Significance of N-Terminal Proteolysis of CCL14a to Activity on the Receptors CCR1 and CCR5 and the Human Cytomegalovirus-Encoded Chemokine This information is current as Receptor US28 of September 29, 2021. Rudolf Richter, Paola Casarosa, Ludger Ständker, Jan Münch, Jean-Yves Springael, Saskia Nijmeijer, Wolf-Georg Forssmann, Henry F. Vischer, Jalal Vakili, Michel Detheux,

Marc Parmentier, Rob Leurs and Martine J. Smit Downloaded from J Immunol 2009; 183:1229-1237; Prepublished online 24 June 2009; doi: 10.4049/jimmunol.0802145 http://www.jimmunol.org/content/183/2/1229 http://www.jimmunol.org/

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2009 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

Significance of N-Terminal Proteolysis of CCL14a to Activity on the Chemokine Receptors CCR1 and CCR5 and the Human Cytomegalovirus-Encoded US281

Rudolf Richter,2*† Paola Casarosa,‡ Ludger Sta¨ndker,† Jan Mu¨nch,§ Jean-Yves Springael,¶ Saskia Nijmeijer,‡ Wolf-Georg Forssmann,† Henry F. Vischer,‡ Jalal Vakili,¶ Michel Detheux,ʈ Marc Parmentier,¶ Rob Leurs,‡ and Martine J. Smit‡

The CC chemokine CCL14a is constitutively expressed in a large variety of tissues and its inactive proform CCL14a(1–74) circulates in high concentrations in plasma. CCL14a(1–74) is converted into CCL14a(9–74) by the proteases urokinase-type plasminogen activator and plasmin and is a highly active agonist for the chemokine receptors CCR1 and CCR5. In this study, a new CCL14a analog, CCL14a(12–74), was isolated from blood filtrate. To elucidate the functional role of the N terminus, a panel Downloaded from of N-terminally truncated CCL14a analogs were tested on the receptors CCR1 to CCR5 and on the human cytomegalovirus (HCMV)-encoded chemokine receptor US28. The order of binding affinity to these receptors and of the activation of CCR1 –ӷ(11(74–10) ؍ (and CCR5-mediated intracellular Ca2؉ concentration mobilization is CCL14a(6–74)<(7–74)<(8–74)Ӷ(9–74 74)ӷ(12–74). The almost identical affinities of CCL14a(7–74), CCL14a(9–74), and CCL14a(10–74) for the US28 receptor and the inhibition of US28-mediated HIV infection of 293T cells by all of the N-terminally truncated CCL14a analogs support the promiscuous nature of the viral chemokine receptor US28. In high concentrations, CCL14a(12–74) did reveal antagonistic activity http://www.jimmunol.org/ on intracellular Ca2؉ concentration mobilization in CCR1- and CCR5-transfected cells, which suggests that truncation of Tyr11 might be of significance for an efficient inactivation of CCL14a. A putative inactivation pathway of CCL14a(9–74) to CCL14a(12– 74) may involve the dipeptidase CD26/dipeptidyl peptidase IV (DPPIV), which generates CCL14a(11–74), and the metalloprotease aminopeptidase N (CD13), which displays the capacity to generate CCL14a(12–74) from CCL14a(11–74). Our results suggest that the activity of CCL14a might be regulated by stringent proteolytic activation and inactivation steps. The Journal of Immunology, 2009, 183: 1229–1237.

