-forming units was converted into a plasmid cDNA library in pTriplEx2 in BM25.8 cells according to the manufacturer's protocol. The pTriplEx2 cDNA library was stored at -80 °C in LB medium containing 50% glycerol. For subcloning of the cDNA library into the mammalian cell expression vector, pEXP-Lib (Clontech), plasmid DNA was prepared from a 5-ml overnight bacterial culture inoculated with 5 μl of the glycerol stock (containing 1 × 108 colony-forming units) using the Wizard® Plus SV Minipreps DNA purification system (Promega, Madison, WI). The resultant plasmid DNA was digested with SfiI, fractionated on a 1.1% agarose gel and cDNA inserts were excised and purified using the QIAquick gel extraction kit (Qiagen, Basel, Switzerland) according to manufacturer's instructions. The cDNA inserts were ligated into SfiI-digested and dephosphorylated, pEXP-Lib vector (Clontech, Palo Alto, CA, USA) using the Quick Ligation™ Kit (New England Biolabs). Ligation reactions were transformed by heat shock into UltraMAX DH5alpha-FT competent cells (Invitrogen, Carlsbad, CA, USA) according to the manufacturer's protocol. The pEXP-Lib cDNA library transformation mix was titered and plated at ∼100 colonies per 10 cm diameter LB-agar plate containing 50 μg/ml ampicillin. A total of 120 plates were prepared and grown for ∼18 h at 37 °C, to yield large colonies (∼2 mm diameter). Bacterial colonies were harvested in 5 ml LB medium by scraping the plates with a sterile, triangular plastic loop. Cells were pelleted by centrifugation at 3500 rpm for 10 min at 4 °C. Plasmid DNA was prepared from each pool using a BioRobot 8000 (Qiagen) and stored at–20 °C in 10 mm Tris-HCl buffer, pH 8. Transfection of HEK293 Cells—HEK293 cells were maintained in a 5% CO2, humidified incubator in Dulbecco's modified Eagle's medium-F-12 Nut medium (Invitrogen) supplemented with 10% heat-inactivated fetal calf serum, 2 mm l-glutamine (Invitrogen), and 1% 100× penicillin-streptomycin solution (Invitrogen). The day before transfection a confluent culture of HEK293 cells was harvested by trypsinization and seeded at ∼2 × 104 cells/well in a 96-well plate that had been precoated with 10 μg/ml poly-d-lysine hydrobromide. The next day cells were transfected with 100 ng of plasmid DNA using the Geneporter2 transfection kit (Gene Therapy Systems) using the manufacturer's protocol for low concentration DNA and adherent cells and incubated at 37 °C for 3–4 days. The conditioned medium was then harvested, cell debris was removed by centrifugation, and the supernatants stored frozen at -80 °C until further use. Expression Cloning—Approximately 80–130 ng of plasmid DNA from each library pool or the positive control plasmid was transfected into HEK293 cells as described above. For the positive controls we used the pEXP-lib vector containing the p35 or vCCI cDNA-coding sequence. Cell culture supernatants (200 μl) were harvested 3 days after transfection and stored frozen. Before use culture supernatants were thawed and concentrated four times using a speed vacuum system. The concentrated culture supernatant (conditioned medium) was tested in a cross-linking assay using 125I-labeled CCL3 as described above. Plasmid DNA from pools that gave a positive signal in the cross-linking assay were retransformed into Escherichia coli, and the transformation mixes plated on 10-cm-diameter LB-amp plates to yield ∼100 colonies per plate. Plasmid DNA was then prepared from 100 individual colonies and re-transfected into HEK293 cells. The conditioned medium from each transfection was harvested after 3 days and retested in the cross-linking assay using 125I-labeled CCL3 as described above. Plasmid DNA derived from individual colonies which gave a positive signal in the cross-linking assay was sequenced on an Applied Biosystems 3700 DNA sequencer using a T7 and pEXP-Lib-3′ reverse primer (Clontech, Palo Alto, CA). The plasmid, which contained the cDNA sequence encoding the CCL3-binding protein (pEXP-Lib-Evasin-1), was subsequently used as a PCR template to generate a six-histidine (His)-tagged version of the cDNA, which was subcloned into the baculovirus vector, pDEST8 (Invitrogen), and into the mammalian cell expression vector pEAK12d (Edge Biosystems) using the Gateway™ cloning system (Invitrogen). Purification of Evasin-1 from Insect and Mammalian Cells—Evasin-1 with a C-terminal His tag was expressed in Trichoplusia ni (TN)5 insect cells using the baculovirus transduction system or was transiently expressed in HEK293 cells. Conditioned