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Department of Immunology, Duke University Medical Center, Durham, NC 27710
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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-heparin interaction suggests a possible structural
difference between the MIP-1ß-heparin and MIP-1-heparin complexes.
To determine whether GAG binding plays an important role in receptor
binding and cellular activation by MIP-1ß, the activities of
wild-type MIP-1ß and R46-substituted MIP-1ß were compared in assays
of T lymphocyte chemotaxis. The two proteins proved equipotent in this
assay, arguing that interaction of MIP-1ß with GAGs is not
intrinsically required for functional interaction of MIP-1ß with its
receptor. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-helix. Four structurally distinct subfamilies of
chemokines are currently recognized. The largest of these, the ß or
CC chemokines (including
MIP-1,5 MIP-1ß,
RANTES, MCP-1, MCP-2, MCP-3, I-309, and others), are distinguished by
the presence of two adjacent cysteine residues that tether the amino
terminus to the framework. In the or CXC chemokine family
(including IL-8, gro, PF4, inflammatory protein-10, and others), these
two cysteines are separated by a single amino acid. Two more recently
defined chemokine subfamilies are each currently composed of only a
single member. The so-called C family (lymphotactin) displays only one
of the two amino-terminal cysteines, whereas the
CX3C family (fractalkine) displays three amino
acids separating the amino-terminal cysteines, and is tethered to the
membrane via a long protein stalk.
Chemokines interact with a large family of
seven-transmembrane-spanning, G protein-coupled receptors on target
cells (1, 3); additionally, they interact with
glycosaminoglycans (GAGs) on cell surfaces, in the extracellular
matrix, and in exocytic granules. Chemokines have been shown to bind to
purified subfractions of heparin in vitro (4), as well as
to naturally occurring GAGs such as heparan sulfate and chondroitin
sulfate in the extracellular matrix in vitro
(5)6, and on the surface of endothelial
cells both in vitro (6, 7, 8) and in vivo (9).
Furthermore, chemokines have been found to be stored in and released
from platelet -granules (10, 11) and T lymphocyte
cytolytic granules (12) in association with GAGs.
The ability of chemokines to bind to GAGs is thought to be critical for
chemokine biology. It has been proposed that the immobilization of
chemokines by GAGs forms stable, solid-phase chemokine foci and
gradients necessary to direct leukocyte trafficking in vivo (13, 14). Consistent with this notion, immobilized IL-8 has been
shown to promote neutrophil migration both in vitro (15)
and in vivo (9). Furthermore, GAG-immobilized MIP-1ß and
RANTES have been shown to promote leukocyte adhesion in vitro (5, 14). GAG binding may also influence chemokine structure and
activity in other ways. For example, the binding of IL-8, MIP-1,
RANTES, and MCP-1 to GAGs has been shown to promote their
multimerization in vitro (8). Binding of chemokines to
cell surface GAGs may also serve to increase their effective local
concentration, and consequently increase their binding to cell surface
receptors (8). Additionally, GAG binding could
potentially influence chemokine t1/2
in vivo. As chemokines vary in the affinity (and perhaps the
specificity) with which they interact with GAGs (4), a
detailed understanding of chemokine-GAG interactions may be critical to
appreciate functional distinctions among chemokines that may have been
judged by other criteria to be functionally redundant.
The molecular determinants of chemokine binding to GAGs have been
studied in several instances. The chemokines carry a highly basic,
amphipathic carboxyl-terminal -helix. In the cases of IL-8 and PF4,
this structure has been shown to be important for binding to acidic
GAGs (16, 17, 40). Additional basic residues within the
PF4 framework have been found to interact with GAGs as well (18, 40). Among ß chemokines, basic residues within the MCP-1
-helix were found to be important for GAG binding (41).
However, we (19) and others (20) previously
noted that other ß chemokines do not have highly basic -helices,
and that other regions of these molecules must be important
determinants of GAG binding. We used in vitro mutagenesis to test the
roles of individual basic amino acids of huMIP-1, and identified
three noncontiguous amino acids (R18, R46, and R48) that were each
essential for heparin binding (19). A fourth residue, K45,
was found to contribute as well. Similarly, a double mutation of K45
and R46 was shown to abrogate heparin binding of murine MIP-1 in
another study (20). Based on a modeled MIP-1 structure,
all four basic residues are predicted to lie on one face of the
molecule, with the side chains of R18, R46, and R48 aligned and
protruding prominently from the surface.