hemokines are a family of cytokines that are involved in cellular infiltrates that appear at the sites of tissue injury. The che- by guest on September 29, 2021 the recruitment and trafficking of immune cell popula- mokines mediate their effect through binding to their cognate che- C tions and are, therefore, essential for the effective orga- mokine receptors, which belong to the class of G -coupled nization of the immune response (1, 2). direct leuko- receptors and are differentially expressed on diverse leukocyte sub- cytes during hematopoiesis in the bone marrow and thymus, during types (5). Impressively, chemokine receptors act as cofactors for initiation of adaptive immune responses in the spleen and lymph HIV-1 entry into CD4 cells and their ligands can suppress HIV nodes, and during the immune surveillance of healthy peripheral replication. The CCR5, which binds CCL3 (MIP-1␣), CCL4 (MIP- tissues (3, 4). Yet, they are also responsible for the inflammatory 1␤), CCL5 (RANTES), and CCL8 (MCP-2), and the CXCR4, which recognizes CXCL12 (SDF-1), are considered to be the ma- jor HIV-1 coreceptors (6–8). Interestingly, the human CMV 3 *Institute of Transfusion Medicine and Immune Hematology, Blood Donation Service (HCMV) -encoded chemokine receptor US28 may serve as a co- of the German Red Cross, Frankfurt, Germany; †Clinic of Immunology and Rheu- receptor for HIV-type 1 entry into cells as well (9). Functionally, ‡ matology, Hannover Medical School, Hannover, Germany; Leiden/Amsterdam Cen- this viral receptor has been suggested to act as a sink by binding ter for Drug Research, Division of Medicinal Chemistry, Faculty of Chemistry, Am- sterdam, The Netherlands; §Institute of Virology, University of Ulm, Ulm, Germany; and sequestering chemokines from the extracellular vicinity of ¶Institute of Interdisciplinary Research, Universite´Libre de Bruxelles, Brussels, Bel- ʈ CMV-infected cells, thereby allowing them to escape immune sur- gium; and Euroscreen S.A., Brussels, Belgium veillance (10). Moreover, it signals in a constitutively active man- Received for publication July 1, 2008. Accepted for publication May 13, 2009. ner, activating proliferative signaling pathways (11). US28 is The costs of publication of this article were defrayed in part by the payment of page structurally similar to the human chemokine receptor CCR1 (12, charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 13) and binds a broad spectrum of chemokines (10, 11, 14, 15). 1 This study was supported in part by a grant from the German Federal Ministry of The hemofiltrate (HF) CC chemokine CCL14a (HCC-1) is a Education and Research (Bundesministerium fu¨r Bildung, Wissenschaft, Forschung member of the CC chemokine family and was originally isolated und Technologie, Grant FKZ 0311815) and the Dutch Organization for Scientific from the HF of patients with chronic renal failure (16). CCL14a Research (to H.F.V. and M.J.S.). This work was also supported by the Actions de Recherche Concerte´es of the Communaute´Franc¸aise de Belgique, the Interuniversity consists of 74 aa, after cleavage of a 19-residue signal sequence, Attraction Poles Programme-Belgian State-Belgian Science Policy, the European Union (LSHB-CT-2005-518167/INNOCHEM), the Fonds de la Recherche Scienti- fique Me´dicale of Belgium, the Walloon Region (Programme d’Excellence 3 Abbreviations used in this paper: HCMV, human CMV; APN, aminopeptidase N; “CIBLES”), the Fe´de´ration Belge contre le Cancer, and the Fondation Me´dicale Reine CD26/DPPIV, CD26/dipeptidyl peptidase IV; ESMS, electrospray mass spectrome- Elisabeth (to M.P.). try; HF, hemofiltrate; uPA, urokinase-type plasminogen activator; RP, reverse phase; 2 Address correspondence and reprint requests to Dr. Rudolf Richter, Institute of TFA, trifluoroacetic acid. Transfusion Medicine and Immune Hematology, Blood Donation Service of the Ger- man Red Cross, 60528 Frankfurt, Germany. E-mail address: [email protected] Copyright © 2009 by The American Association of Immunologists, Inc. 0022-1767/09/$2.00 www.jimmunol.org/cgi/doi/10.4049/jimmunol.0802145 1230 PROTEOLYTIC REGULATION OF CCL14a ACTIVITY and shares a 46% sequence identity with CCL3 and CCL4. Fur- and each was loaded onto a reverse phase (RP) column, 12.5 ϫ 10-cm thermore, an alternative splice variant of CCL14, which is referred inside diameter (i.d.) (Source RPC, 15 ␮m; Pharmacia) and eluted in an 8 to as CCL14b, carries an insert of 16 aa within the amino-terminal L gradient from 100% A (0.01 M HCl in water) to 60% B (0.01 M HCl in 80% acetonitrile). Fractions of 200 ml were collected and tested for their region of the sequence, which is introduced between aa 8 and 9 ability to stimulate CCR5. Fractions containing CCR5-activating material (17, 18). Two unusual features of CCL14a are its constitutive ex- were further purified to homogeneity by four consecutive chromatographic pression in a large variety of tissues and its high plasma concen- steps, A–D, as previously described (20). Step A included preparative RP- tration, which ranges from 1.5 to 10 nM in normal human plasma C18 chromatography using a Vydac PrepPak RP-C18 column, 300 ϫ 47-mm i.d., 15–20 ␮m, 300 Å (Vydac). Separations were performed at a (16). Whereas CCL14a(1–74) and its glycosylated form (19) rep- flow rate of 40 ml/min using a linear binary gradient of 90% solvent A (10 resent inactive precursors, proteolytically activated CCL14a(9– mM HCl in water) to 50% solvent B (10 mM HCl in 80% acetonitrile) in 74) is a high affinity agonist of the chemokine receptors CCR1 and 48 min. Fractions of 40 ml were collected. Step B included preparative CCR5 and, to a lesser extent, of CCR3 (20). Consistent with this RP-C4 chromatography; column Biotek Silica 5 ␮m, 250 ϫ 20-mm i.d. activity, CCL14a(9–74) promotes calcium mobilization and the (Biotek Laboratories). Separation was performed at a flow rate of 7 ml/min using a linear binary gradient of 67% solvent A (0.1% trifluoroacetic acid migration of T lymphocytes, monocytes, and eosinophils (20). (TFA) in water) to 58% solvent B (0.1% TFA in 80% acetonitrile) in 60 CCL14a(1–74) is converted into the active chemokine CCL14a(9– min. Fractions of 7 ml were collected. Step C was analytical RP-C18 chro- 74) by the protease urokinase-type plasminogen activator (uPA) matography; column RP-C18, 250 ϫ 10-mm i.d., 5 ␮m, 300 Å (Biotek and plasmin with high efficiency (21). In accordance with other Laboratories). Separation was performed at a flow rate of 1.8 ml/min using a linear binary gradient from 70% solvent A (0.1% TFA in water) to 45% CCR5 ligands, both CCL14a(9–74) and CCL14a(10–74) possess solvent B (0.1% TFA in 80% acetonitrile) in 45 min. Fractions of 1.8 ml potent anti-HIV-1 activities by virtue of their ability to promote were collected. Step D was analytical RP-C18 chromatography; analytical internalization and the effective intracellular sequestration of the RP-C18 column, 250 ϫ 4.6-mm i.d., 5 ␮m, 300 Å (YMC). Separation was Downloaded from chemokine receptor, which is the major entry cofactor for primary performed at a flow rate of 0.6 ml/min using a linear binary gradient from HIV isolates (22). 77% solvent A (0.1% TFA in water) to 38% solvent B (0.1% TFA in 80% acetonitrile) in 45 min. Fractions of 0.6 ml were collected. Active material This study was performed to further elucidate the impact of the was further separated by step E, which was analytical RP-C18 chromatog- N-terminal amino acid sequence of CCL14a on its biological ac- raphy; analytical RP-C18AQ column, 250 ϫ 1-mm i.d., 3 ␮m, 300 Å tivity and to evaluate the activity of CCL14a(12–74), which is a (Reprosil-pur; Fa. Maisch). Separation was performed at a flow rate of 0.02 ml/min using a linear binary gradient from 77% solvent A (0.1% TFA in CCL14a analog that was identified in this study. We identified http://www.jimmunol.org/ water) to 38% solvent B (0.1% TFA in 80% acetonitrile) in 45 min. Frac- aminopeptidase N (APN) as a putative protease that generates tions of 0.02 ml were collected. CCL14a(12–74). A series of sequential amino-terminally truncated CCL14a analogs was tested on CCR1-, CCR2-, CCR3-, CCR4-, Mass spectrometry CCR5-, and US28 receptor-transfected cells. Moreover, the po- Electrospray MS (ESMS) was conducted on an API III Triple stage qua- tency of CCL14a analogs as inhibitors of HIV infection in US28- druple mass spectrometer that was equipped with articulated ion spray transfected cells was evaluated. Our results suggest that the bio- (PerkinElmer SCIEX). Flow injection was conducted at 5 ␮l/min. The logical activity of CCL14a is regulated by proteolytic steps that masses were determined in the range from 400 to 2300 Da in the positive ion mode, as described by the manufacturer. The peptide masses were form the highly active CCL14a(9–74) and that may inactivate calculated using the MacSpec 3.3 software (PerkinElmer SCIEX). CCL14a(9–74) to CCL14a(12–74). In addition, high concentra- MALDI-MS was performed using a LaserTec RBT MALDI-mass spec- by guest on September 29, 2021 tions of CCL14a(12–74) revealed an antagonistic activity against trometer (Perseptive/Vestec). Specimens were applied to a stainless steel CCL14a(9–74). Proteolytic activation, as well as the inactivation multiple sample tray as an admixture with sinapinic acid using the dried drop technique (24). Measurements were performed in linear mode. The of CCL14a, takes place within the circulating blood stream. This instrument was equipped with a 1.2-m flight tube and a 337-nm nitrogen mechanism may be of importance to limit the biological activity of laser. Positive ions were accelerated at 30 kV and 64 laser shots were CCL14a. automatically accumulated per sample position. The time-of-flight data were externally calibrated for each sample plate and each sample prepara- Materials and Methods tion. Data acquisition and analysis were performed using the GRAMS 386 Materials version 3.04 software that was supplied by the manufacturer (Galactic Industries). CCL14a(1–74) CCL14a(6–74), CCL14a(7–74), CCL14a(8–74), CCL14a(9–74), CCL14a(10–74), CCL14a(11–74), and CCL14a(12– Sequence analysis 74) were prepared by F-moc solid-phase synthesis as previously de- Sequence analyses of the isolated peptides were performed by stepwise scribed (22). Edman degradation using a gas-phase automated sequencer (model 473 A; Purification of CCL14a(12–74) Applied Biosystems). The resulting phenyl-thio-hydantoin amino acids were identified by integrated HPLC. Purification of CCL14a(12–74) was performed from a peptide library that was generated from 10,000 L human HF from 40 adult patients who have Proteolytic processing of CCL14a(1–74) by APN chronic renal failure as previously reported (23). Ultrafilters used for he- To test the ability of APN to process CCL14a(11–74), 1 ␮g of APN (R&D mofiltration had a specified molecular mass cutoff of 20 kDa. The sterile Systems) was incubated with 10 ␮g of CCL14a(11–74) in 10 mM filtrate was immediately cooled to 4°C and acidified to pH 3 to prevent NaH2PO4 (pH 7.4) for 16 h. The inhibition of the proteolytic activity of bacterial growth and proteolysis. For peptide extraction, batches of 1000 L APN was performed with 1 mM EDTA or 10 ␮M 1,10-phenantroline. HF were conditioned to pH 2.7 and applied onto the strong cation ex- ϫ 2ϩ 2ϩ changer Fractogel TSK SP 650(M) (100 250 mm; Merck). Batch elution Intracellular Ca concentration mobilization ([Ca ]i) was performed with 10 L of 0.5 M ammonium acetate. The eluate was measurement stored at Ϫ20°C until further use. For the production of a peptide library, ammonium acetate extracts of 5000 L of HF were pooled and loaded on a CHO-K1 cell lines expressing CCR1, CCR2, CCR3, CCR4, CCR5, G␣16, 10 L cation exchange column (Fractogel SP 650(M); Merck). Bound pep- and mitochondria-targeted apoaequorin were established and a functional tides were eluted using seven buffers with increasing pH, which resulted in assay, which was based on the luminescence of aequorin following intra- seven pH pools. The seven buffers were composed as follows: buffer I, 0.1 cellular Ca2ϩ release, was performed as previously described (22). Briefly, M citric acid monohydrate (pH 3.6); buffer II, 0.1 M acetic acid plus 0.1 M cells were collected from plates with PBS containing 5 mM EDTA, pel- sodium acetate (pH 4.5); buffer III, 0.1 M malic acid (pH 5.0); buffer IV, leted, resuspended at a density of 107 cells/ml in DMEM-F12 medium, and 0.1 M succinic acid (pH 5.6); buffer V, 0.1 M sodium dihydrogen phos- incubated with 5 ␮M coelenterazine H (Molecular Probes) for4hatroom phate (pH 6.6); 0.1 M disodium hydrogen phosphate (pH 7.4); and buffer temperature. The cells were then washed in DMEM-F12 medium and re- VII, 0.1 M ammonium carbonate (pH 9.0). pH pool VIII was generated by suspended at a density of 2 ϫ 106 cells/ml. The cells were subsequently washing the column with distilled water. The eight pH pools were collected mixed with the chemokines, and emission was recorded for 30 s using The Journal of Immunology 1231

