From the British Biotech Pharmaceuticals Ltd., Watlington Road, Oxford OX4 5LY, United Kingdom, the b School of Animal and Microbial Sciences, University of Reading, Whiteknights, P. O. Box 228, Reading RG6 2AJ, United Kingdom, the d Department of Molecular Biology and Biotechnology, University of Sheffield, Sheffield S10 2TN, United Kingdom, the f CRC Department of Experimental Haematology, Paterson Institute for Cancer Research, Christie Hospital NHS Trust, Wilmslow Road, Withington, Manchester M20 9BX, United Kingdom, and the q School of Chemistry, University of Leeds, Leeds LS2 9JT, United Kingdom
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ABSTRACT |
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Human CC chemokines macrophage inflammatory
protein (MIP)-1 Chemokines (chemotactic cytokines) are a family of proteins
primarily involved in the coordination of cellular trafficking and
activation during immune responses (1). They have been characterized
according to the sequence context around the first cysteine residue
giving rise to the C, CC, CXC, and CX3C groups. Chemokines
act via a family of G-protein-coupled receptors
(GPCRs)1 and are unusually
large ligands (>60 amino acids) for this type of receptor. There is
some controversy concerning the quaternary structure of biologically
active chemokines when they bind to receptors, with evidence to suggest
either monomers or dimers (2-4). The three-dimensional structures of
the human CC chemokines MIP-1 Human CC chemokines MIP-1 hMIP-1 The human immunodeficiency virus (HIV) inhibitory activities of
MIP-1 In this paper we describe the identification of two hMIP-1 Construction of Synthetic hMIP-1 Production of Recombinant Chemokine Proteins from S. cerevisiae--
S. cerevisiae strain MC2 was transformed
with DNA containing the wild-type and mutant chemokine genes by the
LiAc/polyethylene glycol protocol (44) and representative clones
identified. All yeast media were as described previously (45).
Transformant cultures were induced after 24 h of growth by
harvesting synthetic complete-Glu broth cultures and resuspension in
synthetic complete-Glu/Gal-broth. All cultures were tested for their
ability to express chemokine protein by reverse-phase HPLC analysis of
cell-free supernatants 2 days after induction. Clones were considered
expression-competent if a clear recombinant protein elution peak was
observed (approximately Native Polyacrylamide Gel Electrophoresis and Sedimentation
Equilibrium Analytical Ultracentrifugation (AUC)--
Nondenaturing
4-20% (w/v) gradient polyacrylamide gels (Novex) were run under
nondenaturing conditions in Tris-glycine buffer. See Blue markers
(Novex) and wild-type hMIP-1
AUC analysis was performed using a Beckman XLA analytical
ultracentrifuge with absorbance optics as described previously (15, 385). Protein samples at 0.1 or 0.5 mg/ml were loaded in a six-sector cell to enable simultaneous measurements and comparisons of variants. PBS "A" was loaded in the reference sector. Samples were analyzed at 15,000 rpm at 25 °C for 18 h to reach equilibrium. Final
solute distributions were recorded at 235, 280, and 295 nm as a
function of radius. Scans were repeated after a further 3 h of
centrifugation to confirm that equilibrium had been achieved. The final
solute distribution ASCII data was fitted using Microcal Origin
software to a model assuming a single ideal species (Ideal1) to provide an estimate of the weight average molecular weight. Data sets shown are
an average of 20 scans in 0.001-cm increments across the radius of the rotor.
Competitive Receptor Binding Assays--
Competitive receptor
binding assays using the FDCP-mix A4 cell line (Spooncer et
al. (37)) have been described previously (38). Briefly, 1 × 106 murine FDCP-mix A4 cells, various amounts of unlabeled
hMIP-1
Competitive receptor binding experiments on human CCR1 were performed
on HEK293 P4 cell membranes (19). Cell membranes (1 µg) were
incubated in 50 mM Tris-HCl pH 7.4 (250 µl), containing 0.05 nM iodinated hMIP-1 Calcium Mobilization Assay--
Human embryonic kidney cell
lines transfected with human CCR1 (P4 cell line, Ref. 19) or CCR5 (8.5 cell line (20)) receptors were propagated in Dulbecco's modified
Eagle's medium (Life Technologies, Inc.), 10% (v/v) fetal calf serum.
P4 cells were supplemented with 0.25 mg/ml hygromycin and 8.5 cells
with 1 mg/ml G418. Cells were harvested with trypsin, washed in growth
medium, and resuspended at 2-3 × 106 cells/ml.