The minimal GAG binding motif found in huMIP-1 is conserved in other
ß chemokines, but may be augmented by additional basic residues. As
an example, huMIP-1ß contains basic residues at positions 18, 45, 46,
and 48, and, in addition, at positions 19 and 22. The solved MIP-1ß
structure (21) indicates that the side chains of all of
these residues lie in proximity on one face of the molecule, leading to
the prediction that MIP-1ß uses an expanded binding motif to bind
more tightly to GAGs. Consistent with this idea, we have found that
MIP-1ß interacts more tightly with heparin than MIP-1, as judged
by the concentration of salt required to disrupt the interaction.
Nevertheless, mutagenesis experiments indicate that only a subset of
these residues contributes detectably to GAG binding. Interestingly,
functional analysis of one MIP-1ß mutant indicates that, as for
MIP-1, GAG binding is not required for functional interaction of
MIP-1ß with its receptor.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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huMIP-1ß full-length coding region cDNA was cloned from PHA/PMA-stimulated HMC-1 human mast cells (22, 23) by RT-PCR using a primer pair derived from the 5' and 3' ends of the published MIP-1ß sequence (24). Primers included EcoRI sites to facilitate cloning. The sequences of the primers used are: 5'-CCCGAATTCCACCAATACCATGAAGC-3' (sense) and 5'-CACGAATTCAGCTCAGTTCAGTTCCAGG-3' (antisense). The cDNA, which included 11 bp of 5' untranslated sequence, was subcloned into the mammalian expression vector pCAGGS (25, 26) and sequenced in its entirety using Sequenase (United States Biochemical, Cleveland, OH). The sequence obtained was identical to the published huMIP-1ß sequence Act-2 (24) with the exception of an S to G substitution at position 70. This substitution has previously been reported for another MIP-1ß isoform (27). pDREF-hyg-MIP-1ß-SEAP and pDREF-hyg-MIP-1ß R46A-SEAP, which encode fusion proteins in which MIP-1ß is linked via a five-amino-acid carboxyl-terminal linker to secreted alkaline phosphatase, were produced as described.6
Site-directed mutagenesis
Mutagenesis was performed by extension overlap amplification (28) using 400 ng of pCAGGS-MIP-1ß as template. Note that the numbering refers to amino acid residues of the mature, secreted protein. The sequences of the mutagenic primers used are as follows: MIP-1ß R18A sense, 5'-TTACACCGCGGCAAAGCTTCCTC-3'; MIP-1ß R18A antisense, 5'-GAGGAAGCTTTGCCGCGGTGTAA-3'; MIP-1ß K19A sense, 5'-CACCGCGAGGGCACTTCCTCGCA-3'; MIP-1ß K19A antisense, 5'-TGCGAGGAAGTGCCCTCGCGGTG-3'; MIP-1ß R22A sense, 5'-GAAGCTTCCTGCAAACTTTGTGG-3'; MIP-1ß R22A antisense, 5'-CCACAAAGTTTGCAGGAAGCTTC-3'; MIP-1ß K45A sense, 5'-ATTCCAAACCGCAAGAGGCAAG-3'; MIP-1ß K45A antisense, 5'-CTTGCCTCTTGCGGTTTGGAAT-3'; MIP-1ß R46A sense, 5'-CCAAACCAAAGCAGGCAAGCAA-3'; MIP-1ß R46A antisense, 5'-TTGCTTGCCTGCTTTGGTTTGG-3'; MIP-1ß K48A sense, 5'-CAAAAGAGGCGCACAAGTCTGCG-3'; MIP-1ß K48A antisense, 5'-CGCAGACTTGTGCGCCTCTTTTG-3'.
PCR products were subcloned into pCAGGS and sequenced in their entirety using Sequenase (Amersham, Arlington Heights, IL).
Transient transfection and metabolic labeling
Transient transfection of COS-P fibroblasts with wild-type and mutant pCAGGS-MIP-1ß constructs and metabolic labeling were performed exactly as described (19). Transient transfection of 293/EBNA-1 cells with pDREF-hyg-SEAP constructs was performed as previously described (29).
Heparin-Sepharose chromatography
Supernatants containing metabolically labeled wild-type or
mutant MIP-1ß were mixed with supernatants containing MIP-1ß-SEAP
fusion protein and chromatographed on 1 ml Hi-trap heparin columns
(Pharmacia Biotech, Piscataway, NJ), as described (19).
