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Originally published online as doi:10.1189/jlb.0406290 on September 8, 2006

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(Journal of Leukocyte Biology. 2006;80:1395-1404.)
© 2006 by Society for Leukocyte Biology

N-terminal proteolytic processing by cathepsin G converts RANTES/CCL5 and related analogs into a truncated 4-68 variant

Jean K. Lim*,{dagger}, Wuyuan Lu*, Oliver Hartley{ddagger} and Anthony L. DeVico*,1

* Institute of Human Virology, University of Maryland Biotechnology Institute, and
{dagger} Department of Microbiology and Immunology, School of Medicine, University of Maryland, Baltimore, Maryland, USA; and
{ddagger} Department of Structural Biology and Bioinformatics, Centre Médical Universitaire, Geneva, Switzerland

1 Correspondence: Institute of Human Virology, University of Maryland, Baltimore, 725 W. Lombard Street, 6th fl., Baltimore, MD 21201, USA. E-mail: devico{at}umbi.umd.edu


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ABSTRACT
 
N-terminal proteolytic processing modulates the biological activity and receptor specificity of RANTES/CCL5. Previously, we showed that an unidentified protease associated with monocytes and neutrophils digests RANTES into a variant lacking three N-terminal residues (4-68 RANTES). This variant binds CCR5 but exhibits lower chemotactic and antiviral activities than unprocessed RANTES. In this study, we characterize cathepsin G as the enzyme responsible for this processing. Cell-mediated production of the 4-68 variant was abrogated by Eglin C, a leukocyte elastase and cathepsin G inhibitor, but not by the elastase inhibitor elastatinal. Further, anti-cathepsin G antibodies abrogated RANTES digestion in neutrophil cultures. In accordance, reagent cathepsin G specifically digested recombinant RANTES into the 4-68 variant. AOP-RANTES and Met-RANTES were also converted into the 4-68 variant upon exposure to cathepsin G or neutrophils, while PSC-RANTES was resistant to such cleavage. Similarly, macaque cervicovaginal lavage samples digested Met-RANTES and AOP-RANTES, but not PSC-RANTES, into the 4-68 variant and this processing was also inhibited by anti-cathepsin G antibodies. These findings suggest that cathepsin G mediates a novel pathway for regulating RANTES activity and may be relevant to the role of RANTES and its analogs in preventing HIV infection.

Key Words: HIV • microbicide • chemokine • antiviral • neutrophils


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INTRODUCTION
 
Chemokines constitute a family of chemoattractant cytokines that bind to seven transmembrane spanning G-protein coupled cell surface receptors [1 ] and regulate the migration of immune cells during inflammatory responses. Chemokine activities are regulated and modulated by multiple mechanisms, including glycosaminoglycan binding [2 , 3 ] and sequestration by decoy receptors [4 5 6 ]. For many chemokines, activity is modulated at the post-translational level by proteases that remove a small number of N-terminal residues. These changes may enhance or diminish biological activity, expand or restrict receptor specificity, or convert a receptor agonist into an antagonist [7 8 9 10 11 12 13 14 15 16 17 18 ]. Recently, several members of the cathepsin family have been shown to process chemokines, including cathepsin G [7 , 19 ], cathepsin L [20 ], and cathepsin D [21 ], resulting in enhancement or inactivation of chemotactic activity. Cathepsins have also been linked with several pathological processes, including cancer [22 23 24 ] and arthritis [25 , 26 ].

RANTES/CCL5 is a CC chemokine that binds to several chemokine receptors, including CCR1, CCR3, CCR5, and DARC [27 , 28 ] and plays a key role in inflammation, cell recruitment, and T cell activation. It is also an antiviral agent that potently suppresses the entry of CCR5-tropic HIV-1 strains by occupying and down-regulating CCR5 [29 30 31 ].

The biological activities of RANTES are modulated by at least two pathways of post translational proteolysis. The first is mediated by CD26 [32 ], which is expressed as either a soluble or cell surface-bound enzyme by activated T cells. This processing removes an N-terminal dipeptide from the chemokine, thus producing a 3-68 RANTES variant that is a selective ligand for CCR5 [33 , 34 ]; a potent suppressor of R5 HIV-1 [35 ]; and a poor inducer of monocyte or eosinophil migration [35 , 36 ]. Recently, we reported a second pathway that involves an unassigned protease associated with neutrophils and monocytes. In this case, RANTES is converted into a 4-68 variant that exhibits less efficient binding to CCR5, lower chemotactic activity, and less potent inhibition of HIV [11 ], compared with unprocessed RANTES.

