(Received for publication, July 24, 1996, and in revised form, December 12, 1996)
From the Department of Immunology, Box 3010 DUMC, Duke University Medical Center, Durham, North Carolina 27701
Chemokines bind to receptors of the
seven-transmembrane type on target cells and also bind to
glycosaminoglycans (GAGs), including heparin. In this study, we
have sought to identify structural motifs mediating binding of the
-chemokine macrophage inflammatory protein-1
(MIP-1
) to GAGs.
Alignment of
-chemokine amino acid sequences revealed the presence
of several highly conserved basic amino acids, and molecular modeling
predicted that the side chains of three of the basic amino acids fold
closely together in MIP-1
. Site-directed mutagenesis was used to
change the conserved basic residues in MIP-1
to alanines, and both
wild-type and mutant proteins were produced in a transient COS cell
expression system. Wild-type MIP-1
bound to heparin-Sepharose, while
three of the mutants, R18A, R46A, and R48A, failed to bind. Mutant K45A
eluted from heparin-Sepharose at lower NaCl concentrations than wild type, while the binding of K61A, with a mutation in the C-terminal
-helix, was indistinguishable from that of the wild-type protein. To
determine whether GAG-binding capacity is required for receptor binding
and cell activation, we performed competition radioligand binding and
calcium mobilization experiments using one of the non-heparin-binding
mutants, R46A. R46A bound as efficiently as wild-type MIP-1
to CCR1
and was equally active in eliciting increases in intracellular free
calcium concentrations. Our data define a GAG binding site in MIP-1
consisting of three noncontiguous basic amino acids and show that the
capacity to bind to GAGs is not a prerequisite for receptor binding or
signaling in vitro.
The initiation of a focal inflammatory response requires a tightly
regulated sequence of events. Circulating leukocytes must attach to
endothelium, become arrested and activated, and undergo extravasation
to reach inflammatory foci. The molecular processes underlying these
events are beginning to be understood (for reviews, see Refs. 1 and 2).
A cascade of events, including selectin-mediated rolling attachment of
leukocytes to endothelium, integrin-mediated arrest of leukocytes, and
the provision of directional cues for migration by chemoattractants all
are crucial for the development of an inflammatory response. The
chemokines are a family of small chemoattractant cytokines implicated
in the attraction and activation of a variety of leukocytes (3-6).
They may be broadly divided into two main classes. -Chemokines, also
known as CXC chemokines due to the spacing of two conserved cysteine
residues by a single amino acid, are mainly active on neutrophils. The
- or CC-chemokines, in which the first two conserved cysteines are
adjacent, are mainly active on monocytes and lymphocytes.
It is clear from both in vivo and in vitro
studies (3-6) that chemokines provide directional cues for the
migration of leukocytes to inflammatory sites. What is not clear,
however, is the mechanism by which the chemokine concentration gradient
required for chemoattraction arises. A soluble chemokine gradient would
not be expected to be stable, particularly under conditions of blood
flow in the circulation. It has been suggested, rather, that an
immobilized, substrate-bound gradient of chemokines is responsible for
the chemoattraction of leukocytes (7, 8). For example, the
-chemokine IL-8,1 in the solid phase,
has been demonstrated to induce migration of neutrophils in
vitro (9). Furthermore, it is known that chemokines bind to
glycosaminoglycans (GAGs) (10); binding of chemokines to GAGs, either
at the surface of endothelial cells or in the extracellular matrix,
might thus serve to establish an immobilized chemokine gradient and
"present" the molecules to leukocytes in vivo. In
support of this idea, the
-chemokine MIP-1
, immobilized by
attachment to solid phase GAG, is capable of stimulating leukocyte
adhesion (11). Similarly, Gilat et al. (12) have shown that
MIP-1
and RANTES bind to an ex vivo extracellular matrix
preparation in a heparinase-sensitive manner and that these bound
chemokines are then capable of stimulating leukocyte adhesion.