The NaCl gradient used for elution was 0–800 mM; salt concentrations
in individual fractions of a sample gradient were determined using a
conductivity meter (VWR Model 1050). Radioactivity in each fraction was
determined by liquid scintillation counting using an LKB 1209 RackBeta
scintillation counter, and by SDS-PAGE (30), followed by
fluorography (31). Alkaline phosphatase activity was
determined by incubation with 1 mg/ml p-nitrophenyl
phosphate at 37°C and measurement of absorbance at 405 nm using an
Anthos Labtech HT-2-2001 plate reader. For the experiment shown in Fig. 4
, radiolabeled chemokines were first partially purified by
chromatography on Hi-trap Q-Sepharose columns (Pharmacia Biotech). Peak
Q-Sepharose eluate fractions were identified by SDS-PAGE and
fluorography, pooled, and applied to 1 ml Hi-trap heparin columns in
buffer containing 150 mM NaCl. Columns were washed in the same buffer,
and bound chemokines were eluted with a linear 150–500 mM NaCl
gradient. Flow-through and eluate fractions were pooled and analyzed by
SDS-PAGE and fluorography.
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Unlabeled wild-type and mutant MIP-1ß proteins were purified from supernatants of transiently transfected COS-P cells by sequential chromatography on Q-Sepharose and heparin-Sepharose columns. Following transfection, cells were cultured for 48 h, as described (19). The plates were then washed twice in PBS and the medium was replaced with phenol red-free DMEM (Life Technologies, Gaithersburg, MD). Cells were cultured for an additional 24 h, and the supernatants were collected. Supernatants (200 ml) were applied to 5 ml Hi-trap Q anion-exchange columns (Pharmacia Biotech) previously equilibrated in buffer B (50 mM Tris-HCl, pH 8, 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 a linear 0–500 mM NaCl gradient in buffer B. Peak chemokine-containing fractions were identified by SDS-PAGE and silver staining and pooled. The partially purified chemokines were diluted to 50 mM NaCl in buffer B and applied to 1 ml Hi-trap heparin columns. Columns were washed with 20-column volumes of buffer, and bound chemokines were eluted with linear NaCl gradients, as described above. Peak fractions were again identified by SDS-PAGE and silver staining, pooled, dialyzed extensively against PBS at 4°C (3000 MWCO), aliquoted, and stored at -70°C. Chemokine concentrations were determined as described (19). Chemokine purity was estimated to be >95% by silver staining (32).
Solid-phase heparan sulfate-binding assay
Wild-type and R46A mutant MIP-1ß-SEAP fusion proteins were partially purified from supernatants (15 ml) of transiently transfected 293/EBNA-1 cells by chromatography over 1 ml Hi-trap Q anion-exchange columns developed with a linear 0–1 M NaCl gradient. Peak chemokine-containing eluate fractions were identified by alkaline phosphatase assay and were dialyzed against 20 mM Tris-HCl, pH 8. A solid-phase heparan sulfate-binding assay was modeled after a previously described solid-phase heparin-binding assay (33). The wells of a polystyrene 96-well plate (Corning, Acton, MA) were coated with 50 µl of 125 µg/ml bovine kidney heparan sulfate (Sigma H7640) or porcine kidney medulla heparan sulfate (34) (kind gift of Dr. Robert Linhardt, University of Iowa, Iowa City, IA, and Dr. Toshihiko Toida, Chiba University, Chiba, Japan) by incubation at 37°C for 16 h. The wells were rinsed twice with 20 mM Tris-HCl, pH 8, containing 0.05% (w/v) BSA, followed by a third rinse that was allowed to stand for 2 h at 23°C before aspiration. A total of 50 µl of 5 nM chemokine-SEAP fusion protein in 20 mM Tris-HCl, pH 8, and varying concentrations of NaCl were introduced into the wells for a 2-h incubation at 23°C. Wells were rinsed three times with 20 mM Tris-HCl, pH 8, containing 0.05% (w/v) BSA, and bound fusion proteins were quantified by alkaline phosphatase assay.
