From the Laboratory of Gene Regulation, Division of Therapeutic Proteins, Center for Biologics Evaluation and Research, Food and Drug Administration, National Institutes of Health, Bethesda, Maryland 20892
Received for publication, July 17, 2000, and in revised form, January 12, 2001
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ABSTRACT |
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Chemokines are secreted proteins that function as
chemoattractants for leukocytes. The chemokines macrophage inflammatory protein 1 Chemokines constitute a family of small secreted proteins that
were initially characterized on the basis of their chemotactic effects
on a variety of leukocytes (1, 2). They are produced locally in tissues
and interact with selective G protein-coupled receptors expressed on
the leukocyte surface. Chemokines also regulate leukocyte maturation,
the trafficking and homing of lymphocytes, and the development of
lymphoid tissues. Human immunodeficiency virus-type 1 (HIV-1)1 also targets
chemokine receptors during entry into cells, and certain chemokines act
as HIV-1 suppressive factors (3-5).
To date, at least 50 chemokines have been identified. Despite its large
size, the chemokine family is remarkably homogeneous. Chemokines are
divided into two subfamilies, The quaternary structures of Human T lymphocytes and monocytes release anti-HIV-1 chemokines,
predominantly the Reagents--
Recombinant human MIP-1 Cell Culture--
Human PBLs and monocytes were isolated by
elutriation from normal blood donors and cultured as described
previously (13, 14). PBLs were cultured under 5% CO2 at
37 °C in RPMI 1640 medium (Life Technologies, Inc.) supplemented
with 10% fetal bovine serum (Hyclone, Logan, UT), 150 units/ml IL-2
(R&D Systems), and 10 ng/ml IL-12 (kindly provided by Genetics
Institute, Boston, MA) or 2 µg/mo PHA (sigma). Monocytes were
cultured in macrophage serum-free medium (Life Technologies, Inc.)
supplemented with 1 µg/ml LPS (Sigma). Cells were plated at a density
of 1 × 106 cells/ml in 6-well plates.
Immunoprecipitation and Immunoblot Analysis--
Cell culture
supernatants were incubated at 4 °C for 2 h with antibodies to
anti-MIP-1 Mass Spectrometry--
Culture supernatants were subjected to
immunoprecipitation with anti-MIP-1 Identification of a Heterodimer of MIP-1
To determine the molecular size of the chemokines precipitated by
anti-MIP-1 MIP-1 MIP-1 Dissociation of Native MIP-1 Specificity of MIP-1 The possible existence and activity of native chemokine dimers
have been controversial (21). The We have now identified a naturally occurring MIP-1 Most studies on chemokine homodimerization have been performed in
vitro, and the natural formation of either homodimers or heterodimers of chemokines has not been described previously. Native
MIP-1 purified from LPS-stimulated mouse macrophage RAW 264.7 migrated
on SDS-polyacrylamide gels as a doublet composed of peptides with
similar physical characteristics. The NH2-terminal sequences of the two peptides identified them as MIP-1 The three-dimensional structures of both A truncation mutant of MIP-1 Most three-dimensional structural determinations have been performed at
high concentrations (>1 mM) of chemokines with
thermodynamics further strongly favoring dimerization. In
contrast, physical data indicate that chemokines are fully dissociated
into monomers at their normal physiological (nanomolar) concentrations.
