Truncation of Macrophage-derived Chemokine by CD26/ Dipeptidyl-Peptidase IV beyond Its Predicted Cleavage Site Affects Chemotactic Activity and CC Chemokine Receptor 4 Interaction*

Paul ProostDagger §, Sofie StruyfDagger , Dominique Scholsparallel , Ghislain OpdenakkerDagger , Silvano Sozzani**, Paola Allavena**, Alberto Mantovani**, Koen AugustynsDagger Dagger , Gunther BalDagger Dagger , Achiel HaemersDagger Dagger , Anne-Marie Lambeir§§, Simon Scharpé§§, Jo Van DammeDagger , and Ingrid De Meester§§

From the Dagger  Laboratory of Molecular Immunology, and parallel  Laboratory of Experimental Chemotherapy, Rega Institute for Medical Research, University of Leuven, Minderbroedersstraat 10, B-3000 Leuven, Belgium, ** Laboratory of Immunology, Istituto di Ricerche Farmacologiche Mario Negri, Via Eritrea 62, I-20157 Milan, Italy, Dagger Dagger  Laboratory of Pharmaceutical Chemistry, and §§ Laboratory of Clinical Biochemistry, University of Antwerp, Universiteitsplein 1, B-2610 Wilrijk, Belgium

    ABSTRACT
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Abstract
Introduction
References

The serine protease CD26/dipeptidyl-peptidase IV (CD26/DPP IV) and chemokines are known key players in immunological processes. Surprisingly, CD26/DPP IV not only removed the expected Gly1-Pro2 dipeptide from the NH2 terminus of macrophage-derived chemokine (MDC) but subsequently also the Tyr3-Gly4 dipeptide, generating MDC(5-69). This second cleavage after a Gly residue demonstrated that the substrate specificity of this protease is less restricted than anticipated. The unusual processing of MDC by CD26/DPP IV was confirmed on the synthetic peptides GPYGANMED (MDC(1-9)) and YGANMED (MDC(3-9)). Compared with intact MDC(1-69), CD26/DPP IV-processed MDC(5-69) had reduced chemotactic activity on lymphocytes and monocyte-derived dendritic cells, showed impaired mobilization of intracellular Ca2+ through CC chemokine receptor 4 (CCR4), and was unable to desensitize for MDC-induced Ca2+-responses in CCR4 transfectants. However, MDC(5-69) remained equally chemotactic as intact MDC(1-69) on monocytes. In contrast to the reduced binding to lymphocytes and CCR4 transfectants, MDC(5-69) retained its binding properties to monocytes and its anti-HIV-1 activity. Thus, NH2-terminal truncation of MDC by CD26/DPP IV has profound biological consequences and may be an important regulatory mechanism during the migration of Th2 lymphocytes and dendritic cells to germinal centers and to sites of inflammation.

    INTRODUCTION
Top
Abstract
Introduction
References

CD26/dipeptidyl-peptidase IV (CD26/DPP IV1; EC 3.4.14.5) is a 110-kDa glycoprotein expressed on the membrane of a variety of cells including epithelial and endothelial cells. Moreover, its expression is up-regulated on activated T cells (1-3). The proteolytic activity of CD26/DPP IV is located in the extracellular domain of the protein, which also occurs in a soluble active form in plasma. CD26/DPP IV has a unique specificity compared with other exopeptidases. It is known to cleave dipeptides from the NH2 terminus of peptides with a penultimate Pro, Hyp, or Ala residue. The penultimate NH2-terminal Pro is present in a number of cytokines (e.g. interleukin-1beta (IL-1beta ), IL-2, IL-5, IL-6, and IL-10), growth factors (e.g. insulin-like growth factor 1, granulocyte colony-stimulating factor, and growth hormone), neuro- and vasoactive peptides (e.g. neuropeptide Y, peptide YY, and substance P) and chemokines (e.g. stromal cell-derived factor-1alpha (SDF-1alpha ) and RANTES). This Pro residue protects these molecules from degradation by most aminopeptidases (4). Some short peptides, including substance P and gastrin-releasing peptide have been known for some time as effective substrates for CD26/DPP IV (3). In contrast, none of the intact cytokines with a penultimate Pro has been identified as a CD26/DPP IV substrate although smaller peptides containing their NH2-terminal sequences were cleaved (5).

A number of observations indicate that CD26/DPP IV plays an important role in immunology, in particular during T cell activation and proliferation (1, 2). The involvement of the enzymatic activity in the immunoregulatory function of CD26/DPP IV is demonstrated under several circumstances. These include the in vitro normalization of impaired responses to recall antigens by the addition of soluble CD26/DPP IV (6, 7) and the in vivo suppression of immune activation upon alloantigen challenge by specific CD26/DPP IV inhibitors (8). Recently, CD26/DPP IV has been shown to process the NH2 terminus of a number of chemokines including RANTES, granulocyte chemotactic protein-2 (GCP-2), and SDF-1, generating naturally occurring truncated molecules with a significantly altered biological activity (9-12). Indeed, truncation of RANTES by CD26/DPP IV into RANTES(3-68) generated a chemotaxis antagonist with enhanced anti-HIV-1 activity against macrophage-tropic (M-tropic) HIV-1 strains. Incubation of SDF-1 with CD26/DPP IV drastically reduced the chemotactic activity of this chemokine but also reduced its anti-HIV-1 activity against T cell-tropic (T-tropic) HIV-1 strains.