RANTES has been used as a template for the development of a class of anti-HIV agents, including analogs AOP- [37 ], PSC- [38 , 39 ], and Met-RANTES [40 ]. These analogs possess N-terminal modifications that are thought to enhance HIV suppressive activity [37 38 39 ] and/or to decrease receptor activation [40 ]. Recently, PSC-RANTES showed efficacy in preventing vaginal infection in a non-human primate model for HIV infection when applied at high doses [41 ]. However, the extent to which RANTES analogs are subject to N-terminal processing in cervicovaginal fluids is not known. Notably, both neutrophils [42 43 44 45 ] and monocytes [46 , 47 ] are located in cervicovaginal lavage fluids collected from humans and nonhuman primates.

In this report, we characterize the enzyme responsible for generating 4-68 RANTES as cathepsin G, a serine protease known to be associated with neutrophils [48 , 49 ] and monocytes [50 , 51 ].We also show that this enzymatic activity converts AOP- and Met-RANTES into the 4-68 variant and that such processing occurs in cell-free and cell-containing cervicovaginal lavage samples.


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MATERIALS AND METHODS
 
Cell preparation and culture
Heparinized peripheral blood was obtained from healthy donors. PBMC were isolated by sedimentation centrifugation using Ficoll-Hypaque (Sigma, St. Louis, MO). Monocytes were isolated from fresh PBMC using a Monocyte Isolation Kit II magnetic cell sorting kit (Miltenyi Biotec), as described previously [11 ]. Purity was assessed by flow cytometry and found to be consistently >90%.

Neutrophils were isolated from healthy donors using dextran sedimentation, as described previously [52 ]. Briefly, fresh blood was collected in sodium heparin tubes and mixed 1:1 with a solution of 3% dextran in 0.9% NaCl for 30 min at 22°C. The neutrophil-rich upper layer was collected and centrifuged on Histopaque solution at 300 g for 30 min at 22°C. Residual erythrocytes in the neutrophil pellet were removed by hypotonic lysis. Neutrophil purity and viability were consistently >95% as determined by microscopy and trypan blue dye exclusion, respectively. Neutrophils were cultured in RPMI 1640 (GIBCO, Gaithersburg, MD) containing 5% serum-free HB101 (Irvine Scientific, Santa Ana, CA) with 10 U/ml penicillin (Invitrogen, Carlsbad, CA), 100 µg/ml streptomycin (Invitrogen), and 2 mM L-glutamine (Invitrogen) and cultured at 37°C. Cell-free supernatants were collected at various time points for further analysis.

Chemokines and protease inhibitors
Recombinant human 1–68 RANTES was purchased from R&D Systems (Minneapolis, MN). Amino-oxypentane RANTES (AOP-RANTES), Methionine-RANTES (Met-RANTES), and PSC-RANTES were generated as described previously [38 , 53 ]. 1-68 RANTES and RANTES analogs were reconstituted in ultrapure water and were kept at –70°C at a stock concentration of 100 µM prior to use. Eglin C was chemically synthesized using native chemical ligation, as described previously [54 , 55 ]. Elastatinal and GM6001 were purchased from Crescent Chemicals (Islandia, NY) and Chemicon (Rosemont, IL), respectively. All other inhibitors were purchased from Sigma-Aldrich.

RANTES ELISAs
An assay that specifically detects the unprocessed 1-68 RANTES (designated N-RANTES ELISA) was constructed, as described previously [11 ]. Briefly, 200 ng anti-RANTES monoclonal antibody MAB678 (R&D Systems) was adsorbed to 96-well microtiter plate wells (Dynex Technologies, Chantilly, VA) for 16 h at 4°C. All subsequent steps were conducted at 22°C. Nonspecific sites were blocked for 1 h with PBS containing 0.1% Tween 20 and 5% dry milk (Blocking Buffer). After washing with PBS containing 0.1% Tween 20 (Wash Buffer), test samples or chemokine standard (100 µl/well) were incubated for 1 h. After washing, N-RANTES Ig (1 µg/ml in blocking buffer) was added for 1 h. Peroxidase-conjugated goat anti-rabbit immunoglobulin (KPL) was then added (125 ng/ml in blocking buffer) for 30 min. The amount of bound immunoglobulin was detected by reaction to tetramethylbenzidine (BioFX Laboratories, Owings Mills, MD), and the resulting absorbance measured at 450 nm. To determine RANTES concentrations in culture supernatants, the absorbance values were compared with a standard curve generated with serial concentrations of recombinant RANTES (starting at 500 pM) in the appropriate buffer or culture media.

A commercial RANTES ELISA system (designated as the standard ELISA) was employed, using MAB678 as a capture antibody and biotinylated anti-RANTES antibody BAF278 (R&D Systems) for detection. The assays were carried out according to the manufacturer’s instructions. The percentage of unprocessed RANTES in a sample was determined by calculating the mean N-RANTES ELISA concentrations/mean standard RANTES concentrations x 100.