Dimer dissociation constants have been determined as 35 µM for RANTES, 33 µM for MCP-1, 58 µM for MCP-2, 40 nM for MIP-1 Comparison of our present data with those of previous studies of
chemokine homodimers indicates that substantial differences exist
between MIP-1 The existence of native chemokine homodimers remains to be
demonstrated with the data having been obtained that are consistent or
inconsistent with homodimers being the functionally active form of
these proteins (12). Mutagenesis and cross-linking studies indicate that the active form of MCP-1 is a dimer (31). However, other
studies have shown that IL-8 and MIP-1 With regard to the functional role of MIP-1 and 1
(MIP-1
and MIP-1
) now have been shown to be secreted from activated human monocytes and peripheral blood
lymphocytes (PBLs) as a heterodimer. Immunoprecipitation and immunoblot
analysis revealed that antibodies to either MIP-1
or MIP-1
precipitated a protein complex containing both MIP-1
and MIP-1
under normal conditions from culture supernatants and lysates of these
cells. Mass spectrometry of the complexes, precipitated from the
culture supernatants of monocytes and PBLs, revealed the presence of
NH2-terminal truncated MIP-1
(residues 5-70)
together with either intact MIP-1
or NH2-terminal
truncated MIP-1
(residues 3-69), respectively. The secreted
MIP-1
/
heterodimers were dissociated into their component
monomers under acidic conditions. Exposure of monocytes or PBLs to
monensin induced the accumulation of heterodimers composed of
NH2-terminal truncated MIP-1
and full-length MIP-1
in
the Golgi complex. The mixing of recombinant chemokines in
vitro demonstrated that heterodimerization of MIP-1
and
MIP-1
is specific and that it occurs at physiological conditions, pH
7.4, and in the range of nanomolar concentrations. The data presented
here provide the first biochemical evidence for the existence of
chemokine heterodimers under natural conditions. Formation of
heterodimers of MIP-1
/
may have an impact on intracellular
signaling events that contribute to CCR5 and possibly to other
chemokine receptor functions.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
(CXC) and
(CC), on the
basis of conserved cysteine residues. Four conserved cysteines form two
essential disulfide bonds, Cys1-Cys3 and Cys2-Cys4, in all chemokines.
The three-dimensional structures of three
chemokines (interleukin
(IL)-8, growth-related oncogene-
, and platelet factor 4) and four
chemokines (macrophage inflammatory protein
(MIP)-1
, MIP-1
, RANTES (regulated on activation normal T cell
expressed), and macrophage chemoattractant protein-1 (MCP-1)) have been
determined either by multidimensional nuclear magnetic resonance (NMR)
or by x-ray crystallography (6-11). These studies have revealed that chemokines possess a short NH2-terminal domain preceding
the first cysteine, a backbone that comprises three antiparallel
strands and a COOH-terminal
-helix. Whereas the backbone exhibits a
well ordered structure, the structure of the NH2 terminus
is disordered. The similarity in the three-dimensional structures of
the chemokine monomers is consistent with the marked sequence homology
of these proteins.
and
chemokines, however, differ
markedly from each other, and the dimer interfaces are formed by
distinct sets of residues. Whereas the IL-8 dimer is globular, the
homodimers formed by MIP-1
, MIP-1
, and RANTES are cylindrical (6,
7, 11, 12). Calculation of the solvation-free energies of dimerization
and analysis of hydrophobic clusters of amino acids suggest that the
formation and stabilization of the two different types of dimers result
from the burial of hydrophobic residues and that the distinct
quaternary structures of
and
chemokine dimers are preserved
throughout the two subfamilies. The biological existence and
significance of dimeric forms of chemokines, especially that of
chemokine heterodimers, have remained unclear.
chemokines MIP-1
, MIP-1
, and RANTES (3, 4,
13), in response to stimulation with mitogens, cytokines, or bacterial
pathogens. We now show that human monocytes stimulated by
lipopolysaccharide, human peripheral blood lymphocytes (PBLs)
stimulated by cytokines (IL-2 and IL-12), or phytohemagglutinin produce
a chemokine heterodimer containing MIP-1
and MIP-1
.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
(full-length), RANTES,
MDC, MCP-1, and IP-10 were obtained from Peprotech (Rocky Hill, NJ);
recombinant human MIP-1
was from Sigma; and recombinant human
(
4)MIP-1
and antigen affinity purified antibodies to chemokines
were from R&D Systems (Minneapolis, MN).