The recently identified CC chemokine macrophage-derived chemokine (MDC), also designated stimulated T cell chemotactic protein-1 (STCP-1), binds to CC chemokine receptor 4 (CCR4). The MDC cDNA was one of the most abundant sequences identified in a macrophage library. MDC is synthesized by macrophages and dendritic cells and is highly expressed in the thymus (13-16). NH2-terminally truncated forms of natural MDC were isolated from a CD8+ T cell clone and have been reported to inhibit HIV-1 infection (17). Here, we show that MDC is a CD26/DPP IV substrate. Surprisingly, the enzyme removes an additional dipeptide from the MDC NH2 terminus proving that the substrate specificity of this dipeptidyl-peptidase is less restricted than anticipated. In addition, this double truncation affects the receptor interaction and chemotactic activity of MDC.

    EXPERIMENTAL PROCEDURES

Reagents and Cells-- Recombinant synthetic MDC and 125I-MDC were obtained from PeproTech (Rocky Hill, NJ), Gryphon Sciences (South San Francisco, CA), and Amersham Pharmacia Biotech (Little Chalfont, UK), respectively. Synthetic NH2-terminal chemokine fragments (GPYGANMED or MDC(1-9), YGANMED or MDC(3-9), ANMED or MDC(5-9), and SPYSSDTTP or RANTES(1-9)) were synthesized using an automated PS3 solid phase peptide synthesizer (Rainin Instrument Company Inc., Woburn, MA) using Fmoc (N-(9-fluorenyl)methoxycarbonyl) chemistry. The peptides were purified by HPLC and analyzed on an electrospray VG Quattro-II triple quadrupole mass spectrometer (Fisons, Manchester, UK). Human CD26/DPP IV was obtained from prostasomes and purified to homogeneity using anion exchange followed by affinity chromatography onto immobilized adenosine deaminase (18). Soluble CD26/DPP IV, without membrane anchor and starting at amino acid Gly31, was obtained from total seminal plasma and purified and characterized as described (19).

Fresh peripheral blood-derived mononuclear cells (PBMC) were obtained from healthy donors and isolated by hydroxyethyl starch sedimentation and Ficoll-sodium metrizoate centrifugation (20). Monocytes were separated from the mononuclear cell fraction with anti-CD14 antibodies coupled to magnetic microbeads (Miltenyi Biotec, Bergisch Gladbach, Germany). Monocyte-derived dendritic cells were purified as described previously (13). Lymphocytic SUP-T1 cells were cultured in RPMI 1640 (BioWhitakker, Walkersville, MD) supplemented with 10% fetal calf serum, and HOS cells transfected with CD4 and CCR4 were grown in Dulbecco's modified Eagle's medium (BioWhitakker), 10% fetal calf serum, and 1 µg/ml puromycin (21).

Chemokine and Peptide Degradation with CD26/DPP IV-- MDC was treated with membrane-bound or soluble CD26/DPP IV and purified by HPLC as described previously (9, 11). NH2-terminal truncation was verified after electroblotting or HPLC on 0.5-2 µg of MDC by Edman degradation on a 477A/120A protein sequencer (Perkin-Elmer).

Degradation of the small synthetic MDC peptides was analyzed by fluorescamine derivatization. The synthetic peptides (1 mM) were incubated at 37 °C with CD26/DPP IV in phosphate-buffered saline, pH 8.0, containing 1 mM EDTA. At indicated time points, 5-µl samples were diluted in 0.5 ml of 100 mM sodium-borate buffer, pH 8.5. Immediately thereafter, 25 µl of fluorescamine (3.4 mg/ml in dry acetone) was added under vortex mixing. Fluorescence measurements were performed on a model RF5000 fluorimeter (Shimadzu, Tokyo, Japan) at excitation and emission wavelengths of 390 and 475 nm, respectively. Linearity of the fluorescence toward the concentration was verified with Phe as a standard and with intact substrates and peptides.

Alternatively, peptide degradation was analyzed by HPLC. The peptides (5 mM) were incubated at 37 °C with CD26/DPP IV in 100 mM Tris-HCl, pH 7.5, containing 1 mM EDTA. At indicated time points, 40-µl samples were taken and diluted in 120 µl of 0.1% trifluoroacetic acid in water. Samples were applied to an Ultrasphere ODS column (4.6 × 250 mm, 5 µm, Beckman, Fullerton, CA) and the peptides were eluted in a linear acetonitrile gradient and monitored at 214 nm. Disappearance curves of MDC analogues were constructed from the integrated peak areas versus time.