To begin to understand the role of GAG association in the function of
chemokines, we have sought to identify GAG binding sites in the
-chemokine MIP-1
(13). Interactions between the acidic GAGs and
proteins are largely electrostatic (14) and thus require basic amino
acids. Often, these interactions involve
-helical structures with
regularly spaced basic residues (15). The structures of several
chemokines, both
and
, have been solved, and all include three
antiparallel
-strands with an overlaying
-helix at the C terminus
of the protein (16-21). In the
-chemokines, the C-terminal helix
tends to be highly basic and has been shown to mediate GAG association
for PF-4 and IL-8 (22, 23). However, the C-terminal helices of at least
some
-chemokines tend to have a lower concentration of basic
residues. Since all of the
- and
-chemokines examined to date
bind to GAGs, and since basic residues often contribute to GAG binding
of proteins, we sought to delineate additional basic structural motifs
conserved among
-chemokines. By aligning the amino acid sequences of
a number of
-chemokines, we identified four conserved basic amino
acids. In MIP-1
, two of these are found at positions 18 and 48, while two are adjacent at positions 45 and 46 and are predicted to lie
in a turn between
-strands. These residues, as well as the single
basic residue in the MIP-1
C-terminal helix, were individually
changed to alanines by site-directed mutagenesis. GAG binding of the
wild-type and mutant MIP-1
proteins was evaluated by
heparin-Sepharose chromatography. The results demonstrate that three
charged residues that are predicted to form a cleft on one face of the
molecule are all essential for heparin binding of MIP-1
. Thus, these
studies define a novel GAG-binding structure that is different from the
amphipathic helix defined for some
-chemokines. We also examined
receptor binding and cell activation by wild-type and
non-heparin-binding mutant MIP-1
. The data support the conclusion
that GAG-binding capability is not a prerequisite for the biological
activity of MIP-1
in solution and allow the design of experiments to
test the importance of GAG binding for chemokine activity in
vivo.
Human MIP-1 full-length
coding region cDNA was cloned from phytohemagglutinin/phorbol
12-myristate 13-acetate-stimulated HMC-1 human mast cells (24, 25) by
reverse transcriptase-polymerase chain reaction using a primer pair
derived from the 5
and 3
ends of the published MIP-1
sequence
(26); primers included EcoRI sites to facilitate cloning.
The sequences of the primers used are 5
-GGGGAATTCAGAATCATGCAGGTCTCC-3
(sense) and 5
-GGGGAATTCAGGCACTCAGCTCCAGGT-3
(antisense). The
cDNA, which included 8 base pairs of 5
-untranslated sequence,
was subcloned into the mammalian expression vector pCAGGS (27, 28) (the
kind gift of T. Yoshimura, NCI-Frederick Cancer Research and
Development Center, Frederick, MD) and sequenced in its entirety using
Sequenase (U.S. Biochemical Corp.). The sequence obtained was identical
to the published human MIP-1
sequence AT464.2 (26).
Mutagenesis was performed by
extension overlap amplification (29) using 100 ng of pCAGGS-MIP1 as
template. The sequences of the mutagenic primers used are as follows:
MIP-1
R18A (sense), 5
-CTACACCTCCGCACAGATTCCA-3
; MIP-1
R18A
(antisense), 5
-TGGAATCTGTGCGGAGGTGTAG-3
; MIP-1
K45A (sense),
5
-CTTCCTAACCGCAAGAGGCCGGC-3
; MIP-1
K45A (antisense),
5
-GCCGGCCTCTTGCGGTTAGGAAG-3
; MIP-1
R46A (sense), 5
-CCTAACCAAGGCAGGCCGGCAG-3
; MIP-1
R46A (antisense),
5
-CTGCCGGCCTGCCTTGGTTAGG-3
; MIP-1
R48A (sense),
5
-CAAGAGAGGCGCACAGGTCTGTG-3
; MIP-1
R48A (antisense),
5
-CACAGACCTGTGCGCCTCTCTTG-3
; MIP-1
K61A (sense), 5
-GTGGGTCCAGGCATACGTCAGTG-3
; MIP-1
K61A (antisense),
5
-CACTGACGTATGCCTGGACCCAC-3
.
