PEDS Advance Access originally published online on October 26, 2005
Protein Engineering Design and Selection 2006 19(1):27-35; doi:10.1093/protein/gzi072
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Generation of GPI-linked CCL5 based chemokine receptor antagonists for the suppression of acute vascular damage during allograft transplantation
1Medizinische Poliklinik, Schillerstrasse 42, 80336 Ludwig-Maximilians-University of Munich, 2Childrens Hospital, Technical University of Munich, Kölnerplatz 1, 80803 Munich and 3Department of Cellular and Molecular Pathology, German Cancer Research Center, Im Neuenheimer Feld 280, 69120 Heidelberg, Germany
4 To whom correspondence should be addressed. E-mail: Roghieh.Djafarzadeh{at}med.uni-muenchen.de
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Abstract |
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Limiting the acute vascular damage associated with leukocyte infiltration is a central issue in solid organ transplantation. The family of chemotactic cytokines (chemokines) helps to regulate leukocyte recruitment. Systemic treatment with the chemokine ligand-5 (CCL5) based antagonist Met-RANTES has previously shown to suppress acute damage to transplanted kidneys by blocking effector cell recruitment. To address problems associated with systemic long-term administration of chemokine receptor antagonists, a chemokine based reagent was designed to be integrated into endothelial surfaces of the organ just before transplantation. Proteins anchored by glycosylphosphatidylinositol (GPI), when purified and added to cells, are efficiently incorporated into their cell surface membranes. A series of modifications were introduced into the CCL5 protein to generate a functional antagonist. These included the addition of an N-terminal methionine group, a mutation to render the protein a dimer and a GPI signal sequence for surface expression. The resultant protein was stably expressed in CHO cells, GPI anchorage was confirmed and the protein purified by FPLC. Exogenously administered Met-CCL5(dimer)–GPI was efficiently inserted into the membrane of microvascular endothelial cells. The reagent is being tested in murine models of renal transplantation. The effect on subsequent immune induced damage will be assessed.
Keywords: CCL5/cell surface engineering/GPI anchor/RANTES/transplant rejection
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Introduction |
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During immune rejection of solid organ transplants, the direct recruitment of effector leukocytes from the peripheral circulation into interstitial tissues is controlled in part by the actions of chemotactic cytokines (Chemokines) (Nelson and Krensky, 2001
; Haskell et al., 2002). Chemokines, sequestered in solid phase on the endothelium (immobilized via electrostatic interactions) act as signposts for directing the selective recruitment of circulating leukocytes (Wells et al., 1999; Weber et al., 2001; Murphy, 2002). In response to chemokine stimulation, leukocytes upregulate integrins and undergo firm adhesion to the endothelial surface (Weber et al., 2001; Murphy, 2002). The leukocytes then migrate through the endothelium and enter the tissue space. Blocking or modifying this process is an important step in the control of transplant rejection (Nelson and Krensky, 2001; Haskell et al., 2002).
The chemokine RANTES/CCL5 is a chemotactic agent for ‘memory’ CD4+ T cells, monocytes and eosinophils, and is produced by many different cell types during allograft rejection (Schall et al., 1990; Nelson et al., 1997; Nelson and Krensky, 2001). CCL5 acts through the receptors CCR1, CCR3 and CCR5 (Murphy, 2002). Human RANTES/CCL5 is bioactive in mouse and rat (Wiedermann et al., 1993; von Hundelshausen et al., 2001; Stojanovic et al., 2002). Blocking analogues of the human CCL5 protein bind to human and rodent CCL5 receptors with high avidity but do not induce signaling (Proudfoot et al., 1996, 1999). In acute and chronic models of allograft rejection, systemic daily treatment with a RANTES based functional antagonist (Met-RANTES) was shown to dramatically suppress the acute tissue damage underlying transplant rejection (Grone et al., 1999; Bedke et al., 2002; Song et al., 2002; Stojanovic et al., 2002; Yun et al., 2004).