Chemotaxis assay
The ability of wild-type and mutant MIP-1ß to stimulate chemotaxis of activated human T lymphocytes was assessed using a 48-well Boyden chamber assay. Human PBMC were isolated with LSM (Organon Teknika, Durham, NC), washed four times in serum-free RPMI 1640 (Life Technologies), and resuspended in RPMI 1640 containing 10% FCS. Adherent cells were removed by two rounds of adherence to plastic tissue culture flasks for 1 h at 37°C. Nonadherent cells were cultured for 16 h at 37°C in tissue culture plates coated with a culture supernatant containing anti-CD3 Ab OKT3 (kind gift of Dr. C. Doyle, Duke University, Durham, NC). Cells were harvested, washed, and resuspended in RPMI 1640 supplemented to 20 mM HEPES-NaOH, pH 7.4, and 2% (w/v) BSA. Purified chemokines in the same medium were added in triplicate to the bottom wells of the chemotaxis chamber (NeuroProbe, Cabin John, MD) in a volume of 28 µl. The wells were overlaid with a 5 µM polyvinylpyrrolidone-free polycarbonate chemotaxis filter (NeuroProbe) that had been precoated for 45 min at 37°C with 5 µg/ml type IV collagen (Sigma, St. Louis, MO), as described (14). Cells (5 x 104/well) were added to the top wells and allowed to migrate for 2 h at 37°C. Filters were stained with Diff-Quik (Baxter Scientific Products, McGaw Park, IL), and cells migrated to the underside of the filter were counted in five randomly selected high power (x400) fields.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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). To determine which of the basic
amino acids contributes to the ability of MIP-1ß to bind to GAGs, we
examined the interaction of wild-type MIP-1ß and single amino acid
mutants of MIP-1ß with heparin-Sepharose. We expressed wild-type
MIP-1ß and alanine substitution mutants R18A, K19A, R22A, K45A, R46A,
and K48A using a COS cell transient expression system, and harvested
the supernatants of metabolically labeled cells. SDS-PAGE analysis of
unfractionated COS cell supernatants revealed a 10- to 12-kDa species
present in the supernatants of transfected cells that was not present
in the supernatants of mock-transfected cells (Fig. 2A). As observed previously in
similar transient expression analysis of MIP-1 (19),
MIP-1ß represented the most abundant radiolabeled species in these
supernatants. We also expressed wild-type MIP-1ß in the form of a
nonradiolabeled fusion protein with SEAP by transient transfection of
293/EBNA-1 cells (29),6 to be used
as an internal control for chromatography experiments.
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). SDS-PAGE analysis across the
profile confirmed that the majority of radioactivity in the peak
fractions represented MIP-1ß (Fig. 2
B). This analysis
confirmed that the SEAP moiety neither contributes to nor interferes
with heparin binding by MIP-1ß. By comparison with MIP-1ß,
MIP-1 elutes at 250 mM NaCl (19). The requirement
for significantly higher salt concentrations to elute MIP-1ß is
consistent with a stronger interaction, perhaps mediated by additional
basic residues unique to MIP-1ß.
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). However, the substitutions in mutants R18A and K45A resulted
in peaks of radioactivity that eluted at salt concentrations (250–300
mM) that were reduced as compared with the wild-type fusion protein.
For each of the mutants, the peak radioactive fraction was confirmed to
include MIP-1ß as a predominant component (Fig. 2C). The
profile for mutant R46A was complex in that two peaks of radioactivity
were identified, one eluting at very low salt concentrations (less than
100 mM), and one eluting at a position only slightly shifted from the
wild-type MIP-1ß fusion protein. For mutant R46A, SDS-PAGE analysis
across the chromatographic profile (Fig. 2D) indicated that
the first radioactive peak eluting from the column represented
MIP-1ß, whereas the second peak represented unrelated background
proteins that were also represented, albeit at lower levels, in the
wild-type elution profile (Fig. 2B). Additional experiments
indicated that the relatively low ratio of MIP-1ß R46A to background
proteins reflected the fact that a portion of MIP-1ß R46A flowed
through the heparin-Sepharose column without binding (data not shown).
We conclude that alanine substitutions at positions R18 and K45 have
small but readily detectable effects on the interaction of MIP-1ß
with heparin, whereas an alanine substitution at position R46 has a
dramatic effect on this interaction.
To further characterize heparin binding by MIP-1ß R46A, we generated
highly enriched preparations of metabolically labeled wild-type
MIP-1ß and MIP-1ß R46A by ion-exchange chromatography on
Q-Sepharose. Analysis of the Q-Sepharose eluates by SDS-PAGE revealed
that radioactivity was associated almost exclusively with the MIP-1ß
proteins (Fig. 4A). The highly
enriched preparations of MIP-1ß and MIP-1ß R46A were then applied
to a heparin-Sepharose column in a buffer containing 150 mM NaCl, and
the column was developed with a linear salt gradient. Under these
conditions, a small amount of radioactivity in the wild-type
preparation flowed through the column, but the vast majority bound and
was eluted at 400 mM NaCl (Fig. 4B). SDS-PAGE analysis
indicated that the eluate was exclusively MIP-1ß, whereas the
flow-through consisted primarily of higher m.w. contaminants (Fig. 4A). In contrast, MIP-1ß R46A was found exclusively in the
flow-through (Fig. 4, A and B). Hence, MIP-1ß
R46A does not stably interact with heparin when binding is assessed
under conditions of physiological salt concentrations.
To ask whether the R46A mutation abrogates interaction between MIP-1ß
and GAGs that occur naturally on cell surfaces and in the extracellular
matrix, we assessed the binding of MIP-1ß and MIP-1ß R46A to
plastic-immobilized heparan sulfate in vitro (Fig. 5). Because sulfation levels vary among
heparan sulfate chains isolated from different sources, we analyzed
binding to both bovine kidney heparan sulfate, which displays a
relatively low degree of sulfation, and porcine kidney medulla heparan
sulfate, which displays a high degree of sulfation (34).