a Microlumat luminometer (PerkinElmer). The results are expressed as relative light units. Chemotaxis assay Murine pre-B .2 cells (provided by Dr. J. Pease, Imperial College Lon- don, London, U.K.) were transfected with 500 ng of CCR5 cDNA/106 cells using a Bio-Rad Pulser Xcell (330 V and 975 ␮F) and subsequently grown overnight in culture medium supplemented with 10 mM sodium butyrate. Twenty-four hours after transfection, the migration of L1.2 cells toward HCC-1 ((9–74), (11–74), (12–74)) or CCL5 was determined using 5-␮m pore ChemoTx 96-well plates (NeuroProbe). First, the ChemoTx plates were blocked using RPMI 1640 medium with GlutaMAX-I and 25 mM HEPES supplemented with 1% (w/v) BSA. CCL5 and HCC-1 were diluted in the same medium supplemented with 0.1% (w/v) BSA and dis- pensed in the bottom wells of the chemotaxis plate. The membrane was placed on top of these wells, and 2.5 ϫ 105 cells in the same buffer were applied to the upper surface and incubated for4hinahumidified chamber ␮ at 37°C and 5% CO2. The number of cells that traversed the 5- m pore membrane and migrated to the bottom wells was quantified on the Victor (3) 1420 multilabel reader (PerkinElmer) after the incorporation of the Calcein AM dye (Invitrogen). Data are shown as the percentage of mi- grated cells. Downloaded from Receptor binding studies Competition binding assays were performed as described on crude mem- brane fractions that were prepared from CHO-K1 cell lines expressing CCR1 or CCR5 (25, 26). Briefly, 1–10 mg of crude membrane extracts

were incubated in binding buffer (50 mM HEPES (pH 7.4), 5 mM MgCl2, 1 mM CaCl2, and 0.5% (w/v) protease-free BSA) containing 0.1 nM ra- FIGURE 1. Final purification step of CCL14a(12–74) and ESMS of http://www.jimmunol.org/ dioligand 125I-CCL3 or 125I-CCL4 as competitors for 90 min at 27°C. fraction 44 from the final purification step of CCL14a(12–74). A, Bound tracer was separated by filtration through GF/B filters (Millipore) CCL14a(12–74) was purified according to Detheux et al. (20). Subse- that were presoaked in 1% BSA or 0.3% polyethyleneimine. Filters were quently, the bioactive material was purified in a final purification step on an then counted by ␥-scintillation counting. The results were normalized for analytical RP-C18 chromatography column. Experimental conditions are total binding in the absence of competitor (100%) and nonspecific binding (0%) in the presence of a 100-fold excess of competitor and were analyzed detailed in Materials and Methods. The hatched bars represent the bioac- by nonlinear regression using a single-site competition model (GraphPad tive fractions (fractions 44–46), which contain CCL14a(9–74) coeluting Prism Software). with CCL14a(12–74). B, CCL14a(12–74) coelutes with CCL14a(9–74). For binding studies on US28, COS-7 cells were grown as previously The long arrows marked with a represent the 6-, 5-, and 4-fold charged described (27). Transfection of the COS-7 cells was performed by DEAE- mass peaks of CCL14a(12–74), with the molecular masses of 1247.5,

