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Originally published In Press as doi:10.1074/jbc.M109309200 on February 6, 2002

J. Biol. Chem., Vol. 277, Issue 17, 14757-14763, April 26, 2002
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Truncation of NH2-terminal Amino Acid Residues Increases Agonistic Potency of Leukotactin-1 on CC Chemokine Receptors 1 and 3*

Jae Kwon LeeDagger , Eun Hwa LeeDagger , Yeo Pyo YunDagger , Kyungjae Kim§, KyuBum Kwack, Doe Sun Na||, Byoung S. Kwon, and Chong-Kil LeeDagger **

From the Dagger  College of Pharmacy and Research Center for Bioresource and Health, Chungbuk National University, Cheongju 361-763, South Korea, the § College of Pharmacy, Sahm Yook University, Seoul 139-742, South Korea, the  Immunomodulation Research Center, University of Ulsan, Ulsan 680-749, South Korea, and the || Department of Biochemistry, University of Ulsan, Seoul 138-763, South Korea

Received for publication, September 26, 2001, and in revised form, January 28, 2002

    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Leukotactin-1 (Lkn-1) is a human CC chemokine that binds to both CC chemokine receptor 1 (CCR1) and CCR3. Structurally, Lkn-1 is distinct from other human CC chemokines in that it has long amino acid residues preceding the first cysteine at the NH2 terminus, and contains two extra cysteines. NH2-terminal amino acids of Lkn-1 were deleted serially, and the effects of each deletion were investigated. In CCR1-expressing cells, serial deletion up to 20 amino acids (Delta 20) did not change the calcium flux-inducing activity significantly. Deletion of 24 amino acids (Delta 24), however, increased the agonistic potency ~100-fold. Deletion of 27 or 28 amino acids also increased the agonistic potency to the same level shown by Delta 24. Deletion of 29 amino acids, however, abolished the agonistic activity almost completely showing that at least 3 amino acid residues preceding the first cysteine at the NH2 terminus are essential for the biological activity of Lkn-1. Loss of agonistic activity was due to impaired binding to CCR1. In CCR3-expressing cells, Delta 24 was the only form of Lkn-1 mutants that revealed increased agonistic potency. Our results indicate that posttranslational modification is a potential mechanism for the regulation of biological activity of Lkn-1.

    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Chemokines are a family of small cytokines that induce migration and activation of leukocytes. They contain 4-6 conserved cysteine residues, and have been classified into four subfamilies based on the configuration of the first two cysteine residues near the amino terminus: CXC(alpha ), CC(beta ), C(gamma ), and CX3C (1-3). Chemokines exert their biological activities via binding to specific surface receptors, which belong to seven-transmembrane G protein-coupled receptors (4-6). Some of the biological activities of chemokines include induction of leukocyte migration, immunoregulation, suppression of hematopoietic stem cell proliferation, and suppression of human immunodeficiency virus (HIV)1 infection (7-10).

Lkn-1 is a human CC chemokine that binds to both CCR1 and CCR3 and induces chemotaxis and calcium influx in human neutrophils, monocytes, eosinophils, and lymphocytes (11). Chemotaxis of neutrophils distinguishes Lkn-1 from other CCR1 agonists such as human macrophage inflammatory protein-1alpha (MIP-1alpha ) (12). Lkn-1 is a member of a human CC chemokine subfamily that contains four conserved cysteines (11). Lkn-1, however, is distinct from other human CC chemokines in that it has two extra cysteines, which may form a third disulfide bond. Lkn-1 is also distinct from other human CC chemokines in that it has long amino acid residues preceding the first cysteine at the NH2 terminus. The mature form of Lkn-1 consists of 92 amino acids and has 31 amino acid residues preceding the first cysteine, whereas most human as well as mouse CC chemokines have 10 or fewer amino acid residues preceding the first cysteine (13).