Partially purified preparations of MIP-1ß-SEAP and MIP-1ß R46A-SEAP
fusion proteins were incubated with the immobilized heparan sulfate
preparations at several salt concentrations, and binding was assessed
by alkaline phosphatase assay. In this binding assay, the signals
detected in the presence of 0.75 M NaCl represent nonspecific,
salt-insensitive binding. Relative to this binding, wild-type MIP-1ß
bound well to both bovine kidney and porcine kidney medulla heparan
sulfate in subphysiological salt (30 mM), but only bound to the more
highly sulfated porcine kidney medulla heparan sulfate in physiological
salt. Importantly, binding of MIP-1ß R46A was undetectable at both
physiological and subphysiological salt concentrations, indicating that
the R46A mutation abrogates the ability of MIP-1ß to bind to a
physiologically relevant GAG ligand.
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A). The
wild-type (as well as a commercially available) MIP-1ß preparation
failed to induce significant increases in cytoplasmic calcium
concentration in either monocytes or lymphocytes, and displayed a
relatively poor chemotactic index in assays of monocyte chemotaxis
(data not shown). Therefore, we assessed the ability of the wild-type
and mutant MIP-1ß preparations to stimulate the chemotaxis of
activated human peripheral blood T lymphocytes. Both preparations
displayed good chemotactic indices and yielded typical bell-shaped
curves with maximal migration at about 10 ng/ml MIP-1ß (Fig. 6B). As the activities of the two preparations were
indistinguishable by this functional assay (the slight difference
between the profiles at the highest concentration tested was not
reproducible), we conclude that the ability of MIP-1ß to bind to GAGs
is unnecessary for functional interaction of MIP-1ß with its receptor
on T lymphocytes. We furthermore conclude that the dramatic effect of
the R46A mutation on GAG binding does not result from any gross
perturbation in the structure of the MIP-1ß protein.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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HuMIP-1, which elutes from heparin-Sepharose at lower salt
concentrations than huMIP-1ß, carries basic amino acids at positions
18, 45, 46, and 48, but lacks the basic amino acids at positions 19 and
22 that are contained within huMIP-1ß. If huMIP-1ß residues K19 and
R22 were responsible for the substantially different salt elution
properties of huMIP-1ß and huMIP-1, we should have detected an
effect of mutations at these positions in our assays. Furthermore, our
data present an apparent paradox in that the more avid interaction of
MIP-1ß with heparin seems to be mediated by fewer basic residues
(three rather than four). Recent experiments by Hoogewerf et al.
(8) may suggest an explanation. These investigators found
that several chemokines bound to immobilized heparin as tetramers,
whereas MIP-1 bound as a dimer. If MIP-1ß (which was not examined
in that study) were to bind as a tetramer, binding of the MIP-1ß
tetramer to GAGs would have contributions from 12 basic residues (3 per
monomer), whereas the MIP-1 dimer would have contributions from 8
basic residues (4 per monomer), providing an adequate explanation for
the distinct heparin-binding properties of the two molecules.
Why might MIP-1ß residues R18, K45, and R46 be selectively involved
in the interaction with heparin? Residues R18 and R46 are notable in
that their side chains are aligned with each other and protrude
directly out from the surface of the MIP-1ß monomer in a fashion that
can easily be envisioned to interact with heparin (Fig. 1). Although
the side chain of K45 is not aligned similarly, R18, K45, and R46 do
form a relatively focused basic patch on the surface of the molecule.
Interestingly, in a MIP-1ß dimer, the basic patches of the two
monomers face away from each other on opposite ends of the molecule. If
MIP-1ß binds to heparin as a multimer (either a dimer or tetramer),
the positioning of the basic patches would suggest that the heparin
chain wraps around the MIP-1ß multimer.
Of the remaining basic residues in MIP-1ß, R22 is probably not
involved in heparin binding because it extends away from the basic
patch formed by R18, R48, and K45. A clear explanation for the apparent
absence of involvement by K19 is less certain. On the other hand, the
absence of a significant role for K48 can probably be best understood
as a function of its orientation in the MIP-1ß dimer
(21). Whereas R18, K45, and R46 create a pair of basic
patches on opposite ends of the dimer, the K48 residues of each monomer
face toward each other on the concave surface of the dimer. As such,
this surface of the dimer most likely plays no role in heparin binding.
Given this conclusion, it is striking that residue R48 plays an
important role in heparin binding by the highly homologous MIP-1
(19). This might be consistent with distinct quaternary
structures for the MIP-1ß-heparin and MIP-1-heparin complexes.