medium from transfected TN5 cells was harvested 72 h post-infection. Conditioned medium from HEK293 cells was harvested 6 days after transfection. The conditioned medium was diluted 8-fold for TN5 expression or 2-fold for expression in HEK293 cells in 50 mm sodium phosphate buffer, pH 7.5, containing 0.3 m NaCl and 10% (v/v) glycerol, respectively, before purification by nickel-affinity chromatography (Ni-NTA-agarose, Qiagen) according to the manufacturer's instructions. Fractions were analyzed by SDS-PAGE, and gels were stained with Coomassie Blue. The pooled peak fractions containing Evasin-1 were dialyzed against PBS and further purified by Sephadex 200 (GE Healthcare) size exclusion chromatography. Fractions of 0.5 ml were collected and analyzed by SDS-PAGE. Two additional gels were run with the same samples and analyzed by Western blotting using anti-His antibodies (Qiagen) according to the manufacturer's instructions and by cross-linking to 125I-labeled CCL3. Evasin-1-containing fractions were pooled, and aliquots were stored at -80 °C until further use or dialyzed against NH4HCO3 and lyophilized. Physicochemical Characterization—N-terminal sequence analysis of Evasin-1 was performed using an Applied Biosystems 475A protein sequenator with on-line phenylthiohydantoin derivative detection. MALDI-TOF spectra were obtained on a Voyager DE-PRO Biospectrometry Work station (Applied Biosystems, Foster City, CA). The instrument was calibrated over the mass range of interest using a standard set of reference proteins provided by the manufacturer. Sinapinic acid was used as the matrix. Size exclusion chromatography was performed by injecting 200 μl of a 1 mg/ml solution onto an analytical Sephadex 75 10/300 GL column (GE Healthcare) previously equilibrated in PBS and eluted at 0.5 ml/min. Elution profiles were monitored by UV absorption at 280 nm. The void volume (V0) was determined with blue dextran, and the column was calibrated with the following standards purchased from GE Healthcare: albumin (67 kDa), ovalbumin (43 kDa), chymotrypsinogen (20.4 kDa), and ribonuclease A (13.7 kDa). Deglycosylation—Evasin-1 produced in TN5 insect cells was subjected to enzymatic deglycosylation with endoglycosidase Hf and peptide-N-glycosidase F (New England Biolabs). A solution of 1 mg/ml Evasin-1 in 50 mm sodium citrate buffer, pH 5.5, was incubated with 25,000 units of endoglycosidase Hf at room temperature for 10 h. A second digestion with peptide-N-glycosidase F (PNGase F) was carried out by incubating 1 mg/ml Evasin-1 in 50 mm sodium phosphate buffer, pH 7.5, containing 10% Nonidet P-40 with 12,500 units of PNGase F at room temperature. The extent of digestion was followed by SDS-PAGE. Surface Plasmon Resonance—Real-time biomolecular interaction analyses were performed using a Biacore 3000 surface plasmon resonance (SPR) system. Recombinant Evasin-1-His was resuspended at 50 μg/ml in 10 mm sodium acetate buffer, pH 4.5, and directly immobilized on the flow cell of a CM4 chip (Biacore) by a standard amine coupling chemistry according to the manufacturer's instructions using the Biacore 3000 Wizard software. Approximately 750 response units of Evasin-1-His were coupled to the cell using this method. A blank cell was prepared using the chemical coupling as a control in the absence of protein. Experiments were performed at 25 °C with a flow rate of 30 μl/min using HBS-P running buffer (0.01 m HEPES, pH 7.4, 0.15 m NaCl, and 0.005% surfactant P20) (Biacore). For all binding experiments chemokines were resuspended at 0.1 μg/ml in running buffer and filtered through a 0.22-μm filter. The injection time was 2 min followed by a dissociation time of 2.5 min after injection. The chip was regenerated using 50 mm glycine buffer, pH 2.0, for 30 s. For each experiment chemokines were injected in triplicate in random order. For the kinetic experiments, serial 2-fold dilutions of CCL3 (ranging from 25 to 1.5 ng/ml), CCL4 (ranging from 50 to 3 ng/ml), CCL18 (ranging from 0.5 μg/ml to 50 ng/ml) were prepared in running buffer, filtered through a 0.22-μm filter, and injected over the experimental blank flow cells. The injection time was 3 min followed by a dissociation time of 15 min. The chip was regenerated using 50 mm glycine buffer, pH 2 for 30 s. Each chemokine dilution was injected in triplicate in random order. For the analysis the sensograms from the blank cell in addition to the sensograms obtained with the running buffer alone were subtracted from the binding to remove the