Recombinant Lkn-1 produced in Escherichia coli also shows several distinguished characteristics. In contrast to other CC chemokines such as monocyte chemoattractant protein 1 (MCP-1), recombinant Lkn-1, which contains additional methionine and six histidines at its NH2 and COOH termini, respectively, shows almost normal biological activities (11). In addition, purified recombinant Lkn-1 undergoes a spontaneous site-specific cleavage producing a 24-amino acid shorter protein than the intact form of Lkn-1 (11). The biological significance of the spontaneous site-specific cleavage is not known yet. In the present study, we produced a series of NH2-terminal deletion mutants of Lkn-1 including the site-specific cleaved form, and the effects of each deletion were investigated in CCR1- or CCR3-expressing cells.

    EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Cells and Cell Culture-- HOS (human osteogenic sarcoma) cells expressing CCR1 and CCR3 and HEK (human embryonic kidney) 293 cells expressing CCR3 were provided by Dr. Ji-Ming Wang (National Cancer Institute, Frederick, MD). The cells were grown in Dulbecco's modified Eagle's medium (Invitrogen) containing 10% fetal bovine serum (Hyclone, Logan, UT), 2 mM glutamine, 100 units/ml penicillin, and 100 µg/ml streptomycin. HOS transfectant cells were selected regularly with 1 µg/ml puromycin (Sigma), and HEK transfectant cells were selected with 400 µg/ml G418 (Invitrogen).

Production and Purification of NH2-terminal Deletion Mutants of Lkn-1-- A plasmid containing full-length cDNA of Lkn-1 was used as a template in PCR reactions to create Lkn-1 mutants with progressive amino acid deletions at the NH2 terminus. The sequences of the oligonucleotide primers used for amplification are shown in Table I. All of the forward primers contained overhanging nucleotide sequences for glycine-isoleucine-glutamic acid-glycine-arginine (GIEGR) at the 5' end of the target gene, and the reverse primer contained stop codon of Lkn-1 cDNA. The PCR products were cloned into the E. coli expression vector, pET30Xa/LIC (Novagen, Madison, WI), and transformed into E. coli strain BL21(DE3). Expression of Lkn-1 mutant proteins was induced by isopropyl-1-thio-beta -D-galactopyranoside (Sigma). The mutant proteins were purified from bacterial lysate with Ni-NTA spin columns (Qiagen, Chatsworth, CA) according to the manufacturer's instructions. Eluted proteins were folded by gradually removing denaturants in the protein preparation by stepwise dialysis. Overhanging extra amino acid residues at the NH2 terminus including the polyhistidine tag were removed by cleavage with factor Xa (Novagen). For cleavage of 1 µg of target protein, 0.04 unit of factor Xa was added, and the mixture was incubated at 22 °C for 7 h. Final purification of the cleaved proteins was performed by chromatographic separation in Superdex Peptide HR 10/30 column (Amersham Biosciences) attached to AKTApurifier (Amersham Biosciences). The proteins were eluted with a 0.02 M phosphate buffer containing 0.25 M NaCl. Finally, purified mutant proteins were free of endotoxin by a Limulus amoebocyte assay (Associates of Cape Cod, Woods Hole, MA). The concentrations of the proteins were determined by micro-bicinchoninic acid assay kit (Pierce) according to the manufacturer's instructions using bovine serum albumin as standard protein.

                              
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Table I
Primers used for the generation of NH2-terminal deletion mutants

SDS-PAGE and Western Blot Analysis-- SDS-PAGE was performed as described previously (14). The gels were either stained with a 0.025% Coomassie Brilliant Blue R-250 or silver nitrate, or they were transferred onto nitrocellulose membrane (Haake Buchler Instruments, Saddle Brook, NJ). The membrane was probed with polyclonal rabbit anti-Lkn-1 and then with alkaline phosphate-labeled goat anti-rabbit Ig (Bio-Rad). The membrane was developed by the addition of enzyme substrates, 5-bromo-4-chloro-3-indolyl phosphate, and nitro blue tetrazolium (Bio-Rad).