The systemic administration of recombinant proteins can be quite expensive. In this instance, for a biological effect to be seen in transplantation experiments, the RANTES based antagonistic protein had to be administered daily over an extended period of time (Grone et al., 1999; Song et al., 2002). This could be problematic as high circulating protein levels could have unwanted side effects on other organ systems. To help address these issues an approach was developed to selectively apply a CCL5-based antagonistic protein only to the vascular cell surface before transplantation. This was achieved by fusing an N-terminal methionine non-aggregating version of CCL5 to a glycosylphosphatidylinositol (GPI) anchor. Proteins that are anchored by GPIs, when purified and added to cells, are incorporated into their cell surface membranes and retain native protein function (Medof et al., 1996; Premkumar et al., 2001). This method was previously referred to as ‘cell painting’ (Medof et al., 1996). The perfusion of a GPI-anchored chemokine based antagonist through the organ before transplantation would allow the insertion of the GPI anchor into the microvascular endothelial cell membranes and thus presentation of the antagonist to the circulating leukocytes. Such an approach could provide protection to the vasculature of the allograft during the critical first few days after transplantation and could thus significantly limit the acute vascular damage associated with poor prognosis for transplant engraftment. We describe here the generation of a GPI-anchored RANTES based antagonist.
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Materials and methods |
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Cell culture
Chinese hamster ovary cells deficient in DHFR (CHO/dhfr–) (ATCC no. CRL 9096; Rockville, MD, USA) were cultured in complete 
-medium (GIBCO BRL, Life Technologies GmbH, Eggenstein, Germany) supplemented with 10% heat-inactivated dialyzed FCS (GIBCO BRL) and HT supplement (GIBCO BRL). Human microvascular endothelial cells (HMVCs) were obtained from Promocell (Heidelberg, Germany) and cultured in the media provided. The THP-1 monocyte cell line was obtained from the American Type Culture Collection and cultured in RPMI 1640 medium (GIBCO BRL) supplemented with 2 mM L-glutamine (Biochrom KG, Berlin) and 10% heat-inactivated FCS (Biochrom KG, Berlin). Fresh medium was provided every third day and the cultures were split when cells were confluent. Recombinant CCL5 protein (#278-RN) was obtained from R&D Systems (Minneapolis, MN, USA).
Construction and stable expression of GPI-anchored CCL5 modifications in CHO/dhfr–
The GPI-anchor attachment sequence of LFA-3 (Kirby et al., 1995) was amplified by PCR and cloned into the pEF-DHFR vector (Mack et al., 1995; Djafarzadeh et al., 2004). The sense primer corresponded to nucleotides 616–633 of LFA-3 and contains an integrated XbaI restriction site to facilitate subcloning (underlined) (5'-TCTTTGGAGARGAGCTCTAGAACAACCTGTATCCCAAGCAG-3'). The antisense primer corresponded to nucleotides 860–877 of LFA-3 and included a SalI site (underlined) (5'-TCCCGCGGCCGCTATTGGCCGACGTCGACTCATAATACATTCATATACAGCACAATACATGTTG-3'). The DNA fragment encoding hCCL5 but excluding the stop codon was amplified by PCR from cDNA (Schall et al., 1988). The sense primer included the initiation codon for human CCL5 with a modification to include an EcoRI restriction site (underlined) (forward primer: 5'-CGGCGGAATTCATGAAGGTCTCCGCGG-3'). The antisense primer introduced an XbaI site (underlined) (reverse primer: 5'-TGGGATACAGGTTGTTCTAGAGCTCATCTCCAAAGAGTTGATG-3'). The GPI anchor signal sequence and fusion contributed nine additional amino acids to the carboxyl end of CCL5 (SSRTTCIPS). The amplified gene sequences were then subcloned sequentially into the pEF-DHFR vector and stably introduced into CHO(dhfr–) using electroporation as described previously (Mack et al., 1995).