dextran using 2 ␮g of DNA of each US28 construct per million cells (27). 1497.5, and 1872.0 Da, respectively. The determined molecular mass of the by guest on September 29, 2021 125 The labeling of CCL5 (PeproTech) with I and binding in COS-7 cells molecule is 7481.8 Ϯ 0.77 Da and is in agreement with the theoretical were performed as previously described (28). Briefly, transfected COS-7 average mass of 7479.5 Da for CCL14a(12–74). The hatched arrows cells were incubated with 0.3 nM 125I-CCL5 in binding buffer (50 mM marked with b represent the 6-, 5-, and 4-fold charged mass peaks of HEPES (pH 7.4), 1 mM CaCl2, 5 mM MgCl2, and 0.5% BSA) in the presence or absence of various concentrations of CCL14a variants for 3 h CCL14a(9–74), with the molecular masses of 1300.5, 1561.0, and 1951.5, Ϯ at 4°C. After incubation, the cells were washed four times at 4°C with respectively. The determined molecular mass of the molecule is 7799.6 binding buffer supplemented with 0.5 M NaCl. Nonspecific binding was 0.77 Da and is in agreement with the theoretical average mass of 7796.8 Da determined in the presence of 0.1 ␮M cold chemokine. for CCL14a(9–74). Infectivity assays ␣ Virus stocks were generated and the p24 Ag levels were quantified as CHO-K1 cell line expressing CCR5, G 16, and mitochondrial previously described (22). HEK293T cells were transiently transfected apoaequorin (20). Extraction of peptides from 10,000 L of human with pcDNA3.1-US28 and pcDNA3.1-CD4 using the calcium phosphate HF by cation exchange chromatography resulted in the generation method and seeded on 96-well dishes 14 h later. The following day, the cells were preincubated with the chemokines for 2 h and subsequently of pH pool fractions that were used to establish a peptide library of infected with virus stocks containing 25 ng of p24 of the luciferase-ex- circulating peptides by RP-HPLC. In the peptide library, CCR5- pressing HIV-1 NL4-3 005pf135 variant (29) in a total volume of 200 ␮l. activating material was detected in fraction 21 of the RP chroma- After overnight incubation, the cells were washed and cultivated in fresh tography of pH pool V. The CCR5-activating material was purified medium. Three days after infection, the luciferase activity was quantified in the Orion II microplate luminometer (Berthold Detection Systems) using a in four subsequent purification steps, as previously described (20). Luciferase Assay System (Promega). After additional analytical purification by high performance RP- C18 chromatography (Fig. 1A), the active fraction was analyzed by Statistical analysis mass spectrometry (Fig. 1B) and Edman sequencing (data not Data were compared by the Student’s t test. Values of p Ͻ 0.05 were shown). ESMS of the purified material revealed two molecular considered to be significant. masses of 7481 Ϯ 0.77 and 7799.6 Ϯ 0.77 Da, which are in ac-

cordance with the theoretical average Mr of 7479.5 Da for Results CCL14a(12–74) and 7796.8 Da for CCL14a(9–74), respectively. Purification of CCL14a(12–74) from human blood filtrate These results were confirmed by amino acid sequencing of the

Various analogs of CCL14a, including CCL14a(1–74), separated material, identifying the NH2-terminal sequences HP- CCL14a(3–74), CCL14a(4–74), and CCL14a(9–74) have been SEXXFTYTTYK and GPYHPSEXXFTYT, which are in accor- described previously (16, 19, 20). In addition to these CCL14a dance with CCL14a(12–74) and CCL14a(9–74), respectively. analogs, we identified another variant, CCL14a(12–74), in human Mass spectrometric analysis of CCL14-containing fractions re- blood filtrate. CCL14a(12–74) was identified in an isolation pro- vealed that CCL14(12–74) is detectable in fractions also contain- cedure for ligands of the chemokine receptor CCR5 using a ing CCL14(9–74). The amount of CCL14(12–74) is ϳ15% of that 1232 PROTEOLYTIC REGULATION OF CCL14a ACTIVITY

with affinities that were comparable to that of CCL5. Other trun- cations that generated longer or shorter forms of CCL14a resulted in a dramatic loss of affinity. CCL14a(8–74) bound CCR1 with a 20-fold reduction of affinity, while a further 2-fold reduction was observed for CCL14a(7–74) and CCL14a(6–74). CCL14a(11–74) displayed a 28-fold reduction of binding affinity when compared with CCL14a(10–74). CCL14a(12–74) binding was the most strongly impaired, with a further 10-fold reduction of affinity com- pared with CCL14a(11–74). The pattern of CCR5 binding for CCL14a truncation variants (Fig. 3B) closely followed that of CCR1, although a few differ- ences could be noted. CCL14a(9–74) and CCL14a(10–74) bound CCR5 with the highest affinity at a level that was comparable to that of CCL5. All other mutants were strongly impaired. Forms (8–74), (7–74), and (6–74) displayed 60-, 80- and 200-fold reduc- FIGURE 2. Processing of CCL14a(11–74) to CCL14a(12–74) by APN. tions of affinity for CCR5, respectively, while CCL14a(11–74) and A, MALDI-MS of CCL14a(11–74) before processing with APN. The CCL14a(12–74) were characterized by 10- and 40-fold reductions marked peaks with the molecular masses of 7646.4 and 3824.5 Da repre- of binding affinities, respectively, compared with CCL14a(9–74) sent the 1- and 2-fold charged mass peaks of CCL14a(11–74), respectively. (Table I). Downloaded from B, ESI-MS of CCL14a(11–74) 16 h after treatment with APN. The marked peaks with the molecular masses of 7482.0 and 3742.0 Da represent the 1- 2ϩ Induction of intracellular calcium mobilization ([Ca ]i) and and 2-fold charged mass peaks of CCL14a(12–74), respectively, which is chemotaxis assays by CCL14a analogs on CCR1-, CCR2-, in agreement with its theoretical average mass of 7479.5 Da. CCR3, CCR4-, or CCR5-expressing CHO-K1 cells In accordance with our earlier investigations, CCL14a(9–74) re- 2ϩ of CCL14(9–74). Given that the CCL14(9–74) concentrations in http://www.jimmunol.org/ vealed a strong [Ca ]i-inducing activity with an EC50 value of HF are ϳ100–200 pM, the concentration of CCL14(12–74) should 2.6 Ϯ 0.8 nM for CCR1 and 6.5 Ϯ 2.9 nM for CCR5 (Fig. 3, C and be ϳ15–30 pM in blood filtrate. 2ϩ D) (20). For [Ca ]i-inducing activity on CCR1-expressing cells, N-terminal truncation subsequent to amino acids Ser5 (CCL14a(6– Proteolytic processing of CCL14a(11–74) by APN 74)), Ser6 (CCL14a(7–74)), and Ser7 (CCL14a(8–74)) is related to 2ϩ CCL14a(9–74) is suggested to be inactivated to CCL14a(11–74) a steady increase of the [Ca ]i-inducing activity, as indicated by Ϯ Ϯ Ϯ by the dipeptidyl peptidase IV (CD26/DPPIV; EC 3.4.14.5) by a the diminished EC50 values of 262 89, 218 80, and 35 6.9 specific processing of the N-terminal amino acids Gly9-Pro10 (30). nM, respectively. A further truncation of the amino acid Arg8