Mass spectrometry
Surface-enhanced laser desorption and ionization (SELDI) mass spectrometry (Ciphergen Biosystems, Fremont, CA) was performed with PS10 protein chips as described [11 ]. MAB678 (R&D Systems) or mouse IgG1 isotype control (Sigma) was spotted on the chips (2 µg/spot in PBS) and incubated overnight at 4°C in a humid chamber. Active sites were blocked by the addition of 1 M ethanolamine in PBS, pH 8 for 1 h at 22°C. Culture supernatants were incubated with each spot overnight at 4°C using a fitted bioprocessor unit (Ciphergen Biosystems). After washing with PBS containing 0.1% Triton-X 100, the samples were crystallized by the addition of 1 µl/spot saturated CHCA (Sigma) in 50% acetonitrile in HPLC-grade water. Bovine ubiquitin (Sigma) with molecular weight of 8564.0 Da was added as an internal standard and analyzed using the Ciphergen Series PBS II reader and software.

Inhibition of cell-associated enzymatic activity
Monocytes (1x107) isolated from healthy donors were lysed in 1 ml of M-Per Cell Lysis Reagent (Pierce Biotechnology, Rockford, IL). Lysates were diluted 1:10 in PBS and incubated with 10 nM recombinant 1-68 RANTES (R&D Systems) in the presence of the following protease inhibitors: 500 nM diisopropyl fluorophosphates (DFP), 1 mM PMSF, 500 nM EDTA, 50 µM GM6001, 100 nM Eglin C, or 20 µM elastatinal for 6 h at 37°C. All inhibitors were preincubated with lysates at 22°C for 30 min prior to the addition of RANTES. Samples were then stored at 4°C after the incubation period. Chemokine processing was assessed by SELDI mass spectrometry as described above.

Experiments with antibodies were performed by mixing neutrophils (1x105 cells/ml in serum-free media) with serial concentrations of polyclonal sheep anti-cathepsin G immunoglobulin (Novus Biological, Littleton, CO) or nonspecific sheep immunoglobulin control (Sigma) for 1 h at 37°C. Recombinant 1-68 RANTES (10 nM) was then added to the cultures and incubated for 12 h at 37°C. Supernatants were harvested and tested by RANTES ELISA and SELDI mass spectrometry as described above.

Chemokine digestion
Cathepsin G (Calbiochem, San Diego, CA) was reconstituted in 50 nM NaOAc, pH 5.5 containing 150 mM NaCl. Recombinant 1-68 RANTES and RANTES analogs were resuspended in RPMI 1640 containing 5% HB101 to final concentrations of 10 nM and then mixed with serial concentrations of enzyme. Control digestions were performed by the addition to buffer alone. The reactions were incubated at 37°C for 12 h. In certain experiments, 100 nM Eglin C was added to the reaction mixtures prior to the addition of RANTES. The resulting cleaved products were analyzed by RANTES ELISA and SELDI mass spectrometry as described above.

Collection of cervicovaginal lavage samples
Three healthy female cynomolgus macaques, weighing between 2.5 and 3.0 kg, were used as donors. During the course of the experiment, animals were housed in accordance with regulations approved by the University of Maryland Biotechnology Institute Institutional Animal Care and Use Committee. Each animal was anesthetized by intramuscular injection of ketamine chloride, avoiding the menstrual period. A speculum was placed intravaginally, and a 5 ml wash of PBS, pH 7.2 was introduced into the canal. After 60 s, the wash was aspirated and immediately placed at 4°C. The wash samples were centrifuged at 300 g for 10 min at 4°C to pellet cells. The cell-free supernatant was retained, and the pelleted cells were resuspended in 5 ml RPMI 1640 supplemented with 5% HB101 serum-free media. The cell-free supernatants and cells were incubated with 5 nM 1-68 RANTES for up to 6 h at 37°C. Samples were tested by SELDI mass spectrometry for detection of RANTES as described above.