Modifications of the CCL5 sequence included the introduction of a N-terminal methionine between the signal sequence of the mature protein, exchange of the glutamic acid at position 66 for alanine (E66A), generating a dimer and exchange of the glutamic acid at position 26 for alanine (E26A) rendering the resultant protein a tetramer (Figure 1) (Czaplewski et al., 1999). The mutations were generated by site-directed mutagenesis using the QuickChangeTM Site-Directed Mutagenisis Kit (Stratagene® Catalog no. 200518), used according to the manufacturer's directions. The primers used were Met-CCL5 forward primer: 5'-CCTGCATCTGCCATGTCCCCATATTCCTCG-3', reverse primer: 5'-GGACGTAGACGGTACAGGGGTATAAGGAGC-3'; E66A forward primer: 5'-CGTGCCCACATCAAGGCGTATTTCTACACCAG-3', reverse primer: 5'-CTGGTGTAGAAATACGCCTTGATGTGGGCACG-3'; and E26A forward primer: 5'-GTACATCAACTCTTTGGCGATGAGCTCTAGAACAACC-3', reverse primer: 5'-GGTTGTTCTAGAGCTCATCGCCAAAGAGTTGATGTAC-3'. All mutants were generated in pUC18 (Yanisch-Perron et al., 1985) and DNA sequenced before subcloning.
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Fluorescence activated cell sorting (FACS) analysis
Expressed human CCL5 and its variants were identified on the CHO cell surface by FACS analysis. Cells were detached with 1.5 mM EDTA (Biochrom A, Berlin, Germany) in 1x PBS and incubated for 60 min on ice with the CCL5 antibody VL3 or VL4 (von Luettichau et al., 1996; Nelson, 2000) (5 µg/ml) or the IgG2b
isotype control (Sigma) (5 µg/ml). The cells were washed three times with 1x PBS and incubated with an FTIC-conjugated donkey anti-mouse (DAKO A/S, Denmark) IgG for 45 min on ice. The cells were washed three times with 1x PBS and analyzed on a flow cytometer (FACS Calibur, Becton, Dickinson and Company, USA) using the CellQuest analysis software.
Purification of Met-CCL5(dimer)–GPI protein
The cells were washed three times with cold 1x PBS and detached with 1.5 mM EDTA. The cells were rotated for 1 h at 4°C with hypertonic lysis buffer [5 M sodium phosphate, 2 mM MgCl, 0.1 mM EDTA, protease inhibitor cocktail tablets (Roche, Basel, Switzerland)]. Cell membranes were isolated with the extraction buffer [100 mM NaCl, 1% Triton X-100 Hydrogenated (Triton X-100 H) (CALBIOCHEM, Merck Darmstadt, Germany), 10 mM sodium phosphate, 5 mM EDTA, protease inhibitor cocktail tablets, pH 7.4] and were rotated for 1 h at 4°C followed by centrifugation at 11 000 g for 20 min at 4°C. The supernatant was used for further purification using FPLC.
Heparin affinity chromatography (Heparin–Sepharose Fast Flow, Amersham Biosciences, Sweden) was used for the initial column purification step. The column was equilibrated with 100 mM NaCl, 10 mM sodium phosphate and 0.1% Triton X-100 H (pH 7.4), and the Met-CCL5(dimer)–GPI protein was eluted from the heparin column with 1200 mM NaCl, 0.1% Triton X-100 H and 10 mM sodium phosphate (pH 7.4). The eluted fractions containing Met-CCL5(dimer)–GPI were pooled and the buffer changed using a desalting column (HiTrap Desalting; Amersham Biosciences, Uppsala, Sweden).
A cation exchange column (SP Sepharose HP; Amersham Biosciences) was used as the next chromatography step. The column was equilibrated with 100 mM NaCl, 10 mM sodium phosphate and 0.1% Triton X-100 H (pH 7.4), and the Met-CCL5(dimer)–GPI protein was eluted from the cationic exchange column using a salt gradient from 900 to 1200 mM NaCl, 0.1% Triton X-100 H and 10 mM sodium phosphate (pH 7.4). The protein pool was concentrated 20-fold using a Centricon concentrator Amicon Ultra with a pore size of 10 000 MWCO (Millopore, Eschborn, Germany). The concentrated protein was then applied to a gel filtration TSK column G3000SWXL (TOSOH Corporation, Tokyo, Japan) using 1x PBS and 0.025% Triton X-100 H. The final step used a desalting column equilibrated with 1x PBS to decrease the concentration of Triton X-100 H. The Met-CCL5(dimer) protein was detected in column fractions by western blot analysis using VL3 monoclonal antibody (von Luettichau et al., 1996).
CCL5 ELISA
A human CCL5 specific ELISA kit was used to detect Met-CCL5(dimer) in the solution using the RANTES/CCL5 protocol applied according to the manufacturer's directions (R&D Systems). The capture anti-human CCL5 mAB (840216), biotinylated goat anti-human CCL5 detection antisera (840217), strepavidin–HRP (890803) and rhCCL5 protein (840218) were purchased from R&D Systems GmbH (Wiesbaden, Germany).