Polymerase chain reaction products were subcloned into pCAGGS and sequenced in their entirety using either Sequenase or Taquence (U.S. Biochemical).
Transient Transfection and Metabolic LabelingCOS-P
fibroblasts, the kind gift of Dr. M. Caron (Duke University), were
maintained at 37 °C/5% CO2 in Dulbecco's modified Eagle's medium (Mediatech, Herndon, VA) supplemented with 10% fetal
calf serum and transiently transfected with wild-type or mutant
pCAGGS-MIP-1 constructs with DEAE-dextran (30)
(Mr 500,000; Pharmacia Biotech Inc.) in 100-mm
plates. Forty-eight hours after transfection, plates were washed twice
with PBS, and the medium was replaced with 10 ml of methionine-free,
cysteine-free Dulbecco's modified Eagle's medium (Life Technologies,
Inc.) containing 0.5 mCi of Tran35Slabel (ICN, Costa Mesa,
CA). Following an additional 24 h of culture,
35S-labeled supernatants were collected.
Metabolically labeled
supernatants containing wild-type or mutant MIP-1 were diluted
4-fold into buffer A (20 mM Tris-HCl, pH 8.0) and applied
to 1-ml Hi-trap Heparin columns (Pharmacia), previously equilibrated in
buffer A, at a flow rate of 0.5 ml/min. Equivalent amounts of labeled
chemokines, normalized by SDS-PAGE (31) and fluorography (32) of
unfractionated supernatants, were loaded onto the columns. Columns were
washed with 20 column volumes of buffer A and developed with a 15-ml
gradient of 0-400 mM NaCl in buffer A at a flow rate of
0.5 ml/min. Control experiments confirmed that no additional chemokine
eluted from the heparin-Sepharose columns at NaCl concentrations up to
1 M. Column flow-through (5 ml), wash (5 ml), and eluate (1 ml) fractions were collected; radioactivity in the eluate fractions was
determined by scintillation counting in an LKB 1209 RackBeta
scintillation counter. Column chromatography was carried out at room
temperature.
Unlabeled wild-type and mutant MIP-1
proteins were partially purified from supernatants of transiently
transfected COS-P cells by chromatography on Q-Sepharose. Following
transfection, cells were cultured for 48 h as described above. The
plates were then washed twice with PBS, and the medium was replaced
with phenol red-free Dulbecco's modified Eagle's medium (Life
Technologies). Cells were cultured an additional 24 h, and the
supernatants were collected. Supernatants were applied to 5-ml Hi-trap
Q anion exchange columns (Pharmacia) previously equilibrated in buffer
B (50 mM Tris-HCl, pH 8.0, 5 mM EDTA) at a flow
rate of 0.5 ml/min. Columns were washed with 20 column volumes of
buffer B, and chemokines were eluted with 400 mM NaCl in
buffer B; control experiments confirmed that concentrations of NaCl up
to 1 M failed to elute additional chemokine from the
column. The partially purified chemokines were dialyzed extensively
against PBS at 4 °C (molecular weight cut-off, 3000), aliquoted, and
stored at
70 °C. Chemokine concentrations were determined by
resolving samples of Q-Sepharose-purified chemokines (see above)
alongside a series of 2-fold serial dilutions of a commercial MIP-1
standard (Peprotech, Rocky Hill, NJ) on 15% SDS-PAGE gels and
comparing band intensities following silver staining (33). We estimate
that the determined protein concentrations must be within half a
dilution of the actual concentration. This level of uncertainty has no
impact on any of the conclusions drawn.