Since we identified CCL14a(12–74), we hypothesized that (CCL14a(9–74)) caused a 15-fold increase of the EC50 value to CCL14a(11–74) is further processed by an exopeptidase. Because 2.6 Ϯ 0.8 nM (Fig. 3C). Whereas CCL14a(9–74) and by guest on September 29, 2021 2ϩ the metalloprotease APN, which has been shown to process the CCL14a(10–74) display almost the same [Ca ]i-inducing activ- ϭ Ϯ N-terminal tyrosine (31), is highly expressed on the surface of ity (EC50 value for CCL14a(10–74) 4.3 0.2 nM), a further 10 2ϩ monocytes, granulocytes, and T cells (32, 33), we tested the ability truncation of the N-terminal amino acid Pro reduced the [Ca ]i- of APN to process CCL14a(11–74). High concentrations of solu- inducing activity to 1/10, with EC50 values of 40.0 nM. ␮ ␮ ϳ 2ϩ ble APN (1 g/300 l) displayed the capacity to specifically pro- CCL14a(11–74) induced 65% of the maximum [Ca ]i-induc- cess the N-terminal amino acid Tyr11, as indicated by MALDI-MS ing activity at 500 nM. In contrast, CCL14a(12–74) did not induce 2ϩ analysis of the APN-treated samples of CCL14a(11–74) (Fig. 2). [Ca ]i in CCR1-transfected CHO-K1 cells in a concentration Within 16 h, ϳ30% of the CCL14a(11–74) were processed to range between 10 pM and 500 nM, which highlighted the signif- CCL14a(12–74), which indicates slow processing under these ex- icance of the proteolytic truncation of Tyr11 for an efficient inac- perimental conditions. The metalloprotease inhibitors EDTA and tivation of CCL14a (Fig. 3C and Table I). 1,10-phenantroline abrogated the processing of CCL14a(11–74) to On CCR5-expressing CHO-K1 cells, N-terminal truncation sub- CCL14a(12–74), which indicates that the APN, rather than some sequent to amino acids Ser5 (CCL14a(6–74)), Ser6 (CCL14a(7– other contaminating protease, is responsible for the proteolytic 74)), and Ser7 (CCL14a(8–74)) resulted in a marginal increase of 2ϩ processing. APN revealed no capacity to process CCL14(9–74). the [Ca ]i-inducing activity. CCL14a(6–74), CCL14a(7–74), and Ϯ Ϯ The leukotriene A4 hydrolase, which is another putative protease CCL14a(8–74) revealed EC50 values of 402 228, 165 48, and with aminopeptidase activity, did not reveal any capacity to pro- 177 Ϯ 94 nM, respectively. In contrast, a further truncation of cess CCL14a(11–74). amino acid Arg8 for CCL14a(9–74) induced a 30-fold increase 2ϩ Ϯ in [Ca ]i-inducing potency, with an EC50 value of 6.5 2.9 Binding of CCL14a analogs to CCR1 and CCR5 nM. In our assays, CCL14a(10–74) appeared slightly more potent Ϯ Since the N-terminal truncation of chemokines is known to affect (mean EC50 of 3.5 2.1 nM) than CCL14a(9–74). A further 10 2ϩ their affinity for their cognate receptors, we examined the affinity truncation of the N-terminal amino acid Pro reduced the [Ca ]i- Ϯ of a panel of N-terminally truncated CCL14a analogs, including inducing activity to an EC50 value of 224 11 nM, whereas the newly identified CCL14a(12–74) analog for CCR1 and CCR5. CCL14a(11–74) at concentrations of 500 nM induced only ϳ50% 2ϩ Heterologous competition binding experiments were performed as of the maximum [Ca ]i-inducing activity. In parallel to the results previously described (20) using 125I-labeled CCL3 as tracer for on CCR1-transfected cells, CCL14a(12–74), up to concentrations CCR1 (Fig. 3A and Table I) and 125I-labeled-CCL4 for CCR5 (Fig. of 500 nM, did not induce calcium mobilization in the CCR5- 3B and Table I). Binding to CCR1 revealed that the CCL14a(9– transfected cells (Fig. 3D and Table I). 74) form was the ligand with the highest affinity among the series In another set of calcium flux assays, the different CCL14a iso- (Fig. 3A). The CCL14a(10–74) form displayed a slightly reduced forms were investigated for their potency to desensitize the CCR1 affinity compared with CCL14a(9–74). Both forms bound CCR1 and CCR5 receptors to a second stimulus that was induced by 10 The Journal of Immunology 1233 Downloaded from http://www.jimmunol.org/