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RESULTS
 
Characterization of the cell-associated proteolytic activity that generates 4-68 RANTES
We previously showed that whole unstimulated monocytes and neutrophils express a proteolytic activity that selectively processes RANTES into a 4-68 variant [11 ], which lacks an N-terminal Ser1-Pro2-Tyr3 tripeptide. To elucidate the type of protease involved, we first tested an array of protease inhibitors for the capacity to block this processing. As many protease inhibitors are toxic to cells, these experiments used monocyte cell lysates, as described previously [7 , 56 ]. Aliquots of cell lysates were preincubated with each inhibitor followed by the addition of 10 nM 1-68 RANTES. Since lysates from several donors were tested, matched donor lysates were tested in the absence of inhibitor for comparison. The samples were then analyzed by SELDI mass spectrometry. As shown in Fig. 1 (bottom panel), 1-68 RANTES was extensively converted into the 4-68 variant in the absence of any protease inhibitor. As we [11 ] and others [57 ] have observed, a fraction of 1-68 and 4-68 RANTES was ~16 Da larger than the expected values (7863.0±2 Da vs. 7847.0 Da for 1-68 RANTES; 7515.6±1 Da vs. 7499.6 Da for 4-68 RANTES). This is most likely due to the oxidation of the methionine residue at position 67 in the RANTES sequence [11 , 57 ]. Such oxidation was routinely observed, although the fraction of the material that exhibited this modification varied between experiments.


Figure 1
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  		Figure 1. Serine protease inhibitors block 4-68 RANTES producton. Aliquots of a monocyte lysate-derived from different donors were either untreated or treated with 500 nM DFP, 1mM PMSF, 500 nM EDTA, 50 µM GM6001, 100 nM Eglin C, or 20 µM elastatinal for 30 min at 22°C followed by the addition of 10 nM recombinant 1-68 RANTES. After 6 h at 37°C, samples were recovered for analysis by SELDI mass spectrometry. Samples treated with inhibitor are shown with matched untreated monocyte lysates from the same donor. Each inhibitor was tested at least twice using lysates obtained from different donors. Mass values and variant assignments are indicated above major peaks.

Compared with the control assays, processing was not affected by the presence of the matrix metalloproteinase inhibitors EDTA or GM6001. In contrast, strong abrogation of processing occurred in the presence of the serine protease inhibitors DFP and PMSF (Fig. 1 , top panel). Processing was also inhibited by Eglin C, which inhibits leukocyte elastase and cathepsin G [58 , 59 ] but not by elastatinal, a potent inhibitor of elastase [60 , 61 ]. These data collectively suggested that cathepsin G may be responsible for generating the 4-68 RANTES variant.

Given these findings, a second series of experiments was carried out to determine whether cathepsin G was responsible for processing RANTES in whole cell cultures. Neutrophils were used, as they are associated with higher levels of processing activity compared with monocytes [11 ]. Recombinant 1-68 RANTES was incubated with whole unstimulated neutrophils (see Materials and Methods) in the presence of serial concentrations of a polyclonal sheep anti-human cathepsin G antibody or a nonspecific sheep immunoglobulin (IgG) control. As shown in Fig. 2A , the ELISAs showed that N-terminal processing of RANTES was reduced in the presence of anti-cathepsin G antibody. Importantly, the percentage of unprocessed chemokine increased in proportion to the anti-cathepsin G antibody concentration. At 300 µg/ml antibody, roughly 80% of the RANTES had an intact N terminus, whereas only 20% was unprocessed in the absence of antibody. In comparison, RANTES processing was not affected by the control IgG. A major limitation of this ELISA approach is the inability to reveal the positions and identities of residues removed by processing. To obtain this information, samples tested by RANTES ELISA were tested in parallel using a SELDI mass spectrometry method, which uses an anti-RANTES antibody-coated chip to selectively capture RANTES from solution. This technique allowed us to evaluate the nature of proteolytic cleavage based on the apparent masses of proteins desorbed from the chip. Through this technique, SELDI analysis confirmed that the amount of 4-68 RANTES produced by the cells was reduced in the presence of anti-cathepsin G antibody (Fig. 2B) . At 300 µg/ml anti-cathepsin G antibody, the intensity of the 1-68 RANTES peak was strongly increased, while 4-68 RANTES peak was diminished compared with experiments carried out with the same concentration of control IgG (Fig. 2B) . Taken together, these data strongly suggested that cathepsin G is responsible for neutrophil-mediated processing of RANTES.


Figure 2
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  		Figure 2. Anti-cathepsin G antibodies block neutrophil-mediated RANTES processing. (A) Human neutrophils were suspended in serum-free media and incubated with 10 nM recombinant 1-68 RANTES in the presence of serial concentrations of sheep anti-cathepsin G antibody (dark gray bars), nonspecific sheep IgG control (light gray bars), or no antibody (white bar). After 12 h, culture supernatants were analyzed for N-terminal processing by standard and N-RANTES ELISAs in order to determine the percentage of unprocessed (1–68) RANTES in the cultures (see Experimental Procedures). All assays were performed in duplicate. Mean percentage values are shown; bars indicate standard deviation. (B) SELDI mass spectrometry was performed on the same supernatant samples analyzed by ELISA. The observed mass values and variant assignments are shown. Samples were tested in parallel on IgG1-coated chips and showed no significant peaks above background. The experiment was repeated twice using blood from two healthy donors with similar results.