Fura-2AM-loaded cells were washed and resuspended in Tyrodes buffer to
give 2-3 × 106 cells/ml. A Perkin-Elmer LS-50B
fluorimeter was used to measure Fura-2 fluorescence emission intensity.
Fura-2-loaded cells (2 ml, 2 × 106 cells/ml) were
transferred to a 4.5-ml UV grade cuvette (Fisons); CaCl2
was added to 1 mM and left to equilibrate for 2 min. The samples were excited at 340 nm with a 10 nm bandwidth and the emission
continuously recorded at 500 nm with a 5 nm bandwidth. Chemokines were
added (20 µl, 100 × final concentration) and the increase in
intracellular calcium noted (Hunter et al. (38)).
Chemotaxis Assay--
Cell migration was evaluated using 24-well
tissue culture plate inserts with polyethylene terephthalate membranes
(8-µm pore size, Falcon). Chemokines were diluted (10 Determination of hMIP-1
Homonuclear assignment was performed as described by Wüthrich
(47), and heteronuclear assignments were performed as reviewed by
Whitehead et al. (48). 15N relaxation data were
acquired as described by Kördel et al. (49).
Constraints for structure calculations were obtained from a 150-ms
homonuclear NOE experiment (50), a 150-ms simultaneous 15N/13C edited NOESY experiment (51), and a
150-ms double-filtered NOESY experiment. All data were collected and
analyzed using FELIX (Micron Separations, San Diego, CA). The
constraints were divided into three ranges with upper bounds of 2.5, 3.5, and 5 Å, based on a calibration from elements of regular
secondary structure. HIV-1 Infectivity Assays--
Neutralization was assessed using
PHA-stimulated PBMC as target cells with determination of soluble p24
antigen production as the end point (56). Chemokines were tested for
their ability to inhibit HIV infection by incubation of a known
concentration with 100,000 PBMC (mixed from two blood donors) in a
final volume of 75 µl of RPMI, 10% fetal calf serum, IL-2 (5 units/ml) for 2 h at 37 °C. Cells were infected with a known
infectious titer of virus, 100 TCID50, and the virus/ligand
incubation mixture incubated at 37 °C overnight. Cells were washed
the following day, the relevant concentration of chemokine added back
to all cultures, and incubated at 37 °C for 5 days.
Construction of a Library of hMIP-1 hMIP-1 Identification of Active Disaggregated hMIP-1 Substitution of Homologous Amino Acid Residues in hMIP-1
The biological activity of the disaggregated chemokines was assessed
using human embryonic kidney (HEK) cells expressing either human CCR1
or CCR5 (19, 20). Disaggregated chemokine variants retained their
ability to bind CCR1 (Fig. 3A)
and to induce signal transduction (Fig. 3B), indicating that
the substitutions did not grossly affect their biological activities.
hMIP-1 Structural Characterization of hMIP-1
The full 1H, 15N, and 13C
heteronuclear assignment of hMIP-1
The structures show that the monomer structures of hMIP-1 A Mechanism for CC Chemokine Disaggregation--
Mutagenesis
studies have identified two acidic residues at positions 26/27 and
66/67 of hMIP-1 Comparison of the HIV-1 Inhibitory Properties of Aggregating and
Nonaggregating Chemokines--
SF-2 (T-tropic, CXCR4 utilizing) and
SF-162 (M-tropic, CCR5 utilizing) viruses were used to evaluate the
specificity of chemokine-mediated inhibition of PBMC infection (Fig.
5A). HIV-1 infection was
quantified by measuring soluble core protein (p24) antigen production
by enzyme-linked immunosorbent assay. Both wild-type and disaggregated chemokines (0.1 µM) inhibited SF-162 infection of two
different donor PBMC cultures (Fig. 5A). All wild-type and
variant chemokines, at a concentration of 0.1 µM, had no
effect on the ability of SF-2 to infect PBMC cultures. Generally,
disaggregated variants substituted at residue 26/27 were less efficient
at neutralizing SF-162 infection than wild-type chemokines or variants
substituted at residue 66/67 (data not shown). Since chemokine
aggregation is concentration-dependent, we evaluated the
effect of higher concentrations of chemokines in the PBMC HIV-1
infection assay (Fig. 5B). Surprisingly, high concentrations
(1.0 µM and greater) of RANTES induced an increase in
extracellular p24 antigen from both SF-2- and SF-162-infected cultures.