FIGURE 3. Binding and functional activity of CCL14a analogs on human chemokine receptors CCR1, CCR2, CCR3, CCR4, and CCR5 expressed in 2ϩ CHO-K1 cells. A, Competition binding assays on CCR1; B, Competition binding assays on CCR5; C, [Ca ]i-inducing activity on CCR1-transfected 2ϩ 2ϩ CHO-K1 cells; D, [Ca ]i-inducing activity on CCR5-transfected CHO-K1 cells; and E and F, desensitization of [Ca ]i-inducing activity on CCR1- transfected CHO-K1 cells (E) and CCR5-transfected CHO-K1 cells (F). Transfected CHO-K1 cells were first stimulated with different concentrations of 2ϩ 2ϩ the CCL14a isoforms. After 30 min, the same cells were subjected to 10 nM CCL14a(9–74) and the [Ca ]i-inducing activity was recorded. G, [Ca ]i- inducing activity on CCR2-, CCR3-, and CCR4-transfected CHO-K1 cells. Calcium mobilization was monitored following stimulation with 500 nM of the different CCL14a isoforms. H and I, Dose-response curves of CCL14a(9–74) and CCL14a(10–74) on (H) CCR2-transfected CHO-K1 cells in comparison by guest on September 29, 2021 to the established ligand CCL2 and (I) CCR3-transfected CHO-K1 cells in comparison to the established ligand CCL11. J, Migration of CCR5-transfected L1.2 cells to CCL5 and CCL14 analogs. Assays were performed with: f, CCL14a(6–74); Œ, CCL14a(7–74); ƒ, CCL14a(8–74); E, CCL14a(9–74); Ⅺ, CCL14a(10–74); छ, CCL14a(11–74); F, CCL14a(12–74); and, ‚, CCL5. Binding and functional assay results are representative of at least three independent experiments. The data represent the mean Ϯ SEM for measurements performed in triplicate. nM CCL14a(9–74). CCL14a(9–74) and CCL14a(10–74) had the on the CCR1- and CCR5-transfected cells (data not shown), pretreat- strongest desensitization on calcium mobilization in CCR1- as ment of the cells with 1000 nM CCL14a(12–74) significantly reduced well as in CCR5-transfected cells. Pretreatment of the cells with the calcium flux mobilization by ϳ85% on the CCR1 receptor and by CCL14a(6–74), CCL14a(7–74), CCL14a(8–74), or CCL14a(11– 50% on the CCR5 receptor (Fig. 3, E and F). 74) reduced the CCL14a(9–74)-induced calcium flux mobilization We also tested the ability of the CCL14a isoforms to stimulate in proportion to their potency to induce calcium flux mobilization. cells expressing CCR2, CCR3, or CCR4. Calcium mobilization Interestingly, although at concentrations of 1000 nM, CCL14a(12– was monitored following stimulation with 500 nM CCL14a(9–74) 74) did not show a significant induction of calcium flux mobilization and CCL14a(10–74) in CCR2- and CCR3-expressing cells but not

Table I. Functional parameters of the different tested CCL14a analogs and of CCL5 on CCR1, CCR5, and US28a

CCR1 CCR5 US28

Binding parameters Calcium mobilization Binding parameters Calcium mobilization Binding parameters Ϯ Ϯ Ϯ Ϯ Ϯ IC50 SEM (nM) EC50 SEM (nM) IC50 SEM (nM) EC50 SEM (nM) IC50 SEM (nM) CCL14a(1–74) Ͼ1000 Ͼ300 CCL14a(3–74) Ͼ300 CCL14a(6–74) 6.52 Ϯ 0.15 262 Ϯ 89.0 21.6 Ϯ 8.81 402 Ϯ 228 CCL14a(7–74) 4.36 Ϯ 0.95 218 Ϯ 80.4 7.72 Ϯ 3.09 165 Ϯ 48 26.93 Ϯ 13.46 CCL14a(8–74) 2.42 Ϯ 0.52 35.2 Ϯ 6.38 5.90 Ϯ 4.82 177 Ϯ 94 CCL14a(9–74) 0.10 Ϯ 0.08 2.62 Ϯ 0.84 0.10 Ϯ 0.04 6.45 Ϯ 2.90 14.94 Ϯ 0.12 CCL14a(10–74) 0.18 Ϯ 0.09 4.32 Ϯ 0.24 0.07 Ϯ 0.03 3.48 Ϯ 2.09 14.02 Ϯ 2.93 CCL14a(11–74) 5.07 Ϯ 0.51 40.3 Ϯ 12.0 1.20 Ϯ 0.57 224 Ϯ 11.2 43.71 Ϯ 3.58 CCL14a(12–74) 50.1 Ϯ 9.93 Ͼ500 4.29 Ϯ 0.31 Ͼ500 Ͼ300 CCL5 0.16 Ϯ 0.03 5.90 Ϯ 2.10 0.12 Ϯ 0.03 8.48 Ϯ 0.2 1.74 Ϯ 0.07

a EC50 values were calculated on estimated sigmoidal dose-response curves using the GraphPad Prism program. 1234 PROTEOLYTIC REGULATION OF CCL14a ACTIVITY

FIGURE 5. N-terminally truncated CCL14a analogs inhibit US28-me- FIGURE 4. Characterization of the binding of CCL14a analogs to the diated HIV-1 infection. 293T cells transiently expressing US28 and CD4 US28 receptor. Competition binding curves for CCL14a analogs were de- were incubated with the indicated concentrations of chemokine and in- termined in US28-expressing COS-7 cells against 125I-labeled CCL5. The fected with the HIV-1 NL4-3 005pf135 luciferase reporter virus. Reporter data are normalized for nonspecific binding (0%) and maximal specific activity was detected 3 days after infection. The luciferase activity of cells

binding in the absence of a competitor (100%). Binding was as follows: Downloaded from infected in the absence of chemokine was considered to be 100% infec- ‚, CCL5; , CCL14a(1–74); ࡗ, CCL14a(3–74); Œ, CCL14a(7–74); tivity. Data represent the mean values Ϯ SEM of two independent exper- E, CCL14a(9–74); Ⅺ, CCL14a(10–74); छ, CCL14a(11–74); and iments, with each performed in triplicate. F, CCL14a(12–74).