nonspecific background. For the kinetic analyses the association (ka) and the dissociation (kd) values were determined simultaneously by globally fitting sensograms for an entire range of chemokine concentrations according to the 1:1 Langmuir fitting model. The apparent equilibrium dissociation constants (KD) were determined from the mean kinetics values with the equation KD = kd/ka. Saturation Binding—A saturation binding experiment was used to determine the affinity constant of 125I-labeled CCL3 binding to Evasin-1 using an scintillation proximity assay (SPA). His-tagged Evasin-1 (40 pm) was incubated with increasing concentrations of 125I-labeled CCL3 with or without a 500-fold excess of unlabeled CCL3 in 75 μl of PBS, pH 7.2 containing 1 mm CaCl2, 5 mm MgCl2, and 0.2% bovine serum albumin. Copper chelate-coated polyvinyl toluene-SPA beads containing scintillant (Amersham Biosciences RPNQ0095) (350 μg) were added in 25 μl of PBS and incubated for 72 h at room temperature with shaking. Competition of heparin for the binding of Evasin-1 to CCL3 was determined by the inclusion of heparin ranging from 1 × 10-3 to 1 × 103 μg/ml in the assay. The amount of bound 125I-labeled CCL3 was determined by measurement of the radioactivity using a β counter. Nonspecific binding, determined in the presence of a 500-fold excess of unlabeled CCL3, was subtracted and represented between 2 and 4.1% of the total counts bound per minute. The ability of Evasin-1 to bind to a chemokine receptor was investigated using iodinated Evasin-1 (custom product from GE Healthcare, specific activity 222 Ci/mmol) and CCR1 expressed in CHO membranes. Data were analyzed using GraphPad Prism software. Measurements were performed in duplicate. Equilibrium Competition Receptor Binding—The ability of Evasin-1 to inhibit binding of radiolabeled CCL3 and CCL4 (GE Healthcare) to their cognate receptors was determined using a SPA. Membranes expressing recombinant CCR1 and CCR5 were prepared as described (16Alouani S. Methods Mol. Biol. 2000; 138: 135-141PubMed Google Scholar). Serial dilutions of Evasin-1 were prepared in binding buffer (50 mm HEPES, pH 7.2 containing 1 mm CaCl2, 5 mm MgCl2, 0.15 m NaCl, and 0.5% bovine serum albumin) to cover the concentration ranges shown in Fig. 5. Wheat germ agglutinin SPA beads (Amersham Biosciences) were suspended in binding buffer at 10 mg/ml, and the final concentration in the assay was 0.25 mg/well. CHO cell membranes expressing CCR1 or CCR5 were diluted in binding buffer to 80 μg/ml. Equal volumes of membrane and bead stocks were mixed before performing the assay to reduce background. The final membrane concentration in the assay was 2 μg/ml, and that of 125I-labeled CCL3 and CCL4 was 0.1 nm. The plates were incubated at room temperature with shaking for 4 h. Measurements were performed in triplicate. Stoichiometry of the Evasin-1-CCL3 Complex—The apparent molecular mass of Evasin-1 in the presence of CCL3 or CXCL8 was analyzed by size exclusion chromatography as described above with a 1 mg/ml solution of Evasin-1 in PBS or a mixture of Evasin-1 and CCL3 or CXCL8, both at 0.5 mg/ml, in PBS. The fractions were analyzed on SDS-PAGE gels stained with Coomassie Blue. Identification of a CCL3-binding Protein in Tick Saliva—The existence of a chemokine-binding protein in tick saliva was demonstrated by the detection of a radiolabeled band migrating between 25 and 35 kDa after autoradiography of an SDS-PAGE gel following incubation of tick saliva with 125I-labeled CCL3 in the presence of the chemical cross-linker, BS3 (Fig. 1A). This cross-linking assay was subsequently used to screen conditioned media from HEK293 cells transiently transfected with a tick salivary gland cDNA library for the presence of the CCL3-binding protein. Construction and Screening of a Tick Salivary Gland cDNA Expression Library—Total RNA was extracted from salivary glands derived from ∼50 female R. sanguineus adult ticks that had engorged on dogs for 5 days. Total RNA was used to prepare a directionally cloned cDNA library in the phage vector, λTriplEx2. The resultant library contained ∼0.575 × 106 independent clones with an average insert size of between 0.3 and 1.5 kilobases. An aliquot of the phage library was then converted into a pTriplEx2 plasmid library. cDNA inserts were excised together from the pTriplEx2 cDNA library using SfiI and ligated into the mammalian cell expression vector, pEXP-lib. Plasmid DNA was prepared from 93 pools of clones, each containing ∼85–100 independent