or anti-MIP-1
, and then for an additional 2 h,
they were incubated with protein G-Sepharose beads (Amersham Pharmacia
Biotech). The beads were washed three times with
phosphate-buffered saline containing 0.3% Triton X-100, after which
proteins were eluted with 25 mM Tris-glycine, pH 3.0, subjected to SDS-polyacrylamide gel electrophoresis under reducing
conditions on a 4-20% gradient gel in the presence of Tricine,
and transferred to a polyvinylidene difluoride membrane. The membrane
was incubated for 2 h at room temperature with Tris-buffered
saline containing 3% bovine serum albumin and then exposed to either
anti-MIP-1
or anti-MIP1
. Immune complexes were detected with
alkaline phosphatase-conjugated goat antibodies to mouse immunoglobulin
G, alkaline phosphatase substrate, and Lumi-Phos TM 530 (Roche
Molecular Biochemicals).
or anti-MIP-1
. The molecular
size of precipitated proteins was determined by matrix-assisted laser
desorption ionization and time-of-flight (MALDI-TOF) mass spectrometry
(PerSeptive Biosystems, Boston, MA).
-Cyano-4-hydroxycinnamic acid
(Sigma) and recombinant MIP-1
or MIP-1
were used as matrix and
internal standards, respectively. For some experiments, cells were
cultured in the presence of monensin (GolgiStop, Pharmingen, San Diego,
CA) for 10 h. They were then washed and lysed for 1 h
on ice at a density of 1 × 108 cells/ml in a solution
containing 1% Triton X-100, 50 mM Tris-HCl, pH 7.4, 1 mM phenylmethylsulfonyl fluoride, 1 µg/ml leupeptin, 1 µg/ml pepstatin, 1 µg/ml E64, and 40 µg/ml bestatin (Roche
Molecular Biochemicals). After centrifugation of lysates at 6700 × g for 15 min, the resulting supernatants were subjected
to immunoprecipitation with either anti-MIP-1
or anti-MIP-1
for
mass spectrometric analysis.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
/
in Culture
Supernatants of Human Monocytes and PBLs--
Supernatants were
collected from cultures of either LPS-stimulated monocytes or PBLs
stimulated with IL-2 and IL-12 after incubation for 1 or 6 days,
respectively, times that correspond to peak chemokine secretion (data
not shown). Enzyme-linked immunosorbent assays revealed that the
culture supernatants contained large amounts of MIP-1
and MIP-1
but only a low concentration of RANTES (data not shown) (13).
Immunoprecipitation of culture supernatants from both cell types with
either anti-MIP-1
or anti-MIP-1
followed by immunoblot analysis
with each of these antibodies revealed that each immunoprecipitate
contained both MIP-1
and MIP-1
(Fig. 1A). The antigen affinity
purified antibodies to each MIP-1 protein were specific and did not
cross-react with the other isoform on Western blot analysis (Fig.
1B) or with immunoprecipitation followed by mass
spectrometric analysis (Fig.
2B).
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Fig. 1.
Coimmunoprecipitation of
MIP-1 and MIP-1
from
culture supernatants of monocytes and PBLs. A, culture
supernatants of either monocytes stimulated with LPS for 1 day or PBLs
stimulated with IL-2 and IL-12 for 6 days were subjected to
immunoprecipitation (IP) with anti-MIP-1
or
anti-MIP-1
. The resulting precipitates were then subjected to
immunoblot analysis with anti-MIP-1
(left
panel) or anti-MIP-1
(right panel). Lanes
1 and 4 contain recombinant human (
4)MIP-1
(7.4 kDa) and recombinant human MIP-1
(7.8 kDa), respectively, probed
with the corresponding antibody. Data in this and subsequent figures
are representative of at least three independent experiments.
B, recombinant human MIP-1
and recombinant MIP-1
probed with anti-MIP-1
or anti-MIP-1
antibody.