GPI-anchor cleavage by phospholipase C
Stably expressing CHO cells were treated with 60 ng/ml of phosphatidylinositol-specific phospholipase C (PLC) (Sigma–Aldrich, Taufkirchen, Germany) in serum-free medium for 60 min at 37°C and 5% CO2, and subjected to FACS analysis.
Incorporation of CCL5–GPI into endothelial cell membranes
HuMVECs (5–10 x 106 cells/ml) were incubated with 150, 300, 450 and 700 ng/ml of purified hCCL5–GPI at 37°C/5% CO2. The cells were then washed three times with ice-cold PBS and analyzed by FACS using the human CCL5-specific monoclonal antibody VL3 (von Luettichau et al., 1996) (see above).
Transendothelial migration assay
Transendothelial migration was measured using a modified Boyden Chamber assay as described previously (Weber et al., 2001). Primary dermal microvascular endothelial cells were grown to confluency on Costar (#3472) 24-well inserts with a pore size of 3 µm and diameter of 6.5 mm. THP-1 cells were labeled with 10 µg/ml Calcein (Molecular Probes, Eugene, OR) and suspended at a concentration of 2 x 105 in 100 µl of assay media (RPMI 1640, 0.1% BSA and 10 mM HEPES). The cells were placed in triplicate in the upper wells of the chamber and media control or recombinant human CCL5 was placed in the lower chamber (0, 50 and 100 ng/ml) (600 µl assay media). Plates were incubated for 2 h at 37°C and increase in fluorescence was measured at emission (535 nm) and excitation (485 nm) using a TECAN GENios Plus ELISA reader (TECAN, Grödig, Austria) and the XFluor 4 software (TECAN). Migration index was evaluated against the control. Results represent two independent experiments performed in triplicates as described previously (Djafarzadeh et al., 2004).
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Results |
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Construction and purification of the CCL5–GPI fusion protein
Human CCL5 was cloned from cDNA (Schall et al., 1990) using hCCL5 specific primers and PCR (see Materials and methods). The CCL5 cDNA sequence (without a translation stop codon) was fused to a GPI signal sequence cloned from LFA-3 cDNA (Kirby et al., 1995).
Modifications were then introduced into the CCL5–GPI construct. A methionine residue was added to the N-terminal of the mature protein, just after the signal sequence. Met-CCL5–GPI was then combined with either the E66A or E26A mutation (see Materials and methods) to generate non-aggregating functional antagonists fused to a GPI anchor. The resulting constructs were then subcloned into the pEF-DHFR vector and stably introduced into CHO cells (Mack et al., 1995).
Surface human CCL5 antigen expression was determined using FACS analysis and the hCCL5 specific antibody VL3 (von Luettichau et al., 1996; Nelson, 2000) (Figure 2A–D). VL3 was selected from a panel of previously characterized anti-CCL5 monoclonal antibodies (von Luettichau et al., 1996; Nelson, 2000). Unlike many of the other anti-CCL5 monoclonal antibodies tested, the modifications made to the CCL5 protein did not disrupt the epitope recognized by VL3.
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PLC digestion confirmed GPI anchorage of CCL5
GPI anchorage of the Met-CCL5(dimer)–GPI protein was confirmed following PI-PCL (phosphatidylinositol-specific phopholipase) digestion. CCL5-specific FACS using the VL3 antibody demonstrated loss of surface signal, which correlated with digestion (Figure 3A). Interestingly, the native CCL5–GPI linked protein proved resistant to PLC digestion (Figure 3B) suggesting that the oligomeric version of the protein may not allow adequate access to the PLC enzyme and that partial digestion of the GPI anchors linking the protein to the surface may not allow efficient release of aggregated protein from the membrane.
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Purification of the CCL5–GPI fusion protein
CCL5–GPI fusion protein was purified from the transfected cells by Triton X-100 detergent extraction followed by column purification using heparin–sepharose, cationic exchange and size exclusion chromatography. The general purification scheme is outlined in Figure 4 (and Materials and methods). The presence of the Met-CCL5(dimer)–GPI protein was followed by western blot analysis using the VL3 antibody (von Luettichau et al., 1996) and silver stain (Figure 4).