21) or infected cells expressing CD4 alone (274 Ϯ 130) (data not

in CCR4-expressing cells (Fig. 3G). Dose-response curves for http://www.jimmunol.org/ CCR2 (Fig. 3H) and CCR3 (Fig. 3I) showed that the potency of shown). First, the effects of CCL14a(9–74) on the replication of CCL14a(9–74) and CCL14a(10–74) on these receptors is much the R5-tropic HIV-1 clone were investigated. CCL14a(9–74) ef- ␮ lower than that observed for CCR1 and CCR5. fectively suppressed replication at 1 and 10 M concentrations. At ␮ In chemotaxis assays with CCR5-transfected, murine pre-B 0.1 M, CCL5 appeared to be more potent, since it significantly L1.2 cells, CCL14a(9–74) displayed a higher potency, with the suppressed replication (data not shown), whereas CCL14a(9–74) ␮ maximal migration observed at 1 nM, than CCL5, with a maximal at a concentration of 0.1 M did not suppress replication. We then migration observed at 32 nM. The efficacy of CCL14a(9–74) at 1 investigated whether different CCL14a analogs suppress the viral nM, with an induction of cell migration of 7%, was significantly entry. The analogs CCL14a(6–74), CCL14a(7–74), and

lower than the efficacy of CCL5 at 32 nM, with an induction of cell CCL14a(12–74) displayed only a moderate but significant inhibi- by guest on September 29, 2021 migration of 14%. Whereas CCL14a(11–74) revealed moderate tion of viral infection and replication at concentrations of 1 and 10 ␮ chemotactic activity with a maximum potency at 32 nM, M, whereas CCL14a(8–74) and CCL14a(10–74) revealed a CCL14a(12–74), in concentrations up to 100 nM, did not induced strong inhibition of HIV infection, which was comparable to that a significant chemotactic response (Fig. 3J). of CCL14a(9–74) (Fig. 5). Binding of CCL14a analogs to the viral chemokine receptor Discussion US28 Proteolytic inactivation of CCL14a N-terminally truncated CCL14a variants dose-dependently dis- Proteolytic activation of CCL14a was shown to be essential to 125 2ϩ placed I-labeled CCL5 binding to US28 in transiently trans- deploy its chemotactic and [Ca ]i-inducing activity on T lym- fected COS-7 cells. Whereas CCL14a(1–74) and CCL14a(3–74) phocytes, monocytes, and eosinophils. Previously, we isolated 125 showed only weak displacement of I-labeled CCL5 with IC50 CCL14a(9–74) as a main effector of CCR5 activity in blood filtrate values Ͼ300 nM, N-terminally truncated CCL14a(7–74), and showed that uPA as well as plasmin are able to process CCL14a(9–74), and CCL14a(10–74) strongly bound to the US28 CCL14a(1–74) into CCL14a(9–74) (20, 21). CCL14a(9–74) has Ϯ Ϯ Ϯ receptor with an IC50 of 26.93 13.46, 14.98 0.12, and 14.02 been suggested to be inactivated to CCL14a(11–74) by CD26/ 2.93 nM, respectively. A further N-terminal deletion in DPPIV by a specific processing of the N-terminal amino acids CCL14a(11–74) and CCL14a(12–74) significantly reduced the af- Gly9 and Pro10 (30). CD26/DPPIV is an abundant peptidase that is Ϯ finity to the US28 receptor with an IC50 of 43.71 3.58 nM and found in blood plasma, tissue, and on the cell surface of various Ͼ300 nM, respectively (Fig. 4 and Table I). cell types. Interestingly, in this study, we identified CCL14a(12– 74), but not the CD26/DPPIV product CCL14a(11–74) in our iso- N-terminally truncated CCL14a analogs inhibit US28-mediated lation procedure for CCR5-activating material as the main coelut- HIV-1 entry ing isoform of CCL14a(9–74). Identical isoelectric points as well Previously, we have shown that US28 may serve as a cofactor for as identical retention times of the different synthetic CCL14a an- HIV-1 entry when expressed in the presence of CD4 (34). To inves- alogs CCL14a(9–74), CCL14a(11–74), and CCL14a(12–74) on an tigate whether CCL14a analogs are able to inhibit viral entry via analytical RP chromatography column (data not shown) suggest US28, a reporter gene assay was used. CCR5-negative HEK293T that, during the isolation procedure performed, the analogs would cells coexpressing US28 and CD4 were infected with a luciferase coelute in the same fractions. The absence of CCL14a(11–74) in encoding R5-tropic HIV-1 NL4-3 variant 005pf135 (29). US28 me- these fractions suggests that, in the blood filtrate, CCL14a(12–74) diates HIV-1 entry (Fig. 5). An ϳ200-fold increase in luciferase is the predominant inactivated form. activity in the presence of US28 (44,874 Ϯ 3,036, relative light A processing pathway that could explain the existence of units per second) was found compared with uninfected cells (64 Ϯ CCL14a(12–74) and the absence of the CD26/DPPIV-derived The Journal of Immunology 1235

CCL14a(11–74) involves the activity of CD26/DPPIV and an ami- nopeptidase. A putative aminopeptidase that has been shown to act in concert with CD26/DPPIV is APN (APN/CD13). APN is ex- pressed on monocytes, neutrophils, activated T cells, and endothe- lial cells and is involved in cell activation and migration (35–37). This pathway was shown to be relevant for the inactivation of FIGURE 6. Alignment of chemokines known to bind CCR1, CCR5, CXCL8 (IL-8) (38). and/or US28. The conserved amino acids are shown in white on a black Our results show that APN specifically, but slowly, truncates background. Tyr11 of CCL14a(11–74). Slow kinetics were also found for the processing of CCL14a(9–74) into CCL14a(11–74) (30) and are known for chemokines, such as CCL5 or CCL3, which are pro- Significance of the N terminus of CCL14a for its biological cessed by CD26/DPPIV. The predominance of the processed activity form of CCL5(3–68) in cell supernatants as well as in blood To perform functional investigations, different N-terminally trun- demonstrates that such slow processing is of relevance in vivo cated CCL14a derivatives containing CCL14a(9–74) and (39, 40). CCL14a(12–74) as well as CCL14a derivatives that have not been Mass spectrometric analyses of CCL14a(9–74)- and identified in natural sources were used. In accordance with other CCL14a(12–74)-containing fractions from our isolation proce- investigations, this study confirms the impact of the N-terminal dure revealed that the CCL14(12–74) concentrations correlate amino acid sequence Thr1-Ser8 of CCL14a for the regulation of its Downloaded from ϳ to 15% of that of the concentration of CCL14(9–74). This activity on chemokine receptors (20–22, 30). Previously, we have indicates that, during renal insufficiency, the proteolytic activa- shown that, compared with CCL14a(1–74), CCL14a(9–74) tion of CCL14a(9–74) from CCL14a(1–74) exceeds the capac- evolves a 3-log increased affinity to and activation of CCR1 and ity of the CCL14a(9–74)-inactivating pathways and may be CCR5 (20). In this study, we demonstrated that successive trun- correlated to the slow kinetics that have been found for the cation of N-terminal Thr1-Ser5, Ser6, Ser7, and Arg8 is correlated