Calcium Influx Assay-- Receptor activation was assessed by real time measurement of intracellular calcium concentration in cells labeled with Fura-2/AM (Molecular Probes, Eugene, OR) in MSIII fluorimeter (Photon Technology International, S. Brunswick, NJ) as described previously (4, 15). Receptor desensitization was tested by monitoring intracellular calcium changes in cells upon repeated chemokine stimulation at 100-s intervals. Results were expressed as excitation ratios at 340 and 380 nm.

Chemotaxis Assay-- Chemotactic activities were performed in a 48-microwell Boyden chamber (Neuroprobe, Cabin John, MD) as described previously (15). The lower wells were filled with 27 µl of buffer alone or with buffer containing chemokines, and the upper wells were filled with 50 µl of cell suspension (6×105 cells/ml). The two wells were separated by a fibronectin (Sigma)-coated polyvinylpyrrolidone-free polycarbonate filter (Neuroprobe) with 10-µm pores. Human MIP-1alpha and human eotaxin were purchased from PeproTech Inc. (Rocky Hill, NJ).

Receptor Binding Assay-- Lkn-1 mutant proteins were labeled radioactively with 125I using the chloramine T method (16). The specific activities of the labeled Lkn-1 mutants were ~ 8×107 cpm/µg protein. CCR1- or CCR3-transfected cells were suspended in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum at a concentration of 2×107 cells/ml; 100 µl of the suspension was added to each tube. Saturation binding assays were performed in a total volume of 0.2 ml containing 2×106 cells and 0.04 to ~10 nM 125I-labeled Lkn-1 mutants. Competition binding experiments were performed under the same conditions using 1 nM 125I-labeled Delta 0 and variable concentrations of competitors. The nonspecific binding was estimated by measuring the binding of the labeled ligand in the presence of 100-fold excess of unlabeled ligand. Samples were incubated at 4 °C for 90 min with continuous rotation. Incubation was terminated by centrifuging the cell suspension over 1 ml of 10% sucrose (Sigma) cushion. Cell pellets were cut from the tubes, and cpm were counted using a gamma counter.

    RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Production and Purification of NH2-terminal Deletion Mutants-- The intact form of Lkn-1 and its mutants lacking NH2-terminal 5, 10, 15, 20, 24, 27, 28, and 29 amino acid residues, respectively, were produced in E. coli using the T7 polymerase-based expression vector, pET-30 Xa/LIC. Scheme I depicts the NH2-terminal deletion mutants of Lkn-1 produced as well as the expression system used in the present study.


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  		Scheme 1.   Schematic diagram of the NH2-terminal deletion mutants of Lkn-1 and the expression vector. A, DNAs for the NH2-terminal deletion mutants were generated by polymerase chain reactions with primers (shown in Table I) from full-length cDNA of Lkn-1. B, the PCR products were cloned into the E. coli expression vector pET30Xa/LIC, which encodes fusion protein that has the 6-amino acid His-tagged and S-tagged sequences at the upstream of the target protein.

Recombinant fusion proteins were readily detected in bacterial lysate (Fig. 1A). The fusion proteins were purified from the bacterial lysate by nickel-chelating resin (Fig. 1B). The purified fusion proteins were strongly reactive with polyclonal rabbit anti-Lkn-1 antibody (Fig. 1C).


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  		Fig. 1.   Identification of progressive deletion of NH2-terminal region. A, recombinant fusion proteins expressed in the bacterial lysate were separated by SDS-PAGE and stained with Coomassie Brilliant Blue. B and C, recombinant fusion proteins purified from the bacterial lysate with Ni-NTA spin columns (Qiagen) were separated by SDS-PAGE and stained with Coomassie Brilliant Blue (B) or probed with rabbit polyclonal anti-Lkn-1 antibody after transfer to nitrocellulose membrane by electroblotting (C). Std indicates the protein size marker. Arrows indicate truncated forms of recombinant Lkn-1.