It has long been argued that chemokine binding to GAGs may be important for stabilization of chemokines in the form of solid-phase gradients for presentation to passing leukocytes (13, 14). However, recent experiments have suggested that there is even greater and more fundamental involvement of GAGs in chemokine function. In support of this notion, enzymatic removal of cell surface GAGs results in a significant reduction in the apparent affinity of RANTES for its cell surface receptor (8). Heparan sulfate synergizes with RANTES to inhibit HIV-1 replication in monocytes (12). Furthermore, removal of cell surface GAGs has been shown to block the inhibitory effect of RANTES on HIV-1 replication in PM1 T cells (38) and to block intracellular Ca2+ signaling in PBL (39). GAG-induced RANTES oligomerization and a GAG-induced increase in local RANTES concentration could be critical to potentiate the interaction of RANTES with its specific receptor (8), or GAGs could represent a fundamental component of the receptor-ligand complex. In contrast, although an IL-8 mutant that binds poorly to GAGs displays reduced activity in a neutrophil chemotaxis assay that is thought to depend on both receptor binding and solid-phase immobilization, the same mutant displays normal activity in a neutrophil elastase release assay that is thought to depend on receptor binding only (9). Further, heparin binding mutants of MCP-1 stimulated normal Ca2+ signaling and chemotaxis of THP-1 cells (41). Different chemokines may therefore be differentially dependent on GAG binding for efficient interaction with their receptors.
We recently showed that a singly substituted MIP-1 mutant that fails
to interact with heparin displays wild-type binding to CCR1 and
wild-type Ca2+ signaling (19).
Although a two-amino-acid heparin-binding mutant of MIP-1 generated
by Graham et al. failed to bind to and signal through CCR1
(20), it seems likely that in this case the mutation
coincidentally disrupted either an adjacent or distal receptor binding
site. Importantly, both studies found wild-type MIP-1 to bind
normally to CCR1 on GAG-deficient CHO cells. Thus, GAG binding is not
critical for the functional interaction of MIP-1 with its
receptor.
The results presented in this work indicate that the same is true for MIP-1ß. Wild-type MIP-1ß and MIP-1ß R46A were indistinguishable in assays of T cell chemotaxis. From this we infer that the two proteins interact indistinguishably with one or more cell surface receptors on activated T cells, and that these interactions do not depend on cell surface GAGs. Interestingly, Oravecz et al. found that heparitinase digestion of PM1 T cells resulted in a dramatic inhibition of the antiviral effect of MIP-1ß, a result that can be interpreted to indicate that cell surface GAGs facilitate the interaction of MIP-1ß with its receptor (38). Assuming that the T cell chemotactic and antiviral effects of MIP-1ß are both mediated by CCR5, the mechanistic implications of the present study and that of Oravecz et al. (38) are clearly distinct. It remains possible that heparitinase treatment of cells modulates the antiviral activity of MIP-1ß by a mechanism that is more complex than that originally envisioned.
We note that our results do not necessarily imply that receptor binding and activation by MIP-1ß occur independently of GAGs in all instances. These parameters may vary according to both the biological assay and the receptor involved. In addition, because we found that MIP-1ß interacts preferentially with highly sulfated GAGs, any dependence of GAGs for binding or activation may vary according to tissue and/or cell type.
In conclusion, the work presented in this study dissects the heparin
binding site of huMIP-1ß, and provides evidence that the cationic
cradle motif previously proposed to mediate the interaction of GAGs
with fibronectin and lactoferrin does not accurately describe the GAG
binding site of MIP-1ß. In addition, the description of a
non-GAG-binding mutant MIP1ß that stimulates chemotaxis in vitro
extends our earlier findings with MIP-1, in that the capacity to
bind to GAGs and to bind to and stimulate chemokine receptors can be
functionally uncoupled for both molecules. GAG binding may nevertheless
be critical for activities of these chemokines that depend specifically
on their immobilization. Experiments designed to address the role(s) of
GAG binding to chemokine biology in vivo are in progress.
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Acknowledgments |
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Footnotes |
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2 W.K. and C.E. contributed equally to this work.
3 Current address: Department of Cell and Molecular Biology, Astra Draco AB, Lund, Sweden.
4 Address correspondence and reprint requests to Dr. Michael Krangel, Department of Immunology, Duke University Medical Center, P.O. Box 3010, Jones Building, Room 313, Research Drive, Durham, NC 27710. E-mail address:
5 Abbreviations used in this paper: MIP, macrophage-inflammatory protein; GAG, glycosaminoglycan; hu, human; MCP, monocyte-chemotactic protein; PF4, platelet factor 4; SEAP, secreted alkaline phosphatase.