Similar effects were not observed with cultures treated with either
hMIP-1 hMIP-1 We have explored the molecular basis for chemokine aggregation, using
hMIP-1 Using wild-type and disaggregated forms of hMIP-1 Our observations have important implications for the clinical use of
chemokines. RANTES may be a key regulator of HIV-1 infection; it is the
most potent chemokine inhibitor of M-tropic HIV-1 infection (32) but as
we show here can also act as a stimulator, enhancing viral infection.
Administration of RANTES to HIV-1 infected individuals could increase
HIV-1 replication and thus accelerate disease progression. In contrast,
administration of disaggregated RANTES is likely to suppress HIV
infection of new target cells if effective concentrations can be
achieved. Epidemiological analysis of correlations between HIV-1 viral
load and RANTES expression or inflammatory disease may be informative.
In conclusion, we have identified amino acid residues critical to CC
chemokine self-association and have identified a biological consequence
of RANTES aggregation. It is likely that there will also be activities
associated with hMIP-1, MIP-1
, and RANTES (regulated on activation
normal T cell expressed) self-associate to form high-molecular mass
aggregates. To explore the biological significance of chemokine
aggregation, nonaggregating variants were sought. The phenotypes of 105 hMIP-1
variants generated by systematic mutagenesis and expression
in yeast were determined. hMIP-1
residues Asp26
and Glu66 were critical to the self-association process.
Substitution at either residue resulted in the formation of essentially
homogenous tetramers at 0.5 mg/ml. Substitution of identical or
analogous residues in homologous positions in both hMIP-1
and RANTES
demonstrated that they were also critical to aggregation. Our analysis
suggests that a single charged residue at either position 26 or 66 is
insufficient to support extensive aggregation and that two charged
residues must be present. Solution of the three-dimensional NMR
structure of hMIP-1
has enabled comparison of these residues in
hMIP-1
and RANTES. Aggregated and disaggregated forms of hMIP-1
,
hMIP-1
, and RANTES generally have equivalent G-protein-coupled
receptor-mediated biological potencies. We have therefore generated
novel reagents to evaluate the role of hMIP-1
, hMIP-1
, and RANTES
aggregation in vitro and in vivo. The
disaggregated chemokines retained their human immunodeficiency virus
(HIV) inhibitory activities. Surprisingly, high concentrations of
RANTES, but not disaggregated RANTES variants, enhanced infection of
cells by both M- and T-tropic HIV isolates/strains. This observation
has important implications for potential therapeutic uses of chemokines
implying that disaggregated forms may be necessary for safe clinical investigation.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
(5), RANTES (6, 7), and MCP-3 (8) and
of the CXC chemokines PF4 (9), IL-8 (10), MGSA (11), and SDF-1 (12)
have been reported. Monomeric chemokine secondary structures are
similar, comprising a triple-stranded antiparallel
-sheet arranged
in a Greek key motif and a C-terminal
-helix. Despite conservation
of secondary structure, two alternative forms of chemokine dimers have
been described. Typically, CC chemokines like MIP-1
are elongated,
whereas CXC chemokines like IL-8 form globular dimers. Exceptions are
the CC chemokine MCP-3, which can form the globular IL-8 type dimer,
and the CXC chemokine PF4, which forms tetramers.
, MIP-1
, and RANTES (13, 14)
self-associate to form high-molecular mass aggregates (6, 15). Not all
chemokines self-associate, leading us to question whether self-association has any biological significance. Such self-association limits the range of purification and formulation conditions available for the clinical development of these proteins. Furthermore, the interpretation of bioassays is complicated, since different protein preparations may vary in their extent of aggregation (16-18). The primary goal of this work was to identify biologically active variants
of hMIP-1
that no longer self-associated, thereby allowing large
scale production of protein for preclinical and clinical investigation.
is a multifunctional CC chemokine that binds with high
affinity to chemokine receptors CCR1 and CCR5 (19, 20). The early
description of MIP-1
as a proinflammatory cytokine (21) has not been
confirmed and it has not restricted its use in a number of in
vitro (22) and in vivo models (23, 24). hMIP-1
has a
number of potential clinical uses: as a myeloprotectant during
aggressive cytotoxic therapy (25-27), for the treatment of psoriasis
(28), for stem cell mobilization (24, 29), and for ex vivo
hematopoietic stem cell expansion (30).