Concentration dependence of
chemokine dimerization was assessed as described by Paolini et
al. (34). Briefly, radiolabeled and unlabeled chemokines in PBS
(see above), partially purified by Q-Sepharose chromatography, were
combined and incubated with 5 mM sulfo-EGS (Pierce) for
1 h at room temperature prior to quenching with SDS-PAGE sample
buffer (31). Reactions were boiled for 5 min and analyzed by 15%
SDS-PAGE. Gels were fluorographed with 2,5-diphenyloxazol/Me2SO and exposed to BioMax MR or X-Omat
AR (Eastman Kodak Co.) film at 70 °C.
cDNA for the
MIP-1/RANTES receptor CCR1 (35, 36), the kind gift of Dr. P. Murphy
(NIH), was subcloned into the expression vector pRc/CMV (Invitrogen,
San Diego, CA). CHO-K1 fibroblasts (the kind gift of Dr. J. Esko, UAB),
were maintained in Ham's F12 medium supplemented with 10% fetal calf
serum and transfected with the pRc/CMV-CCR1 construct using DOTAP
(Boehringer Mannheim) according to the manufacturer's instructions.
Transfectants were selected by growth in G418 (Life Technologies) (0.6 mg/ml), and drug-resistant cells were subcloned by limiting dilution
(0.3 cells/well) in 96-well flat-bottomed plates. Clones expressing high levels of CCR1 mRNA were identified by Northern analysis, and
one clone, CHO-K1/CCR1.12, was selected for further study. HEK-293
human embryonic kidney cells were maintained in Eagle's minimal
essential medium (Life Technologies) supplemented with 10% fetal calf
serum and were transfected, selected, and cloned as described above.
One clone, HEK-293/CCR1.10F6, was selected for further analysis.
Human peripheral blood
mononuclear cells were isolated from normal donors with LSM (Organon
Teknika, Durham, NC), washed three times with serum-free RPMI 1640 (Mediatech), and resuspended at a concentration of 107
cells/ml in serum-free RPMI 1640. Stably transfected HEK-293/CCR1.10F6 cells expressing the MIP-1/RANTES receptor CCR1 (see above) were briefly trypsinized, washed twice in serum-free RPMI 1640, and resuspended at 107 cells/ml in serum-free RPMI 1640;
labeling conditions were identical for monocytes and HEK-293 cells.
Indo-1/AM (Molecular Probes, Eugene, OR) was added to a final
concentration of 2 µg/ml from a freshly prepared 1 mg/ml stock in
Me2SO, 2% pluronic F-127 (Molecular Probes). The cells
were incubated for 30 min at 37 °C in the dark, washed twice in
serum-free RPMI 1640, and resuspended at a concentration of 2 × 106/ml in Hanks' balanced salt solution (with divalent
cations; Life Technologies), 0.1% (w/v) BSA (Boehringer Mannheim) that
had been prewarmed to 37 °C. Cells were equilibrated to 37 °C and
were analyzed for chemokine-stimulated elevations in intracellular free
calcium concentrations using a FACStar Plus (Becton Dickenson, Mountainview, CA) at the Duke University Comprehensive Cancer Center
Flow Cytometry Shared Resource. After a base-line violet/blue fluorescence ratio was established, chemokines in PBS were added to
Indo-1-labeled cells, and cells were analyzed for changes in fluorescence ratio over time. Calcium mobilization was quantified in
terms of the fraction of total cells responding to chemokines. This was
accomplished by generating histograms plotting cell number versus fluorescence ratio before the addition of chemokine
and at the peak of chemokine-stimulated increase in fluorescence ratio and determining the net increase in the percentage of cells that had
fluorescence ratios above base line at the time of peak response. In
experiments with peripheral blood mononuclear cells, analysis was
restricted to monocytes by gating by forward and side scatter with the
FACStar. Control experiments showed that nontransfected HEK-293 cells
did not mobilize calcium in response to chemokines (data not
shown).