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Fig. 2.
Mass spectrometric analysis of
MIP-1 /
complexes
secreted by monocytes and PBLs. A, culture supernatants
of either PBLs stimulated with IL-2 and IL-12 for 6 days (left
panels) or monocytes stimulated with LPS for 1 day (right
panels) were subjected to immunoprecipitation with
anti-MIP-1
(upper panels) or anti-MIP-1
(lower panels). The molecular mass of the precipitated
proteins was then determined by mass spectrometry. B, mass
spectrometric analysis of immunoprecipitation of recombinant MIP-1
and recombinant MIP-1
with anti-MIP-1
or anti-MIP-1
antibody.
or anti-MIP-1
, we analyzed the precipitated proteins
by mass spectrometry. The two antibodies precipitated identical
complexes from the culture supernatant of LPS-stimulated monocytes
(Fig. 2). These complexes consisted predominantly of two polypeptides
with molecular masses of 7459 and 7826 Da, which correspond to MIP-1
lacking the four NH2-terminal residues ((
4)MIP-1
) and
intact MIP-1
, respectively. The complexes immunoprecipitated by each
of the two antibodies from the culture supernatant of IL-2- and
IL-12-stimulated PBLs contained (
4)MIP-1
and a protein with a
molecular mass of 7658 Da, which corresponds to MIP-1
lacking the
two NH2-terminal residues ((
2)MIP-1
). Similar results were obtained with culture supernatants of PBLs stimulated with IL-2
and phytohemagglutinin (data not shown). Immunoprecipitates prepared
from the monocyte and PBL culture supernatants with anti-RANTES or
anti-MDC were also analyzed by mass spectrometry. However, this
approach failed to detect either RANTES or MDC in the supernatants (data not shown). Thus, these data demonstrate that LPS-treated monocytes and PBLs stimulated with IL-2 and IL-12 release a
MIP-1
/
heterodimer into the culture supernatant.
and MIP-1
Are Secreted Predominantly as a
Heterodimer--
We next examined whether secreted MIP-1
and
MIP-1
were present in the culture supernatant of LPS-stimulated
monocytes predominantly as a heterodimer, or whether they were also
present as monomers. The supernatant was subjected to
immunoprecipitation first with anti-MIP-1
and then either with
anti-MIP-1
or again with anti-MIP-1
. The various
immunoprecipitates were then subjected to immunoblot analysis with
anti-MIP-1
or anti-MIP-1
(Fig. 3).
Only a small amount of residual MIP-1
was detected in the second
precipitate prepared with anti-MIP-1
, indicating that the first
immunoprecipitation with this antibody efficiently removed most of the
target antigen. The observation that the second precipitate prepared
with anti-MIP-1
contained little MIP-1
indicated that most of
this protein had been previously precipitated as a heterodimer with
MIP-1
.
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Fig. 3.
Secretion of MIP-1
and MIP-1
from monocytes predominantly
as a heterodimer. Culture supernatant of monocytes stimulated with
LPS for 1 day was subjected to immunoprecipitation (IP)
first with anti-MIP-1
and then either with anti-MIP-1
again or
with anti-MIP-1
. The three precipitates were then subjected to
immunoblot analysis with anti-MIP-1
or anti-MIP-1
as indicated.
Recombinant human MIP-1
(7.4 kDa) or MIP-1
(7.8 kDa) were also
probed with the corresponding antibodies.
/
Heterodimer in the Golgi Complex--
We next
investigated the kinetics and site of MIP-1
/
dimerization.