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Exogenously added CCL5–GPI-anchored protein is incorporated into cell membranes in a dose-dependant manner
To demonstrate reincorporation of the GPI–CCL5 protein into cell membranes 150, 300, 450 and 700 ng/ml of the purified Met-CCL5(dimer)–GPI protein was added to microvascular endothelial cells in cell culture, incubated for 30 min at 37°C. The cells were then washed three times and subjected to FACS analysis (Figure 5A). The addition of increasing concentrations of Met-CCL5(dimer)–GPI shows a dose-dependent incorporation of the GPI-anchored protein into the endothelial cell surface as evidenced by FACS (Figure 5B).
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The efficiency of reincorporation of the Met-CCL5(dimer)–GPI protein was determined using two approaches. In the first, a specific concentration of GPI-anchored agent (700 ng/ml) was incubated with endothelial cells as described in Figure 5A. The cells were washed and the amount of protein left in the wash supernatant was determined using a human RANTES specific ELISA assay. In the second approach, following reincorporation using 700 ng/ml of the protein into endothelial cells, the cells were washed and the GPI anchor was cleaved by treatment with 120 ng/ml PLC. The cells were washed again and the freed protein present in the supernatant was measured using human RANTES ELISA. Using the first method the efficiency of incorporation was determined to be 52% of the starting material. Using the PLC digestion/ELISA approach, 24% of the starting protein was recovered after digestion. Based on these two methods between 25 and 50% of 700 ng/ml of the starting material is incorporated into cell membranes. This is similar to the efficiency of incorporation calculated for other GPI-anchored proteins (Djafarzadeh et al., 2004
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We and others have previously demonstrated that Met-RANTES is a functional antagonist of the chemokine receptors CCR1, CCR3 and CCR5 (Proudfoot et al., 1996, 1999; Grone et al., 1999). In addition, the aggregation mutant (dimer E66A) has also been shown to be an antagonist for CCR1 (Baltus et al., 2003).
Functionality of the Met-CCL5(dimer)–GPI protein was demonstrated using transendothelial migration assays utilizing a modified Boyden Chamber assay and primary HMVCs (Weber et al., 2001) (Figure 5C). HMVC were grown on Transwell inserts. Transendothelial migration of the monocyte cell line was optimal at 50 ng/ml rhCCL5 (data not shown) (Weber et al., 2001). To test the ability of the Met-CCL5(dimer)–GPI agent to block CCL5 receptors, the confluent HMVC/Transwells were incubated for 30 min at room temperature with 100 ng/ml Met-CCL5(dimer)–GPI, which was then washed two times with media lacking FCS. Endothelial cells so treated, effectively blocked transmigration of the monocyte cell line to CCL5 signal and in addition also showed a reduced background transmigration of the cells to media control (Figure 5C).
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Discussion |
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Purified GPI-anchored proteins when added to cells are incorporated into their surface membranes. Once incorporated, the anchored proteins can exert their native biologic functions (Medof et al., 1996
; Djafarzadeh et al., 2004). Such transfer of proteins directly to cells, termed cell painting or surface protein engineering constitutes an alternative to conventional gene transfer for manipulating cell surface composition (Medof et al., 1996; Kooyman et al., 1998; Premkumar et al., 2001; Djafarzadeh et al., 2004).
The chemokines represent a large family of cytokines first characterized by their ability to induce the selective migration of leukocyte subpopulations. Chemokines working in concert with selectins and integrins act as the directional cues to sort, direct and fine tune leukocyte trafficking (Murphy et al., 2000; Nelson and Krensky, 2001; Bacon et al., 2002). The actions of chemokines are mediated through a family of seven transmembrane serpentine Gi/Go-protein coupled receptors. Each chemokine receptor has distinct chemokine specificity and a restricted expression on subclasses of leukocytes (Bacon et al., 2002). CCL5 acts through three receptors CCR1, CCR5 and CCR3. These receptors are expressed on monocytes (CCR1 and CCR5), T cells (CCR1, CCR3 and CCR5), natural killer cells (CCR1 and CCR5) and eosinophils (CCR1 and CCR3) (Nelson and Krensky, 2001; Bacon et al., 2002). CCL5 can also have other effects on cells. It can enhance effector function in specific leukocyte subsets including monocytes, T cells and natural killer cells and can activate respiratory burst in eosinophils (Kuna et al., 1992, 1993; Maghazachi et al., 1994; Nelson and Krensky, 1998).