To compare the relative affinities of CD26/DPP IV for the various peptides and the synthetic substrate Gly-Pro-p-nitroanilide, IC50 values were determined. CD26/DPP IV enzymatic activity was measured at 37 °C using Gly-Pro-p-nitroanilide as a chromogenic substrate. The reaction was monitored at 405 nm, and the initial rate was determined between 0 and 0.25 absorbancy units. The reaction mixture contained 0.5 mM substrate, approximately 1 milliunit of CD26/DPP IV activity, 40 mM Tris-HCl buffer, pH 8.3, and test compounds at final concentrations between 0 and 1.5 mM. Measurements were performed in duplicate. The IC50 value is defined as the concentration of test peptide required to reduce CD26/DPP IV activity to 50% of the control. To verify the specificity of the observed reaction, the enzyme was inactivated by the specific irreversible CD26 inhibitor Pro-Pro-diphenylphosphonate (22) before the start of the experiment.

Detection of Chemotactic Activity, Intracellular Ca2+ Concentrations and Competition for MDC Binding-- The chemotactic activity of chemokines for lymphocytic SUP-T1 cells, monocytes, or monocyte-derived dendritic cells was determined in Boyden chemotaxis chambers as described previously (11, 13). Briefly, SUP-T1 cells (5 × 106 cells/ml) were allowed to migrate at 37 °C for 4 h through 5-µm pore-size fibronectin-coated polycarbonate filters (11). With monocytes and monocyte-derived dendritic cells (106 cells/ml), chemotaxis through polyvinylpyrrolidone-treated polycarbonate filters was stopped after 2 h (13). Filters were removed, cells were fixed and stained and counted microscopically. Results are expressed as chemotactic index corresponding to the number of cells migrated to the sample over the number of cells that migrated to control medium.

The intracellular Ca2+ concentrations ([Ca2+]i) were determined spectrofluorometrically using the fluorescent dye fura-2 (11) and were calculated from the Grynkiewicz equation (23). For desensitization experiments, cells were stimulated first with intact or CD26/DPP IV-truncated MDC and 100 s later with intact MDC at a concentration (3 nM) that induced a significant increase in [Ca2+]i after prestimulation with buffer.

Competition for MDC binding was measured on purified PBMC, monocytes (>95% pure) or lymphocytes (>95% lymphocytes) or CCR4-transfected cells as described (24). Briefly, two (purified monocytes) or five (other cell types) million cells were incubated for 2 h at 4 °C with 0.06 nM 125I-MDC and varying concentrations of unlabeled chemokine. Cells were centrifuged and washed three times with 2 ml of phosphate-buffered saline supplemented with 2% (w/v) bovine serum albumin, and the radioactivity present on the cells was measured in a gamma counter.

Detection of Antiviral Activity-- Purified PBMC from healthy donors were stimulated with phytohemagglutinin at 1 µg/ml for 3 days before infection with the T-tropic HIV-1 strain NL4.3 (obtained through the National Institute of Allergy and Infectious Diseases, AIDS reagent program, Bethesda, MD). The activated cells were washed three times with phosphate-buffered saline to remove nonadsorbed virus and varying concentrations of intact or CD26/DPP IV-truncated MDC were added (11). Cells were cultured in the presence of IL-2 (25 units/ml), and after 8 days, cell supernatants were collected. HIV-1 core antigen was analyzed by a p24 antigen enzyme-linked immunosorbent assay kit (NEN Life Science Products).

    RESULTS

Proteolytic Cleavage of MDC by CD26/DPP IV-- Recombinant and synthetic MDC(1-69) were treated with purified CD26/DPP IV. Incubation of synthetic MDC(1-69) with natural intact membrane-bound CD26/DPP IV for 48 h resulted in the removal of four NH2-terminal residues (Gly-Pro-Tyr-Gly) yielding MDC(5-69) (Table I). This finding was confirmed with recombinant MDC(1-69) incubated with the soluble form of the protease. However, a small amount of MDC(3-69) was also recovered. Prolonged treatment of MDC(3-69) with either membrane-bound or soluble CD26/DPP IV also resulted in complete conversion into MDC(5-69). Specific inactivation of CD26 with Pro-Pro-diphenylphosphonate prevented the formation of MDC(5-69) leaving ±85% of the MDC(3-69) intact (Table I).

                              
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Table I
NH2-terminal truncation of MDC by CD26/DPP IV
MDC was incubated at 37 °C for 1, 24, or 48 h with natural soluble (s) or membrane-bound (m) CD26/DPP IV purified to homogeneity. Detected NH2-terminal sequences after purification by HPLC or after electroblotting on polyvinylidene fluoride membranes and Coomassie Blue staining are shown.