cDNAs. Pools were screened by transfecting HEK293/EBNA cells followed by testing of the conditioned media for cross-linking activity to 125I-labeled CCL3. Plasmid DNA from one of the pools which gave a positive signal in the cross-linking assay was retransformed into E. coli, and plasmid DNA isolated from 96 individual colonies was tested by transfection and cross-linking as described above. This process was repeated until a single cDNA (clone 59) giving rise to a positive signal in the cross-linking assay was identified (Fig. 1b, lane 3). The cDNA insert sequence in clone 59 appeared to be a full-length DNA with an open reading frame of 339 bp encoding a protein of 114 amino acids with a predicted signal peptide of 20 amino acids, which when cleaved gives rise to a mature protein of 94 amino acids, which we called Evasin-1 (Fig. 2). The cDNA contains a single AATTAA polyadenylation site which spans the stop codon. Data base searches indicated that the Evasin-1 cDNA sequence showed no significant homology to any other protein, nucleic acid sequence, or conserved domain by NCBI blast of public databases including available tick genome databases. The predicted mass of Evasin-1 is 10,466 Da, and the predicted isoelectric point is 4.29. The mature protein contains eight cysteine residues, suggesting the presence of four disulfide bonds. There are also three predicted N-linked glycosylation sites in the mature protein sequence. Given that the 125I-labeled chemokine migrates at 8 kDa, it appears that the binding protein produced by the tick is heavily glycosylated, as it migrates with a mass ranging from ∼17–27 kDa, which is considerably larger than the predicted mass. Protein Purification and Characterization of Evasin-1—The cDNA sequence encoding the predicted open reading frame of Evasin-1 was subcloned with a C-terminal His tag for expression of the recombinant protein in insect (TN5) and mammalian (HEK293/EBNA) cells. Evasin-1 was well secreted in both expression systems. Elution from the Ni-NTA column was accompanied by high Mr-contaminating proteins which were easily removed by size exclusion chromatography as shown for the HEK protein in Fig. 1c. This chromatographic step clearly demonstrated the extent of glycosylation resulting in a mass distribution during size exclusion chromatography, but the identity of the different forms was confirmed by Western blot with an anti-His antibody (Fig. 1d), and activity of all the glycosylated forms was demonstrated by the cross-linking assay (Fig. 1e). The recombinant protein expressed in mammalian and insect cell systems showed considerable differences in migration behavior on SDS-PAGE. Recombinant Evasin-1 from HEK293 cells migrated as a broad band between 20 and 30 kDa, whereas Evasin-1 produced in insect cells migrated as a broad band between 15 and 25 kDa (Fig. 3a). Because the His tag at the C terminus was present in both proteins, allowing capture on the Ni-NTA resin, and the N-terminal sequencing showed that the signal sequence was removed in both expression systems according to the in silico prediction (results not shown), the differences in mass were attributed to post-translational modifications, most likely, glycosylation. Treatment of Evasin-1 with a glycosidase (peptide-N-glycosidase F) or Evasin-1 produced by transfected cells cultured in the presence of tunicamycin resulted in a band migrating at 12 kDa, confirming that the mass differences could be solely due to differential glycosylation (Fig. 3a). The apparent molecular mass determined by size exclusion chromatography of insect cell expressed Evasin-1 was 22 kDa, and that of the mammalian expressed Evasin-1 was 31 kDa (data not shown), in accordance with their differential migration pattern on SDS-PAGE. Analysis by isoelectric focusing showed that the major difference in glycosylation appears to be in sialyation. The protein expressed in mammalian cells showed a typical ladder pattern on isoelectric focusing characteristic of sialylated proteins, whereas the insect-der" @default.
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• W2071472192 date "2007-09-01" @default.
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• W2071472192 title "Molecular Cloning and Characterization of a Highly Selective Chemokine-binding Protein from the Tick Rhipicephalus sanguineus" @default.
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• W2071472192 doi "https://doi.org/10.1074/jbc.m704706200" @default.
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