The ability of
wild-type and mutant MIP-1 to compete for binding of
125I-MIP-1
to CCR1 expressed in CHO-K1 cells was
evaluated by incubating 2 × 106 cells, harvested with
20 mM EDTA in PBS, with 0.5 nM
125I-MIP-1
(DuPont NEN; specific activity 2200 Ci/mmol)
in binding buffer (phenol red-free RPMI 1640, 1% BSA (w/v), 25 mM Na-HEPES, pH 7.4) and various concentrations of
unlabeled competitor; the volume of unlabeled competitor added did not
exceed 10% of the final assay volume (200 µl). The reactions were
rotated end over end in 1.5-ml microcentrifuge tubes for 2 h at
4 °C and filtered through Whatman GF/C filters. The filters were
washed twice with 5 ml of ice-cold binding buffer, twice with 5 ml of
ice-cold binding buffer containing 0.5 M NaCl, air-dried,
and counted in an LKB-Wallac CliniGamma 1272
counter. For the
control experiment shown in Fig. 5B, supernatants from
mock-transfected COS-P cells, subjected to Q-Sepharose chromatography
as described above, were added to binding reactions to a final
concentration of 10% (v/v). Incubations and washes were performed as
described above, except that the cold competitor protein was
commercially available pure MIP-1
(Peprotech).
Alignment of the amino acid sequences of several
-chemokines revealed the presence of conserved basic amino acids at
positions 18, 45, 46, and 48 (MIP-1
numbering, Fig.
1A). The sequence at positions 45-48
constitutes an example of the consensus GAG-binding sequence
BBXB, where B represents a basic amino acid and X
represents any amino acid (15). Mapping the amino acid sequence of
MIP-1
onto the coordinates of the closely related chemokine MIP-1
(19) revealed that residues 45 and 46 define a turn between adjacent
-strands (Fig. 1A) and that the side chains of residues
18, 46, and 48 are in close proximity to each other (Fig.
1B). We constructed a set of point mutants in which each of
these residues was mutated to alanine. We also mutated the single basic
residue in the MIP-1
C-terminal helix, Lys61, to
alanine; residue 61 was chosen for mutagenesis because the only two
chemokines for which GAG-binding motifs have been identified, PF-4 and
IL-8, have both been shown to bind via highly basic helices (22,
23).
In initial experiments, we expressed wild-type human MIP-1 in a COS
cell transient expression system and evaluated the binding of
metabolically labeled protein to heparin-Sepharose. SDS-PAGE analysis
of unfractionated supernatants from transiently transfected and
metabolically labeled COS cells revealed a major 8-kDa species, MIP-1
, that was not present in supernatants of mock-transfected cells (Fig. 2A). Heparin-Sepharose
chromatography of supernatants of MIP-1
-transfected cells revealed a
single peak of radioactivity eluting at 250 mM NaCl,
whereas no radioactivity eluted during similar chromatographic analysis
of supernatants of mock-transfected cells (Fig. 2B).
SDS-PAGE analysis of column fractions revealed that the peak of
radioactivity obtained in chromatography of supernatants of
MIP-1
-transfected cells (fraction 12) is almost entirely MIP-1
(Fig. 2C).
Each of the point mutants was then expressed in COS cells,
metabolically labeled, and applied to heparin-Sepharose columns to
evaluate its capacity to bind to heparin. Chemokines bound to and
eluted from the heparin-Sepharose columns were identified by
measurement of radioactivity in the eluate fractions, as described above. Chemokines in the load, flow-through, and wash fractions were
identified by SDS-PAGE analysis, since these fractions contained high
levels of contaminating unincorporated [35S]methionine
and [35S]cysteine. Wild-type MIP-1 bound
quantitatively to the column (Fig. 3B) and
eluted at 250 mM NaCl (Fig. 3A). However,
mutants R18A, R46A, and R48A all failed to stably bind to and elute
from heparin-Sepharose (Fig. 3A). Mutant R46A was recovered
quantitatively in the column flow-through (Fig. 3B),
indicating that interaction with heparin was completely abolished.