To investigate whether the nature of the amino acids in the immediate proximity of the scissile bond exerts an important influence on the cleavage, the NH2-terminal peptides MDC(1-9), MDC(3-9), and RANTES(1-9) were synthesized. The interference of these peptides with the hydrolysis of a commonly used chromogenic CD26/DPP IV substrate, i.e. Gly-Pro-p-nitroanilide, was determined, and the IC50 values for competition between this and the peptide-substrates were calculated. RANTES(1-9) competed more efficiently (IC50: 0.15 ± 0.02 mM) for cleavage of Gly-Pro-p-nitroanilide compared with MDC(1-9) (IC50: 0.94 ± 0.09 mM). In addition, MDC(3-9) (IC50: > 1.5 mM) was a less efficient competitor for CD26/DPP IV-cleavage compared with MDC(1-9).

Peptide hydrolysis by purified CD26/DPP IV was also monitored by fluorescamine derivatization of free NH2 termini of the peptides resulting from incubation with CD26/DPP IV. The NH2-terminal nonapeptides of RANTES and MDC were efficiently cleaved by CD26/DPP IV into RANTES(3-9) and MDC(5-9), respectively. To prove that CD26/DPP IV is responsible for the observed truncations of the nonapeptides, EDTA was included in all incubation buffers to inhibit metallopeptidases. In a control experiment in the presence of the CD26 specific inhibitor Pro-Pro-diphenylphosphonate, the hydrolysis of MDC(1-9) was prevented. CD26/DPP IV was found to metabolize RANTES(1-9) and MDC(1-9) more quickly, generating more free NH2 termini during the same time interval, compared with MDC(3-9) (results not shown). To obtain more information on the difference in kinetics between both cleavage steps at the NH2 terminus, MDC(1-9) and MDC(3-9) were incubated with CD26/DPP IV and the resulting peptides, collected at different time points, were separated by HPLC. Under the experimental conditions used, almost all MDC(1-9) was cleaved within 30 min. Most of the generated MDC(3-9) is further converted into MDC(5-9) within the following hours (Fig. 1A). Using the same incubation conditions and starting with MDC(3-9), 50% of this peptide is processed after about 100 min (Fig. 1B). In addition, 2 µg of MDC(1-69) and MDC(3-69) were incubated with CD26/DPP IV at 37 °C for 1 h, blotted on polyvinylidene difluoride membranes, and the relative amounts of the different forms of MDC were determined by Edman degradation (Table I). The result of this short term incubation indicates that both dipeptides are efficiently and sequentially removed from the MDC NH2 terminus and that the first truncation, resulting in MDC(3-69), is more rapid compared with the second cleavage.


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Fig. 1.   Kinetics of the NH2-terminal truncation of MDC(1-9) and MDC(3-9) by CD26/DPP IV. MDC(1-9) (panel A) and MDC(3-9) (panel B) were incubated with CD26/DPP IV, and cleavage was monitored using HPLC at different time intervals. Fast cleavage of MDC(1-9) (bullet ) to MDC(3-9) (open circle ) was followed by slower conversion of MDC(3-9) to MDC(5-9) (×). The y-axis depicts the integrated peak areas (as a value for the amount of peptide) and the x-axis the incubation time. Linear analysis based on first order kinetics of the disappearance of MDC(1-9) (panel A) and MDC(3-9) (panel B) reveals a half-life of 8.6 min and 108 min, respectively.

Comparison of the Chemotactic Activity of Intact and CD26/DPP IV-cleaved MDC-- CD26-cleaved MDC was compared with intact MDC to stimulate the migration of lymphocytic SUP-T1 cells, peripheral blood monocytes and monocyte-derived dendritic cells. On lymphocytic SUP-T1 cells MDC(1-69) was chemotactic from 10 nM on (Fig. 2A), whereas 10 times higher amounts of MDC(5-69) were necessary to obtain an equivalent chemotactic effect. In contrast, both intact MDC and CD26/DPP IV-truncated MDC(5-69) were equally potent to stimulate monocyte chemotaxis (Fig. 2B), 1 nM of chemokine yielding significant cell migration. CD26/DPP IV treatment of MDC also resulted in reduced chemotactic potency on monocyte-derived dendritic cells (Fig. 2C). Intact MDC significantly induced in vitro chemotaxis of dendritic cells at 0.1 nM, whereas MDC(5-69) remained inactive at concentrations up to 10 nM.


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Fig. 2.   Comparison of the chemotactic activity of MDC(1-69) and MDC(5-69) on lymphocytes, monocytes, and dendritic cells. MDC(1-69) (black-diamond ), MDC(5-69) (black-triangle), and SDF-1alpha (star ) were tested for their chemotactic activity on lymphocytic SUP-T1 cells (panel A), on monocytes (panel B), and on monocyte-derived dendritic cells (panel C). Results represent the mean chemotactic index (CI) ± S.E. of five (SUP-T1), three (monocytes), and two (dendritic cells) experiments.