Immunoprecipitation and mass spectrometric analysis revealed the
presence of the MIP-1
/
heterodimer in the culture supernatants of
monocytes or PBLs as early as 7-10 h after exposure to LPS or to IL-2
and IL-12, respectively (data not shown). To determine whether the
heterodimerization of MIP-1
and MIP-1
occurs intracellularly in
the Golgi complex, we treated cells with monensin to prevent intracellular protein transport by inducing retention of proteins in
the Golgi complex (15, 16). PBLs and monocytes were incubated with IL-2
and phytohemagluttinin A or with LPS, respectively, for 20 h, the
last 10 h of which they were also exposed to monensin. Under these
conditions, chemokines were produced primarily during the last 10 h of culture. The cells were then lysed and subjected to
immunoprecipitation with anti-MIP-1
or anti-MIP-1
, and the resulting precipitates were analyzed by mass spectrometry. The immunoprecipitates prepared from both PBLs (Fig.
4A) and monocytes (Fig.
4B) with either antibody consisting predominantly of
heterodimers of (
4)MIP-1
and full-length MIP-1
. The results
revealed that MIP-1
was processed from the intact form (7826 Da)
detected in the Golgi complex and in the culture supernatant that was
obtained after 1 day (data not shown) to a mixture of both the
full-length protein and (
2)MIP-1
(7658 Da) in the supernatant
obtained after 3 days. After incubation of PBLs for 6 days, almost all
of the MIP-1
had been converted to the NH2-terminal
truncated form. In contrast, analysis of the culture supernatants of
monocytes obtained after 1 day (Fig. 4B) or 3 days (data not
shown) did not reveal proteolytic processing of MIP-1
. Given that
monensin only blocks protein transport to the post-Golgi compartment
from the trans-Golgi network, these data indicate that
MIP-1
/
heterodimers probably form in the endoplasmic reticulum or
cis-Golgi. Other experiments using brefeldin A to disrupt
the Golgi complex also revealed the presence of heterodimers in cell
lysates (data not shown) and suggest that heterodimers are formed in a
pre-Golgi compartment. They also suggest that the proteolytic
processing of MIP-1
released by PBLs may occur at a late or
post-Golgi step during secretion. The MIP-1
/
heterodimer was also
detected in immunoprecipitates prepared from both PBL and monocyte
lysates without monensin treatment (Fig. 4C), indicating
that MIP-1
/
dimerization occurs under normal conditions.
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Fig. 4.
Intracellular formation of the
MIP-1 /
heterodimer. PBLs (A) or monocytes (B)
were stimulated with IL-2 and PHA or with LPS, respectively, for
10 h and then incubated for an additional 10 h in the
presence or absence of monensin (C). The cells were then
either lysed or incubated further in the absence of monensin. Cell
lysates, as well as culture supernatants obtained after incubation of
monocytes for 1 day or PBLs for 3 or 6 days, were subjected to
immunoprecipitation with anti-MIP-1
or anti-MIP-1
as indicated,
and the resulting precipitates were analyzed by mass
spectrometry.
/
Heterodimers under Acidic
Conditions--
To investigate the nature of the interaction between
MIP-1
and MIP-1
, we adjusted the culture supernatants of
stimulated PBLs or monocytes to pH 4.0 before immunoprecipitation with
anti-MIP-1
or anti-MIP-1
. Under these low pH conditions, each
antibody precipitated only the corresponding antigen (Fig.
5). These results suggest that acidic
conditions induce the dissociation of the MIP-1
/
heterodimer and
that electrostatic interactions among charged amino acids therefore are
likely to contribute to formation of the dimer interface.
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Fig. 5.
Dissociation of the
MIP-1 /
heterodimer
under low pH conditions. Culture supernatants of either PBLs
stimulated with IL-2 and IL-12 for 6 days (left panels) or
monocytes stimulated with LPS for 1 day (right panels) were
adjusted to pH 7.0 or 4.0 and then subjected to immunoprecipitation
with anti-MIP-1
or anti-MIP-1
as indicated. The molecular mass of
the precipitated proteins was then determined by mass
spectrometry.