Purified fusion proteins were then cleaved with the protease, Factor Xa, which recognizes GIEGR sequences linked directly to the NH2 terminus of the target protein. Factor Xa cleaved most of the fusion proteins almost completely. Factor Xa, however, was less efficient in cleaving fusion proteins for Delta 27, Delta 28, and Delta 29. Thus, final purification and removal of uncleaved fusion proteins were performed by a chromatographic separation. Fig. 2A shows a representative chromatogram of the Delta 28 fusion protein that was digested with Factor Xa. The protein in the single dominant peak in Fig. 2A was collected and found to be a homogeneous protein that was cleaved with Factor Xa (Fig. 2B, lane 2). Likewise, other fusion proteins were also cleaved with Factor Xa, and then the cleaved proteins were purified by a chromatographic separation. The purified proteins contained undetectable level of endotoxin (data not shown).


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  		Fig. 2.   Removal of tags and final purification of the recombinant proteins. Recombinant fusion proteins were cleaved with Factor Xa and then separated by gel filtration using a Superdex peptide HR 10/30 column (Amersham Biosciences) to remove tagged peptides and uncleaved fusion proteins. Here, we show purification of Delta 28 fusion proteins as a representative example. A, Factor Xa-treated Delta 28 fusion proteins were separated by gel filtration on a Superdex peptide HR 10/30 column, and the eluted proteins were detected at lambda  = 214 nm. B, the protein in the single dominant peak in A was collected and separated in SDS-PAGE, and the gel was stained with silver nitrate. Lane 1 shows total proteins before the chromatographc separation, and lane 2 shows the finally purified Factor Xa-treated proteins. Std indicates the protein size marker.

Induction of Ca2+ Mobilization in CCR1- and CCR3-expressing Cells-- Recombinant proteins of the NH2-terminal deletion mutants were compared for calcium mobilization in CCR1 transfectant cells. When calcium flux-inducing activity was compared at 100 nM concentration, which is the concentration shown to induce maximal calcium flux by Delta 0, it was evident that agonistic potency decreases dramatically between Delta 28 and Delta 29 (Fig. 3A). To further confirm that deletion of one more amino acid from Delta 28 results in an almost complete loss of agonistic potency, calcium flux-inducing activity of Delta 29 was compared at several different concentrations with that of Delta 28 as well as that of MIP-1alpha . As shown in Fig. 3B, calcium flux-inducing activity of Delta 28 was dose-dependent, reaching a plateau at 10 nM. In contrast, Delta 29 elicited maximal response at 100 nM, but the level was at most comparable with that of 0.1 nM of Delta 28. These results show that at least 3 amino acid residues preceding the first cysteine are required for the agonistic activity.


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  		Fig. 3.   Comparison of calcium flux-inducing activity of the NH2-terminal deletion mutants on CCR1 transfectants. Calcium influx was measured in Fura-2/AM-loaded CCR1-HOS cells. Calcium flux-inducing activity was compared at a fixed concentration of 100 nM. A, the arrow indicates the time of addition of the indicated chemokine. B, dose-response curve of calcium flux-inducing activity of Delta 28, Delta 29, and MIP-1alpha . C, flux-inducing activity of the deletion mutants was further compared in a dose-response study, and some of the representative data are shown. D, the EC50 and the potency of the activity for each of the mutants were summarized. Relative activity was calculated from the peak amplitude elicited at the indicated concentration. The results show the mean ± S.D. of three separate experiments.

Potential differences in the agonistic potency were further examined for all of the NH2-terminal deletion mutants by varying protein concentration. We found that deletion of NH2-terminal amino acid residues up to 20 did not change agonistic potency for CCR1; the calcium flux-inducing activities of Delta 5, Delta 10, Delta 15, and Delta 20 were almost the same as that of Delta 0 (Fig. 3, C and D). Lkn-1 lacking NH2-terminal 24 amino acids (Delta 24), however, induced robust calcium flux responses compared with Delta 0. Delta 24 induced maximal calcium flux response at ~1 nM, which is an ~100-fold lower concentration than that required to induce similar level of response for Delta 0 (Fig. 3D). EC50 of Delta 24 and Delta 0 was ~0.2 and ~4.0 nM, respectively. Deletion of 3 or 4 more amino acid residues from Delta 24 (i.e. Delta 27 and Delta 28) also increased agonistic potency to the same level shown by Delta 24. The calcium flux-inducing activity of Delta 28 as well as Delta 24 appears to be stronger than that of MIP-1alpha (Fig. 3B).