Signaling and Receptor Binding Properties of Intact and Truncated MDC-- To explain the altered chemotactic responses, it was verified whether MDC signaling through and binding to CCR4, the only known MDC receptor, was affected by treatment of MDC with CD26/DPP IV. Indeed, intact MDC(1-69) dose-dependently induced a [Ca2+]i rise in CCR4-transfected HOS cells (minimal effective concentration of 0.3 nM), whereas MDC(5-69) was still unable to signal at 30 nM (Fig. 3). Furthermore, in contrast to intact MDC, 30 nM of MDC(5-69) did not desensitize for a subsequent [Ca2+]i increase induced by 3 nM of intact MDC(1-69). Compared with MDC(1-69), the potential of MDC(5-69) to compete for 125I-MDC binding to CCR4 was 100-300-fold reduced. Impaired competition of MDC(5-69) for binding of labeled MDC(1-69) was obtained on CCR4-transfected HOS cells (Fig. 4A), on total PBMC (Fig. 4B), and on CD14-depleted lymphocytes (Fig. 4C). However, on monocytes purified by positive selection with anti-CD14, MDC(5-69) competed for 125I-MDC(1-69)-binding as efficiently as intact MDC (Fig. 4D).


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Fig. 3.   Desensitization of Ca2+-mobilization by MDC(1-69) and MDC(5-69) on CCR4-transfectants. HOS cells transfected with CD4 and CCR4 were loaded with the fluorescent probe fura-2. The [Ca2+]i was monitored spectrofluorometrically. The spectra from one representative experiment of three are shown.


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Fig. 4.   Competition for 125I-MDC(1-69) binding. HOS cells transfected with CD4 and CCR4 (panel A), total peripheral blood mononuclear cells (panel B), purified lymphocytes (panel C) or monocytes separated by magnetic cell sorting using anti-CD14 beads (panel D) were incubated with 125I-MDC(1-69) in 200 µl and varying concentrations of unlabeled MDC(1-69) (black-diamond ) or MDC(5-69) (black-square). Results are expressed as the % of specific binding (mean ± S.E. of two to four independent experiments on cells of different donors) with 0.06 nM 125I-MDC without competition with unlabeled chemokine.

MDC and MDC(5-69) Inhibit Infection of PBMC with HIV-1-- Intact MDC has been reported to have antiviral activity against T- and M-tropic HIV-1 strains (17). CD26/DPP IV-truncated MDC and intact MDC had comparable anti-HIV-1 activity against infection of PBMC with the T-tropic strain NL4.3 (Fig. 5). Although 50% inhibition of HIV-1 infection was not obtained, the inhibition was significant in all four experiments (11 to 28% at 50 nM of intact or CD26/DPP IV-truncated MDC).


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Fig. 5.   Anti-HIV-1 activity of MDC(1-69) and MDC(5-69). Phytohemagglutinin-stimulated PBMC were treated with varying concentrations of intact MDC(1-69) (closed histograms) or CD26/DPP IV-truncated MDC(5-69) (open histograms) at the time of infection with the HIV-1 strain NL4.3. Virus replication was quantified by measuring the viral antigen p24 in a p24 enzyme-linked immunosorbent assay. Results represent the percent inhibition (mean ± S.E. of four independent experiments on purified PBMC of different donors) of viral replication.


    DISCUSSION

CD26/DPP IV is involved in the in vivo metabolization of a number of relatively small natural peptides containing a penultimate Pro (e.g. pancreatic polypeptide family) or Ala (e.g. secretins) (4, 25). Only recently, and in contrast to a number of cytokines, which have a penultimate Pro at their NH2 terminus, some chemokines have been identified as selective substrates for this protease (9-12). Other chemokines, i.e. the monocyte chemotactic protein-1, -2, and -3 were found to be protected from CD26/DPP IV-processing by an NH2-terminal pyroglutamic acid (9, 26). Based on the MDC sequence, containing a penultimate Pro, and on the observation that different natural NH2-terminally truncated MDC forms have been isolated (17), the CC chemokine MDC was investigated as a candidate substrate for CD26/DPP IV.

