From the Laboratory of Molecular Immunology, and
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,
Laboratory of Pharmaceutical Chemistry, and
§§ Laboratory of Clinical Biochemistry,
University of Antwerp, Universiteitsplein 1, B-2610 Wilrijk,
Belgium
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() |
---|
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.
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-1 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.
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).
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).
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.
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.
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).
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).
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-1 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-1 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.
INTRODUCTION
Top
Abstract
Introduction
References
(IL-1
), 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-1
(SDF-1
) 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).
EXPERIMENTAL PROCEDURES
RESULTS
NH2-terminal truncation of MDC by CD26/DPP IV
View larger version (18K):
[in a new window]
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) ( ) to MDC(3-9) (
) 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.
View larger version (18K):
[in a new window]
Fig. 2.
Comparison of the chemotactic activity of
MDC(1-69) and MDC(5-69) on lymphocytes, monocytes, and dendritic
cells. MDC(1-69) ( ), MDC(5-69) (
), and SDF-1
(
) 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.
View larger version (22K):
[in a new window]
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.
View larger version (18K):
[in a new window]
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) ( ) or MDC(5-69) (
).
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.
View larger version (22K):
[in a new window]
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
(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-1
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.
, 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-1
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-1
(3-68), binding to and signaling through its receptor were also strongly reduced. However, this SDF-1
(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).
![]() |
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 |
---|
![]() ![]() ![]() ![]() |
---|