/
Heterodimerization--
Direct
measurements of monomer-dimer equilibrium by NMR or sedimentation
analysis have revealed that
chemokines (IL-8, growth-related oncogene-
, neutrophil-activating protein-2, low affinity
platelet factor 4, and PF4) and
chemokines (MCP-1, MCP-2, MCP-3,
MIP-1
, MIP-1
, RANTES, and I-309) (6-9, 17-20) are monomeric at
physiological (nanomolar) concentrations. To examine the specificity of
MIP-1
/
heterodimerization at physiological concentrations, we
mixed various combinations of recombinant chemokines in
vitro at a 1:1 ratio and concentrations of 25-50 nM
at physiological pH. We paired MIP-1
with MIP-1
, RANTES, MDC,
MCP-1, or IP-10. In parallel, we mixed RANTES with MIP-1
, MDC,
MCP-1, or IP-10; IP-10 was also tested for pairing with MCP-1. The
various mixtures were then subjected to immunoprecipitation with
corresponding specific antibodies, and the resulting precipitates were
analyzed by mass spectrometry. Heterodimerization was detected only
between MIP-1
and MIP-1
and not with RANTES (Fig.
6) or with the other chemokines tested (data not shown), thus indicating that this interaction is highly specific. The antigen affinity purified antibodies to each MIP-1 protein were specific and did not cross-react with the other isoform (Fig. 6).
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Fig. 6.
Specificity of
MIP-1 /
heterodimerization in vitro. Recombinant
(
4)MIP-1
{(7450 Da), MIP-1
(7820 Da), and RANTES (7864 Da)
dissolved in phosphate-buffered saline, pH 7.4, were mixed at final
concentrations of 25 nM in the indicated combinations at
room temperature and then subjected to immunoprecipitation with the
indicated antibodies. The resulting precipitates were analyzed by mass
spectrometry.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
chemokines MIP-1
, MIP-1
, and RANTES tend to self-associate and thereby form homodimers, tetramers, or larger aggregates in vitro. This process is
dynamic and reversible, but it has been thought that chemokine
concentrations in vivo may be too low for the formation of
such multimers to occur. However, high local concentrations of
chemokines may occur in vivo under certain conditions, such
as during platelet degranulation, inflammatory disease, and local
accumulation of chemokines on cell membranes mediated by receptors or
by glycosaminoglycans.
/
heterodimer
produced by activated monocytes and PBLs. Our data demonstrate that the
MIP-1
/
heterodimer forms in the endoplasmic reticulum or Golgi
complex, and that these two chemokines are secreted in the form of the
heterodimer. Furthermore, the combination of MIP-1
with MIP-1
in vitro showed that these two chemokines indeed form heterodimers at physiological (nanomolar) concentrations.
and MIP-1
(22). Although it was not shown that the co-purified mouse chemokines originally existed as a heterodimer, our data now suggest that these
previous results might be explained by the formation of a native mouse
MIP-1
/
heterodimer.
and
chemokines have
been determined mostly with the molecules in the form of crystallized
homodimers. Whereas the monomeric structures of both
and
chemokines are highly similar, the dimeric structures of members of
these two subfamilies, as typified by IL-8 (12, 20, 23) and MIP-1
(9, 11, 20), respectively, differ markedly from each other with the
dimer interfaces being formed by distinct sets of interacting residues.
Whereas the IL-8 dimer is globular, the MIP-1
dimer is cylindrical.
The three-dimensional structure of the
chemokine PF4 revealed it to
be a tetramer composed of two dimers of the IL-8 type (18, 24). NMR
revealed MIP-1
as an end-on-end dimer with the dimer interface
showing a large number of contacts between the two monomers. Other
chemokines, including RANTES, MIP-1
, MCP-1, and MCP-2, exhibit a
similar homodimeric structure (6, 8, 17, 25). MCP-3 remains monomeric
at concentrations of up to 20 mg/ml (7), whereas I-309, another
chemokine, was also shown to exist as a monomer at high concentrations
during sedimentation (17).