Mutant R48A was recovered in both the column flow-through and wash
fractions (Fig. 3C), and mutant R18A was recovered
exclusively in the wash fractions (Fig. 3D), suggesting very
weak interactions between these molecules and the heparin matrix. In
contrast, the K45A mutant bound stably to heparin-Sepharose but eluted
from the column at lower concentrations of NaCl than the wild-type
protein (Fig. 3, A and B). The K61A mutant bound
to heparin-Sepharose and required the same concentration of NaCl as the
wild-type protein for elution (Fig. 3, A and B). Thus, our data identify three amino acids, Arg18,
Arg46, and Arg48, that are absolutely required
for heparin binding of MIP-1
. The side chains of these residues fold
together to form a basic cleft on one face of the molecule (Fig.
1B), defining a novel chemokine heparin-binding motif.
Receptor Binding of a Non-heparin-binding MIP-1
The chemokine presentation hypothesis proposes that
chemokines are presented to leukocytes in the form of a solid-phase
gradient while bound to GAGs. It is possible that chemokine-GAG
interactions are important not only for presentation but also for
chemokine activation of target cells in vivo. The generation
of MIP-1 point mutants in which heparin binding was abolished
allowed us to design experiments to test whether the capacities of
MIP-1
to bind to glycosaminoglycans and to activate cells were
causally linked. We initially addressed this issue by developing a
stably transfected CHO cell line expressing the MIP-1
/RANTES
receptor CCR1 (35, 36). Competition radioligand binding analysis of
wild-type and R46A mutant MIP-1
was performed by incubating the
CCR1-expressing cells with purified iodinated wild-type MIP-1
together with increasing concentrations of partially purified,
unlabeled, COS cell-derived wild-type and mutant MIP-1
(Fig.
4). Competitor chemokine concentrations were determined
as described under "Experimental Procedures." Mutant MIP-1
competed as effectively as wild-type MIP-1
in this assay, indicating
that the mutant protein binds to the receptor with properties that are
indistinguishable from wild type (Fig. 5A).
To ensure that the competition we observed in this assay was due to the
MIP-1 in the COS cell supernatants and that no other proteins in
these partially purified preparations interfered with the competition
assay, we also tested the effects of preparations of mock-transfected
supernatants. These were prepared by mock-transfecting COS-P cells and
subjecting the supernatants to the same protein purification procedures
used for the preparation of unlabeled chemokines. Mock-transfected cell
supernatants were added to binding assays to a final concentration of
10% (v/v), since this was the maximum volume contributed to the
binding assay by unlabeled chemokine preparations in the competition
binding assays. Such preparations did not compete for the binding of
125I-MIP-1
and did not affect competition by pure
unlabeled MIP-1
(Fig. 5B). Therefore, the results of the
competition binding experiments establish that the capacity to bind
GAGs is not a prerequisite for receptor binding. However, they do not
address a possible relationship between GAG-binding capacity and cell
activation.
To investigate a possible linkage between
GAG-binding capacity and cell activation, partially purified MIP-1
wild-type and R46A mutant proteins were tested for their ability to
stimulate increases in intracellular free calcium concentrations in
human monocytes in a dose-response experiment. Indo-1-loaded peripheral blood mononuclear cells were incubated with varying concentrations of
chemokine, and the fraction of monocytes displaying elevations in
intracellular free calcium concentrations was determined. We noted no
significant differences between the mutant and wild-type MIP-1
(Fig.
6, top panel). Since human monocytes may bear
a number of chemokine receptors capable of transducing signals by
MIP-1
(37), we also examined signaling via a single receptor by
using stably transfected HEK-293 cells expressing the MIP-1
/RANTES receptor CCR1. The stably transfected cells also responded equivalently to wild-type MIP-1
and the R46A mutant (Fig. 6, bottom
panel). Taken together, the receptor binding and calcium
mobilization experiments indicate that the capacity to bind GAG is a
prerequisite for neither receptor binding nor signaling by MIP-1
in
solution.