6 D. D. Patel, W. Koopmann, T. Imai, L. P. Whichard, O. Yoshie, and M. S. Krangel. A subset of chemokines bind to extracellular matrix and mast cells in rheumatoid arthritis synovium via electrostatic interactions with glycosaminoglycans. Submitted for publication.
Received for publication January 21, 1999. Accepted for publication May 27, 1999.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-helix of monocyte chemoattractant protein-1 are essential for glycosaminoglycan binding. J. Biol. Chem. 273:29641.This article has been cited by other articles:
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 D. Krilleke, A. DeErkenez, W. Schubert, I. Giri, G. S. Robinson, Y.-S. Ng, and D. T. Shima Molecular Mapping and Functional Characterization of the VEGF164 Heparin-binding Domain J. Biol. Chem., September 21, 2007; 282(38): 28045 - 28056. [Abstract] [Full Text] [PDF]
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H. Jin, X. Shen, B. R. Baggett, X. Kong, and P. J. LiWang The Human CC Chemokine MIP-1beta Dimer Is Not Competent to Bind to the CCR5 Receptor J. Biol. Chem., September 21, 2007; 282(38): 27976 - 27983. [Abstract] [Full Text] [PDF]
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L. Zhang, M. DeRider, M. A. McCornack, S.-c. Jao, N. Isern, T. Ness, R. Moyer, and P. J. LiWang Solution structure of the complex between poxvirus-encoded CC chemokine inhibitor vCCI and human MIP-1beta PNAS, September 19, 2006; 103(38): 13985 - 13990. [Abstract] [Full Text] [PDF]
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T. T. Murooka, M. M. Wong, R. Rahbar, B. Majchrzak-Kita, A. E. I. Proudfoot, and E. N. Fish CCL5-CCR5-mediated Apoptosis in T Cells: REQUIREMENT FOR GLYCOSAMINOGLYCAN BINDING AND CCL5 AGGREGATION J. Biol. Chem., September 1, 2006; 281(35): 25184 - 25194. [Abstract] [Full Text] [PDF]
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 S. Ali, H. Robertson, J. H. Wain, J. D. Isaacs, G. Malik, and J. A. Kirby A Non-Glycosaminoglycan-Binding Variant of CC Chemokine Ligand 7 (Monocyte Chemoattractant Protein-3) Antagonizes Chemokine-Mediated Inflammation J. Immunol., July 15, 2005; 175(2): 1257 - 1266. [Abstract] [Full Text] [PDF]
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R. Colobran, P. Adreani, Y. Ashhab, A. Llano, J. A. Este, O. Dominguez, R. Pujol-Borrell, and M. Juan Multiple Products Derived from Two CCL4 Loci: High Incidence of a New Polymorphism in HIV+ Patients J. Immunol., May 1, 2005; 174(9): 5655 - 5664. [Abstract] [Full Text] [PDF]
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 V. Petkovic, C. Moghini, S. Paoletti, M. Uguccioni, and B. Gerber I-TAC/CXCL11 is a natural antagonist for CCR5 J. Leukoc. Biol., September 1, 2004; 76(3): 701 - 708. [Abstract] [Full Text] [PDF]
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 E. Billick, C. Seibert, P. Pugach, T. Ketas, A. Trkola, M. J. Endres, N. J. Murgolo, E. Coates, G. R. Reyes, B. M. Baroudy, et al. The Differential Sensitivity of Human and Rhesus Macaque CCR5 to Small-Molecule Inhibitors of Human Immunodeficiency Virus Type 1 Entry Is Explained by a Single Amino Acid Difference and Suggests a Mechanism of Action for These Inhibitors J. Virol., April 15, 2004; 78(8): 4134 - 4144. [Abstract] [Full Text] [PDF]
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F. C. Peterson, E. S. Elgin, T. J. Nelson, F. Zhang, T. J. Hoeger, R. J. Linhardt, and B. F. Volkman Identification and Characterization of a Glycosaminoglycan Recognition Element of the C Chemokine Lymphotactin J. Biol. Chem., March 26, 2004; 279(13): 12598 - 12604. [Abstract] [Full Text] [PDF]
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 T. Baltus, K. S. C. Weber, Z. Johnson, A. E. I. Proudfoot, and C. Weber Oligomerization of RANTES is required for CCR1-mediated arrest but not CCR5-mediated transmigration of leukocytes on inflamed endothelium Blood, September 15, 2003; 102(6): 1985 - 1988. [Abstract] [Full Text] [PDF]