lacking the NH2-terminal
five residues forms a dimer similar to that formed by the wild-type protein, whereas a mutant lacking the NH2-terminal eight
residues exists only as a folded monomer (11). An MCP-1 mutant lacking the NH2-terminal eight amino acids exists predominantly as
a monomer (7). IL-8 and melanocyte growth stimulating activity
each dimerize by the formation of a central six-stranded
sheet,
three strands of which are contributed by each subunit (26-30). The
calculation of the solvation-free energies of dimerization and analysis
of clusters of hydrophobic amino acids indicates that the formation and
stabilization of the two main types of chemokine homodimers result from
the burial of hydrophobic residues and that the distinct quaternary
structures are preserved throughout the two subfamilies (12, 21). Such
a scenario would explain the lack of receptor cross-binding and
cross-reactivity, which is apparent between the
and
chemokine subfamilies.
, 18 µM for IL-8, 73 µM for melanocyte growth
stimulating activity/growth-related oncogene-
, and 100 µM for neutrophil-activating protein-2. MIP-1
also tends to aggregate, but this process is reversible, and MIP-1
exists as a monomer under physiological conditions (6, 7, 9,
17-20).
/
heterodimers and chemokine homodimers in terms of
physical properties: (i) the formation of homodimers requires high
concentrations of chemokines, whereas the MIP-1
/
heterodimer
forms at physiological concentrations of monomers; (ii) the
dissociation of homodimers occurs under physiological conditions,
whereas dissociation of MIP-1
/
heterodimers is apparent only at
low pH; (iii) homodimers have been detected only in solution under
in vitro conditions, whereas MIP-1
/
heterodimers are
secreted from primary monocytes and PBLs; and (iv) the formation of the MIP-1
/
heterodimer appears to be mediated by electrostatic
interactions, whereas the formation of chemokine homodimers is thought
to be mediated by hydrophobic interactions.
derivatives that do not
dimerize are fully active (32, 33).
/
heterodimerization,
it is possible that the formation of stable heterodimers protects these
chemokines from enzymatic digestion and, thus, increases or stabilizes
their activity. Preliminary data indicate that MIP-1
/
heterodimer-containing mixtures have potent activity in inducing
down-regulation of the CCR5
receptor.2 The formation of
the MIP-1
/
heterodimer under natural conditions may induce
(possibly heterologous) receptor dimerization that may have an impact
on intracellular signaling events, which contribute to CCR5 and
possibly other chemokine receptor functions. Production of pure
homogeneous preparations of heterodimers is required for further
characterization of heterodimer activities and receptor binding properties.
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ACKNOWLEDGEMENTS |
---|
We thank K. Fields and V. Calvert for assistance with the isolation of human PBLs and monocytes and R. Boykins for help with mass spectrometry.
![]() |
FOOTNOTES |
---|
* 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 may be addressed: Div. of Therapeutic
Proteins, Ctr. for Biologics Evaluation and Research, FDA, NIH, Bldg.
29B, Rm. 4E12, HFM 535, 8800 Rockville Pike, Bethesda, MD 20892. Tel.:
301-827-0793; Fax: 301-480-3256; E-mail: guan@cber.fda.gov or
norcross@cber.fda.gov.
Published, JBC Papers in Press, January 16, 2001, DOI 10.1074/jbc.M006327200
2 E. Guan, J. Wang, M. A. Norcross, unpublished observations.
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ABBREVIATIONS |
---|
The abbreviations used are: HIV-1, human immunodeficiency virus-type 1; IL, interleukin; MIP, macrophage inflammatory protein; RANTES, regulated on activation normal T cell expressed; MCP, macrophage chemoattractant protein; LPS, lipopolysaccharide; PBL, peripheral blood lymphocyte; MDC, macrophage-derived chemokine; IP-10, gamma interferon inducible protein-10; anti-, antibodies to; Tricine, N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine; PHA, phytohemagglutinin A.
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REFERENCES |
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