proteolytic inactivation of CCL14a(9–74) by CD26/DPPIV and with a stepwise increase in the affinity to CCR1 and CCR5 and http://www.jimmunol.org/ APN. An increase in the plasma concentration of CCL14a(9– 2ϩ with a stepwise increase in the [Ca ]i-inducing activity that is 74) during renal insufficiency has been suggested by a rise in mediated via CCR1 and CCR5. The most distinctive increase in the concentration of CCL14a and its activating protease, uPA, 2ϩ affinity and [Ca ]i-inducing activity is induced by the deletion of in the plasma of these patients (41–43), whereas the plasma amino acid Arg8, which forms CCL14a(9–74). We also found a 2ϩ concentrations of APN and CD26/DPPIV are not significantly moderate [Ca ]i-inducing activity in CCR2- and CCR3-trans- increased during renal insufficiency (44, 45). In hemodialysis, fected CHO-K1 cells that was effected by CCL14a(9–74) and patients also experience an increase in other chemokines. A CCL14a(10–74), but not by the other N-terminally truncated dysregulation of the CCR system has been described (42, 46), CCL14a derivatives. This finding underscored the significance of which indicates that dysregulation of the CCL14a pathway may optimal proteolytic trimming of the N terminus of CCL14a. by guest on September 29, 2021 be part of the pathology of renal insufficiency. Under physio- Our data further demonstrated that the amino acid sequence logical conditions, proteolytic inactivation of CCL14a(9–74) Gly9-Pro10 of CCL14a is essential for high-affinity binding to and by CD26/DPPIV and APN may be sufficient for the adequate effective activation of CCR1 and CCR5 in the low nanomolar 9 10 inactivation of CCL14a(9–74). Such a mechanism may be of range. Deletion of the amino acids Gly -Pro (CCL14a(11–74)) significance to maintaining CCL14a(9–74) plasma concentra- caused a significant reduction in the affinity for CCR1 and CCR5, 2ϩ tions in a physiological range, which would avoid systemic ef- the [Ca ]i-inducing activity in CCR1- and CCR5-transfected fects, such as systemic inflammatory responses, that have been cells, and the chemotactic activity in CCR5-transfected pre-B L1.2 known to occur during situations with increased chemokine and cells. But the moderate activity of CCL14a(11–74) indicates that Tyr11 is also of significance in the mediation of activity via CCR1 cytokine plasma concentrations (42, 47). 11 Functionally, proteolytic inactivation of CCL14a by CD26/ and CCR5. However, the truncation of Tyr , which results in CCL14a(12–74), abrogates [Ca2ϩ] activity and chemotactic ac- DPPIV and APN should be considered within the network of i tivity, suggesting that this processing is important for an efficient chemokines and cytokines that regulate the immune response. inactivation of CCL14a. Interestingly, CCL14a(12–74) preserves a Besides the expression of CD26/DPPIV and APN in solid or- moderate affinity for CCR1 and CCR5. Therefore, it is reasonable gans like the prostate, pancreas, liver, and kidney, CD26/DP- to infer that, in high nanomolar concentrations, CCL14a(12–74) PIV is abundantly expressed in T 1 cells and APN in mono- H exhibits significant antagonistic activity against CCL14(9–74) in cytes and myelocytes. These cells might use CD26/DPPIV and calcium flux assays. However, CCL14a(12–74) concentrations in APN to precisely modulate the immune response by processing blood are probably in the lower picomolar range, which would chemokines and cytokines. CD26/DPPIV was found to process suggest that CCL14a(12–74) does not display antagonistic activity CCL3, CCL4, CCL5, CCL11, and CCL22, leading to a change against CCR1 and CCR5 in vivo. in the agonistic or antagonistic potential or receptor specificity. According to the two-site model that describes chemokine-re- CD26/DPPIV-processed chemokines predominantly impair sig- ceptor interactions, the N-loop region of chemokines confers spec- naling through CCR3 and impair chemotaxis to eosinophils. In ificity for receptor binding. Once the chemokine is correctly po- contrast, signaling through CCR1 and/or CCR5 is restored for sitioned on the receptor, the residues in the N terminus are utilized CCL3, CCL4, and CCL5, thus maintaining the chemotactic ac- for triggering (49, 50). In the N-loop region, tivity of the monocytes and lymphocytes. Truncation of CXCL CCL14a carries a number of amino acids that are conserved in chemokines by CD26/DPPIV or APN primarily impairs T cell other CCR1 and CCR5 ligands, as well as in CCR2 and CCR3 function. These results demonstrate a clear hierarchy, with the ligands and, therefore, might be involved in specific interactions protease dictating the function of a chemokine (see also with these receptors (Fig. 6). The aromatic nature of the amino Ref. 48). acid phenylalanine, which is located proximate to the C-C motif, is 1236 PROTEOLYTIC REGULATION OF CCL14a ACTIVITY essential for the binding of CCL4 to CCR5, for the binding of that were found for CCL14a(7–74), CCL14a(9–74), CCL5 to CCR3 and CCR5, and for the activation of CCR1 (49, 51, CCL14a(10–74), and CCL14a(11–74). In contrast, only 52). The conserved amino acids, Lys19 and Arg22, in CCL4 are CCL14a(9–74) and CCL14a(10–74), but not the other CCL14a also of significance for its CCR5-binding properties and are also length variants, revealed anti-HIV-1 activity on CCR5-trans- conserved in CCL14a (50). Arg24, Lys38, and Lys45 of CCL2 are fected cells (22). of significance for its binding to CCR2 and are also conserved in Taken together, these data indicate that the biological activity of CCL14a. On the other hand, Tyr13 and Lys35, which are important CCL14 analogs is tightly regulated by proteolytic processing of for the binding of CCL2 to CCR2, are not conserved in CCL14a, CCL14a. Truncation of CCL14a to CCL14a(9–74) appears to be which might explain the reduced affinity of CCL14a for CCR2 crucial for proper binding to CCR1, CCR2, CCR3, CCR5, and (53). US28, while further cleavage leads to loss of activity. In contrast to the two-site model, our results clearly show that the N-terminal amino acids Gly9, Pro10, and Tyr11 of CCL14a are Acknowledgments not only involved in the activation of but also in the binding to We thank Jutta Barras-Akhnoukh, Rolf Koppitke, Wilfried Hahn, Jane van CCR1 and CCR5. Our binding data indicate that the N-loop region Heteren, and Richard Groen for excellent technical assistance. accounts for a moderate affinity of CCL14a for CCR1 and CCR5, but that the N-terminal amino acids Gly9, Pro10, and Tyr11 poten- Disclosures tiate the affinity for both receptors and induce signaling in the cell. The authors have no financial conflict of interest.

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