Purified cathepsin G processes RANTES into the 4-68 variant
To confirm our observations in the cell-based experiments, reagent cathepsin G was directly tested for its capacity to process 1-68 RANTES. Proteolytic processing was assessed by N-RANTES/Standard ELISA (see Materials and Methods) and SELDI mass spectrometry of reaction mixtures containing 10 nM RANTES and serial concentrations of enzyme. Control assays were carried out in the absence of enzyme. As shown in Fig. 3A , the total amount of RANTES detected by the standard ELISA was not significantly altered by cathepsin G treatment, indicating that cathepsin G was not degrading RANTES non-specifically. However, the addition of enzyme resulted in a dose-dependent decrease in the amount of RANTES containing an intact N terminus. Only a minor fraction (8.4%) of chemokine possessed an intact N terminus after incubation with 5 mU of enzyme. In comparison, equivalent amounts of total and unprocessed RANTES were detected in the absence of enzyme.


Figure 3
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  		Figure 3. 1-68 RANTES is processed into 4-68 RANTES by cathepsin G. (A) Recombinant 1-68 RANTES (10 mM) was untreated or treated with the indicated concentrations of reagent cathepsin G for 12 h at 37°C and analyzed by N-RANTES (light bars) and standard ELISAs (dark bars) to quantify unprocessed and total RANTES, respectively (see Experimental Procedures). All assays were performed in duplicate. Mean ELISA values are shown; bars indicate standard deviation. (B) The same samples were tested by SELDI mass spectrometry. No peaks were recovered from chips spotted with isotype control IgG1 antibody (data not shown). The experiment was repeated twice using blood from two donors with similar results. (C) The experiments were repeated in the presence or absence of 100 nM inhibitor Eglin C and 0.5mU Cathepsin G and analyzed by SELDI mass spectrometry. Mass values and variant assignments are indicated above major peaks.

Samples from the enzymatic reaction mixtures were also tested by SELDI mass spectrometry. As shown in Fig. 3B , only one peak was detected in the absence of enzyme (7863.3 Da), which corresponded with the predicted mass of 1-68 RANTES (7863.0 Da expected for the oxidized form). However, enzyme treatment yielded a second species with a molecular weight (7499±2 and 7515±1 Da) corresponding to the size (7499.6 Da and 7515.6 Da for the oxidized form) predicted for the 4-68 RANTES variant. The peak intensity for this variant was directly related to the amount of enzyme added to the reaction mixture. Thus, most of the RANTES treated with 5 mU enzyme was detected as the 4-68 variant, in agreement with the ELISA results, indicating that extensive N-terminal processing had occurred [Fig. 3A ]. Importantly, 4-68 RANTES was not produced in assays that included 0.5 mU Cathepsin G and the Eglin C protease inhibitor [Fig. 3C ]. These data confirmed that cathepsin G is capable of converting 1-68 RANTES into the 4-68 variant, in accordance with the neutrophil experiments.

Neutrophils and reagent cathepsin G process the N termini of AOP-RANTES and Met-RANTES
To further investigate cathepsin G-mediated processing of RANTES, we tested whether the N terminally modified RANTES analogs AOP-RANTES [37 , 62 , 63 ], PSC-RANTES [39 ], and Met-RANTES [40 , 64 ] could serve as substrates. Each RANTES analog was treated with reagent cathepsin G or added to neutrophil cultures (1x105 cells/ml) as shown in Fig. 4 . 1-68 RANTES was tested as a control. Reagent cathepsin G enzyme converted AOP- and Met-RANTES into species with sizes corresponding to the predicted mass of the 4-68 variant [Fig. 4 , bottom panel]. However, cathepsin G was unable to digest PSC-RANTES, as incubation with cathepsin G did not produce any truncated variants. In the neutrophil cultures, all three RANTES variants showed evidence of proteolytic modification similar to what was observed in the presence of purified cathepsin G. Incubation of AOP- and Met-RANTES (with expected molecular weights of 7902.0 and 7978.2 Da, respectively) with neutrophils produced one predominant processed variant matching the predicted molecular weight of the 4-68 variant (observed 7499.6±1 and oxidized variant 7515.6±2). In comparison, PSC-RANTES (7895.0 Da expected) was converted into a variant with an observed molecular weight of 7343.7 Da, which corresponded to the removal of 5 N-terminal residues, which was designated as 6-68 RANTES (7341.5 Da expected for the oxidized form). These data indicated that neutrophil-associated cathepsin G is capable of processing AOP- and Met-RANTES, as well as 1-68 RANTES into a 4-68 variant, whereas PSC-RANTES is resistant to cathepsin G and subject to proteolysis by an unidentified neutrophil-associated enzyme.