Agonistic potency on CCR3 was also examined for all of the NH2-terminal deletion mutants using CCR3 transfectant cells, and some of the data are shown in Fig. 4. In CCR3 transfectant cells, Delta 24 was the only form of the NH2-terminal deletion mutants that showed enhanced agonistic potency; the EC50 of all the other NH2-terminal deletion mutants as well as Delta 0 was in the range of 40 to ~55 nM. In contrast, the EC50 of Delta 24 was ~22 nM. The agonistic potency of Delta 24, however, was lower than that of eotaxin, which showed a peak response at 50 nM with an EC50 of ~18 nM. At a 50 nM concentration, Delta 24 exhibited ~50% of the calcium flux response to eotaxin. It is noteworthy that the EC50 of Delta 24 on CCR1 was ~ 0.2 nM, which is ~100-fold lower than that on CCR3.


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  		Fig. 4.   Comparison of calcium flux-inducing activity of the NH2-terminal deletion mutants on CCR3 transfectants. Calcium influx was measured in Fura-2/AM-loaded CCR3-HOS cells. A, calcium flux-inducing activity was compared at a fixed concentration of 100 nM. The arrow indicates the time of addition of the indicated chemokine. B, calcium flux-inducing activity of Delta 0, Delta 10, Delta 24, and Delta 28 was compared in a dose-response study with that of eotaxin. C, the EC50 and the potency of the activity for each of the mutants were summarized. Relative activity was calculated from the peak amplitude elicited at the indicated concentration. The results show the mean ± S.D. of three separate experiments.

Desensitization Experiments-- To test whether loss of agonistic activity of Delta 29 is due to inability to bind to CCR1, desensitization experiments were performed in CCR1 transfectant cells. As shown in the upper panel of Fig. 5A, MIP-1alpha was not able to desensitize CCR1 transfectant cells completely to subsequent exposure to Delta 0 or Delta 28. However, stimulation of CCR1 transfectant cells with Delta 0 or Delta 28 completely abolished responsiveness to a second stimulation with MIP-1alpha . These data indicate that Delta 28, as well as Delta 0, shares receptors with MIP-1alpha and has a stronger desensitizing capability than MIP-1alpha . Then, we examined whether Delta 29 could desensitize Delta 28. Stimulation of CCR1 transfectant cells with Delta 29 did not affect the ability of Delta 28 to induce the subsequent calcium response, even at a concentration as high as 400 nM (Fig. 5A). These results suggest that Delta 29 may not be able to bind to CCR1.


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  		Fig. 5.   Cross-desensitization of calcium mobilization in CCR1- and CCR3-HOS cells. Calcium flux was measured in Fura-2/AM-loaded cells, which were stimulated sequentially at 100-s intervals. A, Fura-2/AM-loaded CCR1-HOS cells were exposed sequentially to the indicated chemokine at 100 nM (upper panel) or exposed first with increasing concentration of Delta 29 and then with 100 nM Delta 28 (lower panel). B, Fura-2/AM-loaded CCR3-HOS cells were stimulated sequentially with eotaxin and Delta 24 at the same concentration (100 nM). The arrows indicate the time of addition of the indicated chemokine. The results are representative of three separate experiments.

Desensitization experiments were also performed in CCR3 transfectant cells to compare desensitizing capability of Delta 24 with that of eotaxin. As shown in Fig. 5B, stimulation of CCR3 transfectant cells with eotaxin abolished responsiveness to a subsequent stimulation with Delta 24. In contrast, stimulation with Delta 24 did not completely abolish the ability of eotaxin to induce a calcium response.