Dimerization of Wild-type and R46A Mutant MIP-1
Although the reported structures of the two CC
chemokines for which primary data are available, RANTES and MIP-1,
are dimeric (19-21), the association states in which CC chemokines
exist in vivo are not known. We reasoned that if MIP-1
binds heparin as a dimer, mutations that interfere with dimerization
could inhibit heparin binding, due to the loss of half of the heparin
binding sites per molecule. The predicted structure of MIP-1
reveals that two of the residues implicated as critical for heparin binding, Arg18 and Arg48, are unlikely to be involved in
dimer formation. However, the basic turn region of one monomer is in
close contact with the amino terminus of the other. Residue
Arg46 may, in fact, form part of the dimer interface. To
address the possibility that the R46A mutation has a direct effect on
dimerization, we assessed the fraction of MIP-1
in monomeric and
dimeric form as a function of MIP-1
concentration (Fig.
7). To do so, a constant amount of radiolabeled
wild-type or R46A mutant MIP-1
was mixed with graded amounts of
unlabeled wild-type or R46A mutant, respectively, and radiolabeled
dimeric species were detected by trapping with the chemical
cross-linker sulfo-EGS. The results indicate that R46A dimerizes with a
concentration dependence similar to that of wild-type MIP-1
.
Although the proportion of MIP-1
in dimers appears higher for
wild-type than the R46A mutant at the highest concentration tested,
this difference is not apparent at lower concentrations. More
importantly, this partial effect is unlikely to account for the
complete abolition of heparin binding by the R46A mutation. While we
cannot exclude the possibility that there may be differences in the
association states of wild-type and R46A mutant MIP-1
that are
indistinguishable by the cross-linking technique used, we suggest that
the R46A mutation disrupts GAG binding because R46 interacts with GAGs
directly.
The work presented here implicates basic amino acids in a
conserved three-dimensional motif as critical for the association of
the CC-chemokine MIP-1 with glycosaminoglycans. Mutation of any one
of the three residues in the motif (Arg18,
Arg46, or Arg48) to alanine resulted in a
complete loss of heparin binding. Our data suggest that residue
Lys45 plays a lesser role in heparin binding and that the
single basic residue in the MIP-1
helix, Lys61, plays no
detectable role. The R46A mutant bound as efficiently as wild-type
MIP-1
to CCR1 stably expressed in CHO-K1 cells and exhibited
signaling activity in solution that was indistinguishable from
wild-type MIP-1
as measured by calcium mobilization assays on both
human monocytes and stably transfected cells expressing CCR1. Thus, our
results define a novel heparin-binding motif in MIP-1
and indicate
that the capacity to bind GAGs is essential for neither receptor
binding nor signaling by MIP-1
.
Numerous studies have focused on the role of charged residues within
-helical domains in protein-GAG interactions (for review, see Ref.
15), but there is evidence for involvement of nonhelical basic motifs
in such interactions as well. As one specific example, recent basic
fibroblast growth factor-heparin co-crystallization experiments (40)
have demonstrated the importance of a series of residues in loop
regions connecting
-strands for heparin binding. These residues are
widely separated in primary structure but fold together to form a
functional heparin-binding site. Similarly, a series of six basic
residues in lactoferrin fold together to form a structure, denoted a
cationic cradle, that has been shown to make up a functional
heparin-binding site (41). A comparable cationic cradle, with six basic
side chains forming a cleft with three residues per side, has been
demonstrated to make up a heparin-binding site in fibronectin (42). The
MIP-1
motif defined in this study is composed of fewer basic
residues. However, a similar cleft is formed, with a single basic side
chain on one side and two on the other.
Related basic motifs are found in other -chemokines. For example,
the three basic residues identified in this study are conserved in
MIP-1
, RANTES and MCP-2. MIP-1
contains, in addition, a basic residue at position 19 that is predicted to augment the basic cleft by
providing a fourth residue, two on each side. An additional basic
residue at position 22 may contribute as well. The basic residue at
position 19 is also found in MCP-2, and the basic residue at position
22 is also found in RANTES. Therefore, we would predict that all of
these chemokines might require higher concentrations of salt than
MIP-1
for elution from heparin-Sepharose; in preliminary studies, we
have found this to be the case for
MIP-1
.2 Thus, MIP-1
appears to carry
a minimal cationic cradle, which is represented in more substantial
form in some other
-chemokines.