The role of RANTES/CCL5 and its receptors in the propagation of allograft rejection has been previously demonstrated in diverse models of acute allograft rejection (Grone et al., 1999; Bedke et al., 2002; Song et al., 2002; Yun et al., 2004). Met-RANTES treatment was shown to dramatically reduce acute and chronic tissue damage in mouse models of heart transplantation (Song et al., 2002) as well as in acute and chronic rat renal allograft rejection models (Grone et al., 1999; Song et al., 2002; Yun et al., 2004). Mechanisms of action of Met-RANTES and CCL5 receptors were demonstrated in experiments using monocyte and CD4+ T cell attachment assays on microvascular endothelium under physiological flow conditions, where leukocyte firm adhesion was shown to be suppressed by the antagonist (Grone et al., 1999; Weber et al., 2001; Baltus et al., 2003). This effect leads in vivo to a pronounced suppression of vascular injury in the Met-RANTES treated animals compared with the untreated animals. Limiting of this early damage to vessels was linked to a general suppression in the development of chronic allograft rejection (Grone et al., 1999; Song et al., 2002). These results have demonstrated that chemokine receptor antagonists can influence discrete stages in the initiation and propagation of inflammatory processes and that they represent important targets for therapeutic intervention (Nelson and Krensky, 2001; Weber et al., 2001).
NMR analysis of the structure of CCL5 shows that the N-terminus is unconstrained and is presented out from the core protein. The integrity of the N-terminus of CCL5 is crucial for receptor binding and cellular activation. The extension of human CCL5 by a single methionine residue at the N-terminus is sufficient to produce a potent and selective antagonist (Skelton et al., 1995; Proudfoot et al., 1996). The C-terminus appears well suited for a GPI anchor allowing apical presentation of the antagonistic N-terminus from the endothelial surface (Skelton et al., 1995). In addition, there is a clear difference in the signaling properties of aggregated and disaggregated CCL5 forms. The CCL5 protein forms higher-order oligomers that have been shown to be essential for CCR1-mediated arrest (Baltus et al., 2003). Single charged residues either at position 26 or 66 play key roles in this aggregation (Czaplewski et al., 1999). CCL5 mutants deficient in oligomerization have been shown to block recruitment of monocytes and CD45RO+ CD4+ T cells triggered by RANTES immobilized on activated endothelium under flow conditions (Baltus et al., 2003).
To generate a GPI-anchored version of a CCL5-receptor antagonist two additional modifications were introduced into the CCL5 sequence. The first was the addition of an N-terminal methionine interspersed between the signal sequence and the mature protein. Second, a mutation of the glutamic acid residue at amino acid 66 to an alanine, which has been previously shown to render the protein dimeric (Czaplewski et al., 1999). These changes insured that the resultant protein would be antagonistic (Proudfoot et al., 1996; Czaplewski et al., 1999). In addition, the resultant protein should be easier to work with as a non-aggregating version of the protein would be easier to purify, quantify and administer.
We describe here the generation, purification and preliminary characterization of Met.CCL5(dimer)–GPI. GPI anchorage was demonstrated through PLC digestion. The purified protein was found to be efficiently incorporated into the surface of endothelial cells and to block the transendothelial migration of a monocyte cell line. The agent Met-CCL5(dimer)–GPI is currently being evaluated in murine and rat renal allograft transplant models. It is hoped that that the protein represents a viable alternative to the systemic administration of recombinant antagonists for the suppression of acute vascular damage to allografts.
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5 These authors contributed equally to this work.
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The work was supported by the grants SFB 571 (C2), GK 455 and DFG NE 648/2-1 to P.J.N. and SFB 405 (B10) to H.J.G. Madleine Schickedanz Kinder Krebsstiftung and Mucos-Stifung support was given to I.v.L.
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Received May 9, 2005; revised August 30, 2005; accepted September 26, 2005.
Edited by Andreas Kungl
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