Recombinant and synthetic MDC(1-69) were treated with the purified CD26/DPP IV to verify whether MDC was cleaved after the penultimate Pro as previously shown for the chemokines RANTES, GCP-2 and SDF-1alpha (9-12). It was observed that incubation of MDC(1-69) with CD26/DPP IV resulted in the unexpected removal of four NH2-terminal residues (Gly-Pro-Tyr-Gly) yielding MDC(5-69) instead of the predicted MDC(3-69). A small amount of MDC(3-69) was also recovered indicating that this form is generated as an intermediate. The conversion of MDC(3-69) into MDC(5-69) was prevented by the specific CD26/DPP IV inhibitor Pro-Pro-diphenylphosphonate. This confirmed that the generation of MDC(5-69) was specific for CD26/DPP IV and that the cleavage involved the sequential removal of the two NH2-terminal dipeptides Gly1-Pro2 and Tyr3-Gly4. The efficient cleavage after Gly4 is unexpected based on previous reports on the specificity of this protease (1-4, 27). Interference by other aminopeptidases in the purified natural membrane-bound or soluble CD26/DPP IV preparations was found to be unlikely, because no further truncation of RANTES, GCP-2, or SDF-1alpha was obtained with the same CD26/DPP IV preparations using identical incubation conditions (9, 11). Investigation of the kinetics of the cleavage of intact MDC and of NH2-terminal MDC-fragments by CD26/DPP IV confirmed that both dipeptides were subsequently removed and that the first hydrolysis proceeds more rapidly compared with the second one.

To our knowledge, this is the first report on the CD26/DPP IV-mediated release of an NH2-terminal Xaa-Gly dipeptide from a natural peptide. A study by Bongers et al. (27) on growth hormone-releasing hormone and synthetic analogs revealed that the introduction of a Gly or Ser as the penultimate residue still allowed degradation by CD26/DPP IV although at a slower rate compared with the natural hormone containing a penultimate Ala. In contrast, the synthetic glucagon-like peptide 2 analog with an Ala to Gly substitution at position 2 was resistant to cleavage by CD26/DPP IV (28). Along the same line, we did not observe the generation of RANTES(5-68) by CD26/DPP IV, which means that Tyr3-Ser4 is not released from RANTES(3-68), although the same dipeptide was released from the synthetic growth hormone-releasing hormone analog with a penultimate Ser (27). It was observed for other peptidases that the less the residue subject to cleavage is preferred, the more the extended substrate recognition influences the hydrolysis. The nature of the surrounding amino acids clearly influences the efficiency with which CD26/DPP IV removes dipeptides from substrates with an unusual penultimate residue (29).

The CC chemokine MDC is chemotactic for Th2 lymphocytes, monocytes, dendritic cells, and natural killer cells (13, 15, 30). Both intact MDC and MDC(5-69) were equally potent monocyte chemotactic proteins. In contrast, cleavage of MDC into MDC(5-69) resulted in reduced chemotactic activity on lymphocytic and dendritic cells. A similar reduction of the chemotactic activity has been observed for the chemokines RANTES and SDF-1alpha , whereas CD26/DPP IV processing had no effect on the chemotactic activity of GCP-2. Removal of the Xaa-Pro dipeptide from the NH2 terminus of SDF-1alpha and RANTES caused the loss of chemotactic activity on lymphocytes (11, 12) and monocytes or eosinophils (31), respectively. The reduced binding of MDC(5-69) to CCR4 and the reduced signaling of CD26/DPP IV-truncated MDC through this receptor explained the lower chemotactic activity on lymphocytes and dendritic cells. Moreover, MDC(5-69) did not desensitize for a subsequent [Ca2+]i increase induced by the intact chemokine. In concert with the chemotaxis data on monocytes, CD26/DPP IV processing had no influence on the binding properties of MDC to monocytes. The weak antiviral activity of MDC against HIV-1 infection of PBMC with a T-tropic strain also remained essentially unaltered. In this respect, it is interesting to notice that MDC(5-69) and MDC(3-69) (the intermediate reaction product of MDC cleavage by CD26/DPP IV) are the two most abundant MDC forms that were purified from CD8+ T cells based on their antiviral activity against the T-tropic HIV-1 strain IIIB (17). However, our results with the CD26/DDP IV-truncated MDC indicate that NH2-terminal truncation of MDC cannot explain the conflicting results between several laboratories concerning the absence or presence of anti-HIV-1 activity for this chemokine. For SDF-1alpha (3-68), binding to and signaling through its receptor were also strongly reduced. However, this SDF-1alpha (3-68), in contrast to MDC, lost most of its antiviral activity against T-tropic HIV-1 strains (11, 12, 32). Similarly, RANTES(3-68) lost its signaling properties through two receptors, i.e. CCR1 and CCR3 (10, 31). However, RANTES(3-68) remained a strong ligand for CCR5 and, because of the processing by CD26/DPP IV, obtained strong antiviral activity against M-tropic HIV-1 strains (9, 24, 31).

In conclusion, MDC is a CD26/DPP IV substrate with an unexpected second cleavage site resulting in the generation of MDC(3-69) and MDC(5-69). Processing of MDC by this dipeptidyl-peptidase results in reduced biological activity on lymphocytes and dendritic cells, but not on monocytes. Thus, in addition to the regulation of chemokine and chemokine receptor expression by endogenous and exogenous immunomodulation, the NH2-terminal processing of MDC by CD26/DPP IV may have an important down-regulatory function in Th2 lymphocyte and dendritic cell trafficking without affecting its monocyte chemotactic and antiviral activity.