Induction of Chemotaxis in CCR1- and CCR3-expressing Cells-- Chemotactic activities of the NH2-terminal deletion mutants of Lkn-1 were evaluated on CCR1 transfectant cells, and the results are shown in Fig. 6A. Consistent with the calcium flux results, chemotactic activity decreased dramatically when 29 amino acids were deleted. Delta 29 did not show significant chemotactic activity even at a concentration of 10 nM.


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  		Fig. 6.   Chemotactic activity of the NH2-terminal deletion mutants. Chemotaxis assays were performed using a Boyden chamber. A, chemotactic activity of the deletion mutants was compared in a dose-response study using CCR1-HOS cells, and some of the representative data are shown. MIP-1alpha was served as internal standards. B, chemotactic activity of Delta 0, Delta 10, Delta 24, and Delta 28 was compared in a dose-response study with that of eotaxin. The results show the mean ± S.D. of three separate experiments.

Potential differences in the chemotactic activity were also examined for all of the NH2-terminal deletion mutants at different protein concentrations, and some of the results are shown in Fig. 6A. Chemotactic activities of Delta 5, Delta 10, Delta 15, and Delta 20 were almost the same as that of Delta 0. In contrast, consistent with the calcium flux results, Delta 24, Delta 27, or Delta 28 exhibited increased chemotactic activity on CCR1 transfectants. EC50 values for Delta 0, Delta 5, Delta 10, Delta 15, and Delta 20 were within a range of 0.51 to ~0.58 nM, whereas EC50 values for Delta 24, Delta 27, and Delta 28 were 0.09 ± 0.01, 0.17 ± 0.01, and 0.14 ± 0.02, respectively.

In CCR3 transfectant cells, Delta 24 was again the only form of the NH2-terminal deletion mutant of Lkn-1 that showed increased agonistic activity (Fig. 6B). The chemotactic activity of Delta 24, however, was lower than that of eotaxin. The EC50 of Delta 24 was 33.0 ± 2.5, whereas that of eotaxin was 13.0 ± 1.2. Furthermore, at a concentration of 100 nM, at which eotaxin showed a peak response, Delta 24 exhibited 58% of the chemotactic activity of eotaxin.

Receptor Binding Assay-- Binding affinities of Lkn-1 mutant proteins with CCR1 and CCR3 were determined with 125I-labeled ligands (Fig. 7). In this experiment, we focused mainly on three proteins: Delta 0, which is the intact form of Lkn-1; Delta 24, which is the deletion mutants with enhanced agonistic activities on CCR3 as well as CCR1; and Delta 29, which essentially lacks the biological activity. Fig. 7, A and B, depicts the binding isotherm. Conversion of the data by Scatchard analysis revealed a Kd of 1.00 ± 0.02 nM (Delta 0) and 0.38 ± 0.01 nM (Delta 24) to the CCR1 (Fig. 7A), and 2.41 ± 0.08 nM (Delta 0) and 1.05 ± 0.09 nM (Delta 24) to the CCR3 (Fig. 7B). The relative affinity of the Lkn-1 mutants was investigated further in a cross-competition binding assay using CCR1 transfectant cells (Fig. 7C). Delta 24 was more effective in inhibiting the binding of 125I-labeled Delta 0 with CCR1 than Delta 0. The IC50 of Delta 24 was ~26 nM, whereas that of Delta 0 was ~42 nM. Delta 29, at the highest concentration tested, inhibited the binding of 125I-labeled Delta 0 by only 20%.