Figure 4
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  		Figure 4. RANTES analogs are processed by neutrophils and reagent cathepsin G. 1–68 RANTES, AOP-RANTES (AOP), PSC-RANTES (PSC), or Met-RANTES (Met) were incubated alone (top), in the presence of neutrophils (middle), or reagent cathepsin G (bottom). After 12 h, samples were collected for analysis of processing by SELDI mass spectrometry. The observed mass values and variant assignments are shown. Samples were tested in parallel on IgG1-coated chips and show no significant peaks above background (data not shown). Experiments were repeated with cells from three donors and produced similar results; a representative experiment is shown.

N-terminal processing of RANTES analogs by cervicovaginal lavage samples from cynomolgus macaques
RANTES analogs have been evaluated as vaginal microbicides in non-human primate models for vaginal HIV-1 transmission [41 ]. Since the vaginal mucosa is known to contain chemokine-processing enzymes, including elastase [65 , 66 ], and cathepsin D [67 ], and is known to contain resident neutrophils [42 43 44 45 , 68 ] and monocytes [46 , 47 ], it is possible that RANTES analogs are subject to proteolytic degradation when placed in the cervicovaginal environment. To examine this possibility, cervicovaginal lavage samples were acquired from 3 cynomolgus macaques and tested for RANTES processing activities. The samples were separated into cell-free and cellular fractions by centrifugation; the recovered cells were resuspended in serum-free medium. RANTES and RANTES analogs were incubated with each fraction and analyzed by SELDI mass spectrometry after 1 and 6 h.

As shown in Fig. 5A , the cell-free lavage fraction contained proteolytic activities that processed 1-68 RANTES into truncated variants corresponding to 3-68 RANTES and 4-68 RANTES, respectively, that was detectable after incubation for 1 h. The observed molecular weights (7678.2±0.6 and 7514.6±0.1 Da) corresponded to the expected molecular weights (7678.8 and 7515.6 Da) of the oxidized variants, suggesting that the soluble fraction of these lavage samples contain both soluble CD26 and cathepsin G. After the 6 h incubation, these species comprised an even greater proportion, as the intensities of the peaks increased significantly. AOP-RANTES and Met-RANTES were similarly processed by the cell-free fraction, with the 4-68 variant being the most prominent product. Notably, the CD26-processed form (3-68 RANTES) was not generated for either AOP- or Met-RANTES variants, as these alterations to the N-terminal region will disrupt the specificity of CD26, which cleaves dipeptides and requires the proline residue at position 2 of native RANTES. In contrast, PSC-RANTES appeared intact at both time points.


Figure 5
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  		Figure 5. RANTES is processed by cervicovaginal enzymes. Cervicovaginal lavage samples obtained from female cynomolgus macaques were separated into cellular and cell-free fractions by centrifugation. Recovered cells were resuspended in serum-free media to the original volume of the lavage. The cell-free (A) or cellular (B) fraction was treated with a 5nM final concentration of 1-68 RANTES, AOP-RANTES, PSC-RANTES, or Met-RANTES for 1 or 6 h at 37°C. After incubation, samples were collected for analysis by SELDI mass spectrometry. The observed mass values and variant assignments are shown. A representative experiment is shown; experiments were repeated with lavage samples collected from three animals and produced similar results.

In assays carried out with the cellular fraction, 1-68 RANTES, AOP, and Met-RANTES were again processed into the 4-68 RANTES variant over time [Fig. 5B ]. Notably, unprocessed Met-RANTES was detected as three distinct molecular weights (7979.8, 7991.1±0.2, and 8010.5) corresponding to an unoxidized form (7978.2 Da expected), or the oxidized variants (7994.2 Da and 8010.2 Da expected) with a mass increase of 16 and 32 Da, respectively, which is expected given the additional N-terminal methionine residue available for oxidation. PSC-RANTES appeared to be intact after the 1-h incubation period but was apparently processed into a 6-68 RANTES variant (7339.5 Da observed, 7341.5 expected) by 6 h after incubation with the cellular fraction, similar to what was observed in the neutrophil cultures [Fig. 4 ].

To verify that cathepsin G contributed to the lavage-associated processing, the experiments were repeated with 1-68 RANTES in the presence of sheep neutralizing anti-cathepsin G antibody. The cellular fraction was used as the soluble fraction exhibited significant soluble CD26 activity [Fig. 5A ], which would confound the ELISA analyses. As shown in Fig. 6 , RANTES processing by the cellular fraction was inhibited by anti-cathepsin G antibody in a concentration-dependent manner as determined by ELISA and SELDI analyses.