    ACKNOWLEDGEMENTS

We thank Dr. Jan Balzarini and Dr. Anja Wuyts for critically reading the manuscript and Lizette van Berckelaer, Sandra Claes, René Conings, Erik Fonteyn, Jean-Pierre Lenaerts, and Willy Put for technical assistance. The chemokine receptor-transfected HOS cells were obtained from Dr. Nathaniel Landau through the AIDS Research and Reference Program, Division of AIDS, NIAID, National Institutes of Health.

    FOOTNOTES

* This work was supported by the AIDS project from the Istituto Superiore di Sanità, Italy, the Flemish Fund for Scientific Research, the Concerted Research Actions of the Regional Government of Flanders, the Interuniversity Attraction Pole of the Belgian Federal Government, and the BioMed Program of the European Commission.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.: 32-16-337348; Fax: 32-16-337340; E-mail: Paul.Proost{at}rega.kuleuven.ac.be.

Recipient of fellowship of the Flemish Fund for Scientific Research.

The abbreviations used are: CCR or CXCR, CC or CXC chemokine receptor; CD26/DPP IV, dipeptidyl-peptidase IV; GCP-2, granulocyte chemotactic protein-2; IL, interleukin; MDC, macrophage-derived chemokine; RANTES, regulated on activation normal T cell expressed and secreted; HPLC, high performance liquid chromatography; SDF-1, stromal cell-derived factor-1; HIV-1, human immundeficiency virus-1; PBMC, peripheral blood-derived mononuclear cells.
    REFERENCES
Top
Abstract
Introduction
References