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  		Fig. 7.   Binding characteristics of 125I-labeled ligands to CCR1-and CCR3-HOS cells. Saturation binding of 125I-labeled ligands to CCR1 (A) and CCR3 (B) was shown. A total of 2×106 cells were incubated for 90 min at 4 °C with the indicated concentration of 125I-labeled ligand. Nonspecific binding, determined by the addition of a 100-fold excess of the corresponding unlabeled ligand, was subtracted. The results show the mean ± S.D. of three separate experiments. The Kd and the number of binding sites were determined by Scatchard analysis of the binding data. C, competition binding experiments were performed in the same conditions, using 1 nM 125I-labeled Delta 0 and variable concentrations of competitors. The results show the mean ± S.D. of three separate experiments.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

It has been found that recombinant Lkn-1 produced in insect cells undergoes a spontaneous site-specific cleavage at the NH2 terminus, producing a 24-amino acid shorter protein than the intact form (11). The significance of this autolysis, however, has remained unknown. In the present study, the role of the NH2-terminal domain for agonistic activity was studied with a series of NH2-terminal deletion mutants of Lkn-1, including the spontaneously cleaved form (Delta 24). Our preparations of NH2-terminal deletion mutants of Lkn-1 do not have any foreign amino acid residues at either the NH2 or COOH terminus of the recombinant proteins. Although recombinant Lkn-1, which has additional methionine and six histidines at its NH2 terminus, has biological activities (11), we found that truncation of the foreign amino acid residues at the NH2 terminus of the recombinant proteins increases the biological activity by ~10-fold (data not shown).

In contrast to other CC chemokines such as MCP-1 and RANTES, deletion of NH2-terminal amino acids up to 20 residues from the natural form of Lkn-1 did not cause noticeable alterations in agonistic potency on CCR1. This feature is unique for Lkn-1, because for CC chemokines such as MCP-1 and RANTES, minimal truncation or modification of the first few NH2-terminal amino acids leads to significant changes in receptor binding and functional activity (17-20). Deletion of the pyroglutamate residue at the NH2 terminus of the natural form of MCP-1 results in an at least 50-fold decreases in agonistic activity on monocytes and basophils (17, 18). Deletion of 2 amino acid residues, MCP-1-(3-76), leads to total loss of agonistic activity on eosinophils and basophils (18). RANTES loses agonistic potency and becomes a potent antagonist of chemokine binding when the first amino acid residue has been modified artificially by the addition of methionine or treatment with aminooxypentane (19, 21). Deletion of NH2-terminal amino acid residues can even result in changes in receptor specificity (18, 22). In particular, MCP-1 acquires agonistic activity on CCR3 only when the first residue at the NH2 terminus, pyroglutamate, is deleted (18). Similar to artificially modified chemokines, posttranslationally modified natural forms of MCP-1 such as MCP-1-(5-76) and MCP-1-(6-76) are also devoid of bioactivity (20). The naturally cleaved form of MCP-2, MCP-2-(6-76), is also devoid of bioactivity and blocks the chemotactic effects of MCP-2 as well as that of MCP-1, MCP-3, and RANTES (20). Thus, the integrity of the NH2-terminal region of CC chemokines appears to be critical for receptor binding and biological function. In fact, the search for NH2-terminal variants as receptor antagonists has been one of the major areas of interest since the discovery that chemokines inhibit HIV-1 infection (9, 23-25).

The situation is quite different for Lkn-1. Our results show that deletion of up to 20 amino acids from the intact form of Lkn-1 does not affect agonistic potency on CCR1. More interestingly, recombinant Lkn-1, which contains additional methionine and six histidines at its NH2 terminus, is only 10-fold less active the than intact form of Lkn-1. These observations suggest that the receptor binding and biological function of Lkn-1 is more dependent on the downstream amino acid residues of the NH2-terminal domain. Deletion of more amino acid residues, 24 amino acids (Delta 24), increases the calcium flux-inducing activity almost 100-fold on CCR1 transfectants. These results may explain why recombinant Lkn-1 undergoes a spontaneous site-specific cleavage at the NH2 terminus producing a 24-amino acid shorter protein than the intact form (10). For Lkn-1, posttranscriptional modification may be a mechanism to augment chemokine potency on CCR1. This is also true for CCR3; Delta 24 shows increased calcium flux-inducing activity in CCR3 transfectants compared with that of the intact form of Lkn-1. Deletion of 27 or 28 amino acids (Delta 27 or Delta 28) also produces a protein that has increased calcium flux-inducing activity on CCR1 transfectants. Deletion of 29 amino acid residues (Delta 29), however, abolishes the calcium flux-inducing activity almost completely, showing that at least 3 amino acid residues preceding the first cysteine are essential for the biological activity of Lkn-1.