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M. A. McCornack, C. K. Cassidy, and P. J. LiWang The Binding Surface and Affinity of Monomeric and Dimeric Chemokine Macrophage Inflammatory Protein 1beta for Various Glycosaminoglycan Disaccharides J. Biol. Chem., January 10, 2003; 278(3): 1946 - 1956. [Abstract] [Full Text] [PDF]
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J. Middleton, A. M. Patterson, L. Gardner, C. Schmutz, and B. A. Ashton Leukocyte extravasation: chemokine transport and presentation by the endothelium Blood, December 1, 2002; 100(12): 3853 - 3860. [Abstract] [Full Text] [PDF]
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B. Nardelli, L. Zaritskaya, M. Semenuk, Y. H. Cho, D. W. LaFleur, D. Shah, S. Ullrich, G. Girolomoni, C. Albanesi, and P. A. Moore Regulatory Effect of IFN-{kappa}, A Novel Type I IFN, On Cytokine Production by Cells of the Innate Immune System J. Immunol., November 1, 2002; 169(9): 4822 - 4830. [Abstract] [Full Text] [PDF]
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 S. Ali, S. J. Fritchley, B. T. Chaffey, and J. A. Kirby Contribution of the putative heparan sulfate-binding motif BBXB of RANTES to transendothelial migration Glycobiology, September 1, 2002; 12(9): 535 - 543. [Abstract] [Full Text] [PDF]
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S. E. Stringer, M. J. Forster, B. Mulloy, C. R. Bishop, G. J. Graham, and J. T. Gallagher Characterization of the binding site on heparan sulfate for macrophage inflammatory protein 1alpha Blood, August 13, 2002; 100(5): 1543 - 1550. [Abstract] [Full Text] [PDF]
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C. W. Frevert, R. B. Goodman, M. G. Kinsella, O. Kajikawa, K. Ballman, I. Clark-Lewis, A. E. I. Proudfoot, T. N. C. Wells, and T. R. Martin Tissue-Specific Mechanisms Control the Retention of IL-8 in Lungs and Skin J. Immunol., April 1, 2002; 168(7): 3550 - 3556. [Abstract] [Full Text] [PDF]
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H. Lortat-Jacob, A. Grosdidier, and A. Imberty Structural diversity of heparan sulfate binding domains in chemokines PNAS, February 5, 2002; 99(3): 1229 - 1234. [Abstract] [Full Text] [PDF]
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 N. Bannert, S. Craig, M. Farzan, D. Sogah, N. V. Santo, H. Choe, and J. Sodroski Sialylated O-Glycans and Sulfated Tyrosines in the NH2-Terminal Domain of CC Chemokine Receptor 5 Contribute to High Affinity Binding of Chemokines J. Exp. Med., December 3, 2001; 194(11): 1661 - 1674. [Abstract] [Full Text] [PDF]
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S. Ali, A. C. V. Palmer, B. Banerjee, S. J. Fritchley, and J. A. Kirby Examination of the Function of RANTES, MIP-1alpha , and MIP-1beta following Interaction with Heparin-like Glycosaminoglycans J. Biol. Chem., April 14, 2000; 275(16): 11721 - 11727. [Abstract] [Full Text] [PDF]
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M. Jones, L. Tussey, N. Athanasou, and D. G. Jackson Heparan Sulfate Proteoglycan Isoforms of the CD44 Hyaluronan Receptor Induced in Human Inflammatory Macrophages Can Function as Paracrine Regulators of Fibroblast Growth Factor Action J. Biol. Chem., March 10, 2000; 275(11): 7964 - 7974. [Abstract] [Full Text] [PDF]
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K. Rajarathnam, Y. Li, T. Rohrer, and R. Gentz Solution Structure and Dynamics of Myeloid Progenitor Inhibitory Factor-1 (MPIF-1), A Novel Monomeric CC Chemokine J. Biol. Chem., February 9, 2001; 276(7): 4909 - 4916. [Abstract] [Full Text] [PDF]
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R. Sadir, F. Baleux, A. Grosdidier, A. Imberty, and H. Lortat-Jacob Characterization of the Stromal Cell-derived Factor-1alpha -Heparin Complex J. Biol. Chem., March 9, 2001; 276(11): 8288 - 8296. [Abstract] [Full Text] [PDF]
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A. E. I. Proudfoot, S. Fritchley, F. Borlat, J. P. Shaw, F. Vilbois, C. Zwahlen, A. Trkola, D. Marchant, P. R. Clapham, and T. N. C. Wells The BBXB Motif of RANTES Is the Principal Site for Heparin Binding and Controls Receptor Selectivity J. Biol. Chem., March 30, 2001; 276(14): 10620 - 10626. [Abstract] [Full Text] [PDF]
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