Several -chemokines display a series of basic residues in the
C-terminal
-helix that may contribute to GAG binding as well. In
some instances (for example, RANTES and MCP-2) basic cleft and helical
residues coexist in the same molecule. Hence, these molecules may bind
GAG even more tightly than MIP-1
and MIP-1
, using both helical
and extrahelical residues. Interestingly, the chemokine I-309 has an
extremely high concentration of basic residues in its C-terminal
-helix. Using the same techniques outlined in this paper, we have
obtained evidence that mutating the basic turn region of I-309
(residues 43-48 in MIP-1
numbering) from KLKRGK to ALAAGA has no
effect on heparin binding.2 Thus, I-309 and MIP-1
bind
heparin by different motifs. Presumably, I-309 binds GAGs via its
C-terminal helix, similar to IL-8 and PF4, or perhaps by a combination
of helical and nonhelical residues. Experiments to determine the
contributions of basic cleft residues to GAG binding of other
chemokines are currently under way.
Our results suggest that the ability to bind glycosaminoglycans is
essential for neither receptor binding nor signaling by MIP-1.
However, it is possible that chemokine binding to heparin in
vitro does not fairly reflect binding to glycosaminoglycans in vivo. To attempt to address the importance of
cell-surface heparan sulfate proteoglycan for receptor binding and/or
signaling by MIP-1
, we generated stable transfectants of the heparan
sulfate proteoglycan-deficient CHO-677 mutant cell line (Ref. 43; the kind gift of J. Esko) expressing CCR1. Although we were unable to
measure signaling in either wild-type CHO or CHO-677 transfectants, we
were able to show that wild-type MIP-1
bound as efficiently to CCR1
expressed in the mutant cells as it did to CCR1 expressed in wild-type
CHO cells.2 This indicates that the binding of MIP-1
to
CCR1 is independent of heparan sulfate proteoglycan, confirming, in
part, our conclusions from the R46A mutant.
The relationship between chemokine-GAG interactions and activity has
been addressed in two other instances. PF-4 appears to bind to heparin
by charged residues in the C-terminal helix (22), although a recent
study by Mayo et al. (44) suggests that residues outside the
helix may be important for PF-4-heparin interactions, as well. Maione
et al. (22) have presented evidence that a
non-heparin-binding PF-4 mutant in which four basic residues in the
C-terminal helix are changed to acidic or neutral residues retains both
in vitro antiangiogenic activity and in vivo
antitumor activity. These results indicate that for PF-4,
heparin-binding capacity and biological activity, both in
vitro and in vivo, are not directly linked. In
contrast, a version of the -chemokine IL-8 that has a truncated C-terminal helix fails to bind heparin (23) and has impaired cell
activation and receptor binding properties (45). However, helix
truncation is a rather drastic change of the molecule that may have
perturbed the folding of other functional domains as well, limiting the
certainty of any conclusions regarding the relationship between heparin
binding and biological activity. Our experiments provide a
straightforward test of this relationship, because GAG binding can be
abolished by a single amino acid mutation. Whether the same
relationship can be established for other chemokines that bind more
tightly to glycosaminoglycans remains to be seen.
In conclusion, the data presented here identify a novel three-amino
acid motif, composed of residues Arg18, Arg46,
and Arg48, required for the binding of MIP-1 to GAGs.
Since the R46A mutant is active in receptor binding and cell activation
experiments in vitro, it may be an appropriate reagent to
test the presentation hypothesis for MIP-1
and evaluate the
importance of MIP-1
-GAG association for biological activity in
vivo.
We thank A. Fisher and the Duke University Comprehensive Cancer Center Flow Cytometry Shared Resource for Indo-1 analysis and A. Sanfridson for careful reading of the manuscript.