Because deletion or proteolytic cleavage of the NH2-terminal region usually results in derivatives that still recognize the receptor but do not induce functional responses, we were curious to know whether Delta 29 acts as an antagonist of Lkn-1. This issue was addressed by receptor binding experiments as well as desensitization experiments. As shown in Fig. 6A, Delta 29 was unable to desensitize calcium flux-inducing activity of Delta 28 even at 400 nM concentration. Furthermore, receptor binding experiments showed that Delta 29 was not able to bind to CCR1 effectively. Thus, it was obvious that deletion of 29 amino acids at the NH2 terminus inactivates the receptor binding capability of Lkn-1. This feature is unique for Lkn-1 in that it does not produce antagonists by truncations of the NH2-terminal domain.

Although cleavage of the NH2-terminal region increases the agonistic potency on CCR1, the length of the NH2-terminal region does not proportionally affect agonistic potency; Delta 24, Delta 27, and Delta 28 show similarly potent biological activity in both calcium flux assays and migration assays. This feature is in contrast with that of hemofiltrate CC chemokine (HCC)-1, a recently cloned CC chemokine that is structurally similar to MIP-1alpha (26). Comparison of the three NH2-terminal truncated variants of HCC-1 revealed that the rank order of potency was inversely correlated with the length of the protein. Thus, the shortest form of HCC-1 was the most potent, and the longest form of HCC-1 was the least potent (27).

To examine whether increased agonistic potency shown by the deletion mutants is due to increased binding affinity to receptors, we also performed receptor binding assays for Delta 0 and Delta 24. Based on the Kd values, Delta 24 appeared to have an ~2.6-fold higher binding affinity on CCR1 than Delta 0. Cross-competition experiments using labeled Delta 0 also showed that Delta 24 had higher binding affinity on CCR1 than Delta 0. Because both Delta 27 and Delta 28 have similarly strong agonistic potency as Delta 24 on CCR1, it is reasonable to assume that Delta 27 and Delta 28 would also have similar binding affinity as Delta 24 on CCR1. In CCR3 transfectants, Delta 24 showed an ~2.3-fold higher binding affinity than Delta 0. Thus, it appears that the increased agonistic potency of the NH2-terminal deletion mutants is due, at least in part, to the increased binding affinity to receptors.

    ACKNOWLEDGEMENT

We thank Dr. Ji-Ming Wang for providing transfectant cell lines and for critical comments on this manuscript.

    FOOTNOTES

* This work was supported by Science Research Center funds to the Immunomodulation Research Center, University of Ulsan from the Korea Science and Engineering Foundation (KOSEF) and Korea Ministry of Science and Technology.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

** To whom correspondence should be addressed. Tel.: 82-43-261-2826; Fax: 82-43-268-2732; E-mail: cklee@chungbuk.ac.kr.

Published, JBC Papers in Press, February 6, 2002, DOI 10.1074/jbc.M109309200

    ABBREVIATIONS

The abbreviations used are: HIV, human immunodeficiency virus; CCR, CC chemokine receptor; HCC, hemofiltrate CC chemokine; Lkn-1, leukotactin-1; MCP, monocyte chemoattractant protein; MIP-1alpha , macrophage inflammatory protein-1alpha ; Ni-NTA, nickel-nitrilotriacetic acid; RANTES, regulated on activation normal T cell expressed protein..

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
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Copyright © 2002 by The American Society for Biochemistry and Molecular Biology, Inc.
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