  1. von Bonin, A., Hühn, J., and Fleisher, B. (1998) Immunol. Rev. 161, 43-53[Medline] [Order article via Infotrieve]
  2. Morimoto, C., and Schlossman, S. F. (1998) Immunol. Rev. 161, 55-70[Medline] [Order article via Infotrieve]
  3. Yaron, A., and Naider, F. (1993) Crit. Rev. Biochem. Mol. Biol. 28, 31-81[Abstract]
  4. Vanhoof, G., Goossens, F., De Meester, I., Hendriks, D., and Scharpé, S. (1995) FASEB J. 9, 736-744[Abstract/Free Full Text]
  5. Hoffmann, T., Faust, J., Neubert, K., and Ansorge, S. (1993) FEBS Lett. 336, 61-64[CrossRef][Medline] [Order article via Infotrieve]
  6. Tanaka, T., Duke-Cohan, J., Kameoka, J., Yaron, A., Lee, I., Schlossman, S. F., and Morimoto, C. (1994) Proc. Natl. Acad. Sci. U. S. A. 91, 3082-3086[Abstract]
  7. Schmitz, T., Underwood, R., Khiroya, R., Bachovchin, W., and Huber, B. (1996) J. Clin. Invest. 97, 1545-1549[Abstract/Free Full Text]
  8. Korom, S., De Meester, I., Stadlbauer, T. H. W., Chandraker, A., Schaub, M., Sayegh, M. H., Belyaev, A., Haemers, A., Scharpé, S., and Kupiec-Weglinski, J. W. (1997) Transplantation 63, 1495-1500[Medline] [Order article via Infotrieve]
  9. Proost, P., De Meester, I., Schols, D., Struyf, S., Lambeir, A.-M., Wuyts, A., Opdenakker, G., De Clercq, E., Scharpé, S., and Van Damme, J. (1998) J. Biol. Chem. 273, 7222-7227[Abstract/Free Full Text]
  10. Oravecz, T., Pall, M., Roderiquez, G., Gorrell, M. D., Ditto, M., Nguyen, N. Y., Boykins, R., Unsworth, E., and Norcross, M. A. (1997) J. Exp. Med. 186, 1865-1872[Abstract/Free Full Text]
  11. Proost, P., Struyf, S., Schols, S., Durinx, C., Wuyts, A., Lenaerts, J.-P., De Clercq, E., De Meester, I., and Van Damme, J. (1998) FEBS Lett. 432, 73-76[CrossRef][Medline] [Order article via Infotrieve]
  12. Shioda, T., Kato, H., Ohnishi, Y., Tashiro, K., Ikegawa, M., Nakayama, E. E., Hu, H., Kato, A., Sakai, Y., Liu, H., Honjo, T., Nomoto, A., Iwamoto, A., Morimoto, C., and Nagai, Y. (1998) Proc. Natl. Acad. Sci. U. S. A. 95, 6331-6336[Abstract/Free Full Text]
  13. Godiska, R., Chantry, D., Raport, C. J., Sozzani, S., Allavena, P., Leviten, D., Mantovani, A., and Gray, P. W. (1997) J. Exp. Med. 185, 1595-1604[Abstract/Free Full Text]
  14. Imai, T., Chantry, D., Raport, C. J., Wood, C. L., Nishimura, M., Godiska, R., Yoshie, O., and Gray, P. W. (1998) J. Biol. Chem. 273, 1764-1768[Abstract/Free Full Text]
  15. Chang, M.-S., McNinch, J., Elias, C., III, Manthey, C. L., Grosshans, D., Meng, T., Boone, T., and Andrew, D. P. (1997) J. Biol. Chem. 272, 25229-25237[Abstract/Free Full Text]
  16. Chantry, D., DeMaggio, A. J., Brammer, H., Raport, C. J., Wood, C. L., Schweickart, V. L., Epp, A., Smith, A., Stine, J. T., Walton, K., Tjoelker, L., Godiska, R., and Gray, P. W. (1998) J. Leukocyte Biol. 64, 49-54[Abstract]
  17. Pal, R., Garzino-Demo, A., Markham, P. D., Burns, J., Brown, M., Gallo, R. C., and DeVico, A. L. (1997) Science 278, 695-698[Abstract/Free Full Text] and Comments in (1998) Science 281, 487
  18. De Meester, I., Vanhoof, G., Lambeir, A.-M., and Scharpé, S. (1996) J. Immunol. Methods 189, 99-105[CrossRef][Medline] [Order article via Infotrieve]
  19. Lambeir, A.-M., Diaz-Peirera, J. F., Chacon, P., Vermeulen, G., Heremans, K., Devreese, B., Van Beeumen, J., De Meester, I., and Scharpé, S. (1997) Biochim. Biophys. Acta 1340, 215-226[Medline] [Order article via Infotrieve]
  20. Proost, P., Wuyts, A., Conings, R., Lenaerts, J.-P., Put, W., and Van Damme, J. (1996) Methods Companion Methods Enzymol. 10, 82-92[CrossRef]
  21. Deng, H., Liu, R., Ellmeier, W., Choe, S., Unutmaz, D., Burkhart, M., Di Marzio, P., Marmon, S., Sutton, R. E., Hill, C. M., Davis, C. B., Peiper, S. C., Schall, T. J., Littman, D. R., and Landau, N. R. (1996) Nature 381, 661-666[CrossRef][Medline] [Order article via Infotrieve]
  22. De Meester, I., Belyaev, A., Lambeir, A.-M., De Meyer, G. R. Y., Van Osselaer, N., Haemers, A., and Scharpé, S. (1997) Biochem. Pharmacol. 54, 173-179[CrossRef][Medline] [Order article via Infotrieve]
  23. Grynkiewicz, G., Poenie, M., and Tsien, R. Y. (1985) J. Biol. Chem. 260, 3440-3450[Abstract]
  24. Schols, D., Proost, P., Struyf, S., Wuyts, A., De Meester, I., Scharpé, S., Van Damme, J., and De Clercq, E. (1998) Antiviral Res. 39, 175-187[CrossRef][Medline] [Order article via Infotrieve]
  25. Mentlein, R., Gallwitz, B., and Schmidt, W. (1993) Eur. J. Biochem. 214, 829-835[Abstract]
  26. Van Coillie, E., Proost, P., Van Aelst, I., Struyf, S., Polfliet, M., De Meester, I., Harvey, D., Van Damme, J., and Opdenakker, G. (1998) Biochemistry 37, 12672-12680[CrossRef][Medline] [Order article via Infotrieve]
  27. Bongers, J., Lambros, T., Ahmad, M., and Heimer, E. (1992) Biochim. Biophys. Acta 1122, 147-153[Medline] [Order article via Infotrieve]
  28. Drucker, D., Shi, Q., Crivici, A., Sumner-Smith, M., Tavares, W., Hill, M., De Forest, L., Cooper, S., and Brubaker, P. L. (1997) Nat. Biotechnol. 15, 673-677[Medline] [Order article via Infotrieve]
  29. Keil, B. (1992) Specificity of Proteolysis, pp. 19-42, Springer-Verlag, Berlin, Germany
  30. Bonecchi, R., Bianchi, G., Bordignon, P. P., D'Ambrosio, D., Lang, R., Borsatti, A., Sozzani, S., Allavena, P., Gray, P. A., Mantovani, A., and Sinigaglia, F. (1998) J. Exp. Med. 187, 129-134[Abstract/Free Full Text]
  31. Struyf, S., De Meester, I., Scharpé, S., Lenaerts, J.-P., Menten, P., Wang, J. M., Proost, P., and Van Damme, J. (1998) Eur. J. Immunol. 28, 1262-1271[CrossRef][Medline] [Order article via Infotrieve]
  32. Crump, M. P., Gong, J.-H., Loetscher, P., Rajarathnam, K., Amara, A., Arenzana-Seisdedos, F., Virelizier, J.-L., Baggiolini, M., Sykes, B. D., and Clark-Lewis, I. (1997) EMBO J. 16, 6996-7007[Abstract/Free Full Text]


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