Figure 6
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  		Figure 6. Anti-cathepsin G antibodies block the generation of 4-68 RANTES in cervicovaginal lavage samples. (A) Cells recovered from macaque cervicovaginal lavage were suspended in serum-free media and incubated with the indicated concentrations of sheep anti-cathepsin G antibody (dark gray bars) or nonspecific sheep control IgG (light gray bars) for 1 h at 37°C followed by addition of 5 nM recombinant 1-68 RANTES. After 6 h, supernatants were analyzed by standard and N-RANTES ELISAs in order to determine the percentage of unprocessed 1–68 RANTES in the cultures (see Experimental Procedures). All assays were performed in duplicate. Mean percentage values are shown; bars indicate standard deviation. (B) SELDI mass spectrometry was performed on the same samples analyzed by ELISA in (A). The observed mass values and variant assignments are shown. Samples were tested in parallel on IgG1-coated chips and show no significant peaks above background (data not shown).


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DISCUSSION
 
The N-termini of many chemokines play critical roles in the recognition and functional activation of cognate receptors [69 ]. These domains typically form disordered structures and as such are prone to proteolytic digestion [69 70 71 72 ]. Consequently, the immune system is able to exploit proteolysis as a means for modulating the immunological properties of chemokines.

Recently, we showed that an enzymatic activity associated with neutrophils and monocytes removes three N-terminal residues from RANTES to produce a 4-68 variant with decreased chemotactic activity and lower anti-HIV potency [11 ]. Neutrophils contain several serine proteases, including human leukocyte elastase, proteinase-3, and cathepsin G, which are stored at high concentrations within azurophilic granules [49 , 73 ]. These proteases are released upon activation by stimulants such as formyl peptides and PMA [74 ] and are primarily involved in the breakdown of engulfed pathogens [75 ]. More recently, it has been shown that enzymatically active forms of these proteins are bound to the surfaces of unstimulated neutrophils and monocytes [74 , 76 77 78 ] where they are available to interact with extracellular substrates. Processing by neutrophil-derived proteases has been reported to alter the biological activities of IL-8 [79 ] and ENA-78 [19 ] and to abrogate the functions of SDF-1 [7 ]. Although neutrophil-derived serine proteases were shown to digest the CCR5 ligand LD78 [56 ], they have not been shown to act on RANTES.

This study revealed two lines of evidence to demonstrate that cathepsin G is the neutrophil-derived enzyme that processes RANTES into the 4-68 variant. First, the production of this variant in neutrophil cultures was effectively inhibited by a neutralizing anti-cathepsin G-specific antibody [Fig. 2 ]. Second, RANTES processing by cell lysates was blocked by Eglin C, a known inhibitor of neutrophil elastase and cathepsin G. Importantly, processing was not inhibited by elastatinal, a specific inhibitor of elastase [Fig. 1 ], which by elimination indicated that cathepsin G was the pertinent enzyme inhibited by Eglin C.

In accordance with these findings, the 4-68 variant was also produced by enzymatic digestion of RANTES with reagent cathepsin G [Fig. 3 ]. Since this enzyme has dual specificities with a preference for both bulky hydrophobic and cationic residues at the P1 position of the target polypeptide [80 ], the P1 residue (Tyr) of the cleavage site that yields 4-68 RANTES fits the expected specificity profile. While both Met-RANTES and AOP-RANTES feature Tyr at residue P1, PSC-RANTES features L-cyclohexylglycine, a non-natural amino acid, at this position [38 ]. Although cyclohexylglycine has a bulky hydrophobic side chain, this discrepancy might be the basis of the resistance shown by PSC-RANTES to degradation by cathepsin G. A previous study by Delgado et al. [7 ] failed to detect RANTES processing by reagent cathepsin G; however, it is possible that the analytical techniques used in that study (SDS-PAGE and Coomassie blue staining) were not as sensitive as the ones used here and were unable to distinguish the 1-68 and 4-68 variant forms, which differ by only three residues. Low sensitivity might also explain why Delgado and colleagues did not detect cathepsin G-mediated cleavage of IL-8; a chemokine known to be modified by the enzyme [79 ].

Taken together, our findings indicate that there are at least two proteases that act on RANTES to differentially modulate its activity. One is soluble or cell surface CD26, which removes two N-terminal residues from the chemokine to produce 3-68 RANTES [11 , 32 ]. The second, as defined here, is neutrophil-associated cathepsin G, which removes three N-terminal residues to produce a 4-68 variant. The impact of these two enzymes on the biological activities of RANTES is readily distinguishable despite the rather subtle differences in processing (removal of two vs. three N-terminal residues). Compared with 3-68 RANTES, the 4-68 variant has roughly 10-fold lower CCR5 binding efficiency, 100-fold lower chemotactic activity, and 10-fold lower HIV-suppressive activity [11 ]. Notably, 3-68 RANTES appears to be resistant to processing into the 4-68 variant [11 ]. Thus, it is reasonable to expect that RANTES exhibits variable biological functions during an immune response, depending on the local concentrations of CD26 and/or cathepsin G, the magnitude of neutrophil infiltration, and the relative processing kinetics of the two enzymes.

It is noteworthy that RANTES processing into the 4-68 variant occurred in cultures of intact unstimulated cells [11 ]. We attribute this to the membrane-associated cathepsin G [74 , 76 77 78 ], which would be accessible to neutralizing antibody. However, we cannot eliminate the possibility that some of the processing activity was mediated by enzyme released from granules in response to plastic adherence or other manipulations during the cell isolation procedures.

RANTES and other CCR5 ligands potently suppress R5 HIV-1 infection [81 , 82 ] and may play a role in protecting against mucosal HIV infection [83 , 84 ]. Consequently, RANTES has been used as a basis for developing analogs such as AOP [37 ], PSC [38 , 39 ], and Met-RANTES [40 ] that might be used as topical microbicides to prevent sexual transmission of the HIV [41 ]. These analogs possess N-terminal modifications designed to greatly enhance HIV suppressive activity [37 38 39 ], and/or to decrease receptor activation [40 ]. Because the potential value of these agents requires the maintenance of an intact N terminus, stability in the cervicovaginal environment may be a concern. Neutrophils are the most abundant leukocyte cell type in vaginal fluid [42 , 68 ] and are recruited to the vaginal mucosa in response to sexually transmitted viral and bacterial infections, including HSV-2 [68 ] and Chlamydia trachomatis [85 ]. In accordance, our experiments show that RANTES, AOP-RANTES, and Met-RANTES are converted into the 4-68 RANTES variant when incubated with the soluble and cellular fractions of cervicovaginal lavage samples taken from cynomolgus macaques [Fig. 5 ], which are routinely used as models for sexual HIV transmission [86 87 88 ]. Further, RANTES processing by the cells was inhibited by neutralizing anti-cathepsin G antibody [Fig. 6 ].

Collectively, our data suggest that RANTES and some of the related analogs may exist primarily as truncated variants in the vaginal mucosa. We suspect that the proteolytic processing observed in our experiments underestimates what occurs in vivo, as the sample collection method greatly dilutes the soluble enzymatic activity and densities of resident cells. Such findings indicate that the prophylactic efficacy of CCR5 ligands may be significantly affected by proteolytic conversion into the 4-68 variant upon vaginal application, resulting in reduced efficacy. In comparison, PSC-RANTES was resistant to cathepsin G-mediated proteolysis, although it was subject to a different form of N-terminal processing when incubated with either neutrophils or the cellular fraction of cervicovaginal lavage fluid. In both cases, N-terminal proteolysis generated a variant apparently missing 5 N-terminal residues along with the PSC moiety [Fig. 4 and 5 ]. Nevertheless, because of its greater stability, PSC-RANTES seems better suited than the other analogs for prophylactic use against HIV in agreement with a recent study showing that high doses of the molecule protected against vaginal SHIV infection in a nonhuman primate model [41 ]. This study brought to light a striking discrepancy between the antiviral potency of PSC-RANTES in vitro and its potency in this model. Hence, degradation in the vaginal environment may have reduced the efficacy of PSC-RANTES in this study, although it should be noted that other entry inhibitors have shown similar discrepancies between their potencies in vitro and the efficacy in this model.

CCR5 ligands, including RANTES, have been detected in cervicovaginal lavage fluid from HIV-positive, as well as uninfected women [89 90 91 ], at levels capable of suppressing HIV-1 [91 ]. Recently, it was shown that Kenyan commercial sex workers who resist infection have roughly 10-fold higher expression of RANTES in genital mucosa than matched control individuals [92 ]. Our results suggest the 4-68 variant represents the predominant form of RANTES in these subjects. In accordance, the native RANTES present in cervicovaginal lavage samples collected from rhesus macaques exists primarily as the 4-68 RANTES form (data not shown). However, it will be important to determine whether there are significant differences in RANTES processing at the genital mucosa of patients at high or low risk of sexually transmitted HIV infection.


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ACKNOWLEDGEMENTS
 
The authors thank Drs. Tina Kish and Alan Cross for guidance in cervicovaginal lavage collection and neutrophil isolation techniques, respectively. We also thank Engin Erdogan for graphical assistance. This work was supported by grants RO1 HL063647 and RO1 HD39108 to A. L. D. and by grants PO1 AI 51649-01 and 3100A0-110042 (Swiss National Science Foundation) to O. H.

Received April 25, 2006; revised July 4, 2006; accepted July 20, 2006.


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