, MIP-1
, and RANTES (31, 32) and the identification of
chemokine receptors as the essential co-factors for HIV infection of
CD4+ cells (33, 34) suggested that chemokines, or small
molecule mimics, could be considered as potential antiviral agents. HIV is generally classified according to its ability to replicate in
macrophages (M-tropic) or established T-cell lines (T-tropic). Viruses
isolated over the course of disease progression have been reported to
change their in vitro properties from M- to T-tropic, suggesting that M-tropic (CCR5-utilizing) viruses are responsible for
infection and the T-tropic (CXCR4-utilizing) viruses may be associated
with the rapid CD4+ cell decline and onset of symptoms
(35). Thus a second goal of our study was to compare the antiviral
properties of aggregating and nonaggregating hMIP-1
, hMIP-1
, and
RANTES variants.
amino
acid residues critical for aggregation, substitution of which yielded
biologically active disaggregated proteins. Furthermore, we show that
substitution of the homologous residues in hMIP-1
and RANTES
inhibits their aggregation without loss of bioactivity. Solution of the
three-dimensional structure of hMIP-1
by NMR has enabled comparison
of these residues in hMIP-1
, hMIP-1
, and RANTES. The
G-protein-coupled receptor-mediated biological activities of the
disaggregated chemokines, including inhibition of HIV-1 infection, are
unaffected by these substitutions. However, RANTES was found to
stimulate M- and T-tropic HIV infection in a
concentration-dependent manner. In contrast
nonaggregating RANTES did not enhance HIV-1 infection.
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
, hMIP-1
, and RANTES Genes,
Yeast Expression Vectors, and Mutants--
Synthetic genes for
hMIP-1
, hMIP-1
, and RANTES were designed by back-translation from
their protein sequences with codon usage optimized for
Saccharomyces cerevisiae (41). The genes were divided into
10 oligonucleotides (synthesized by R+D Systems Ltd.), and the
oligonucleotides were annealed and cloned into M13 vectors. M13 clones
were used as templates for mutagenesis to create the hMIP-1
D27A,
hMIP-1
E67S, RANTES E26A, RANTES E66S, and the library of hMIP-1
mutants. Mutagenesis was performed according to the method of Kunkel
et al. (42). Oligonucleotides used for mutagenesis were
17-36 nucleotides in length (41). Dideoxy sequencing was used to
identify the correct clone for each variant in the M13 vectors. The
wild-type and variant genes were transferred as
HindIII/BamHI DNA fragments into the yeast expression vector pSW6 to drive protein expression under the control of
the inducible galactose promoter (43). Recombinant pSW6 clones containing the mutated chemokine genes were sequenced using the dideoxy
sequencing method for double-stranded DNA.
20% of wild-type chemokine yield). Three
days after induction the proteins were purified from the yeast culture
supernatants according to previously published methods (15). Both
ion-exchange and reverse-phase HPLC chromatography were used to obtain
the protein at 95% purity.
(2 µg) were analyzed on each gel. All
variants were scored according to their mobility relative to the
wild-type protein.
or variants, and 3.85 ng/ml 125I-hMIP-1
(250 µCi/µg; Amersham Pharmacia Biotech) were incubated in a total
volume of 250 µl of binding medium (RPMI 1640, 20 mM HEPES, 1 mg/ml bovine serum albumin) at 25 °C for 60 min.
Phosphate-buffered saline (1 ml) was added, mixed, and the cells
harvested by centrifugation. The supernatants were removed and the
cells washed twice with PBS. Radioactivity in the cell pellet was
determined and IC50 values for the variants estimated.
(Amersham Pharmacia Biotech)
and various concentrations of unlabeled hMIP-1
mutant proteins for
30 min at 37 °C. The membranes were harvested onto Whatman GFB
filters (presoaked in 0.3% polyethyleneimine) on a Brandel harvester
and washed with 5 × 1 ml of ice-cold wash buffer (50 mM Tris-HCl, 0.5 M NaCl, 5 mM
MgCl2, 1 mM CaCl2, 0.05% bovine
serum albumin, pH 7.4). Filters were counted on a Beckman Cobra counter
and the data fitted using Prism (GraphPad Software, Inc., San Diego,
CA) and IC50 values determined automatically.
7
to 10
12 M) in fetal calf serum-free RPMI 1640 medium and 0.5 ml of each dilution placed into the companion plate.
Freshly purified human mononuclear cells were suspended at 2.5 × 106 cells/ml in fetal calf serum-free RPMI 1640 and 0.2 ml
placed onto the insert filters prior to setting the inserts into the wells. The filters were removed after 2-h incubation, the medium discarded, and the filters dried. The cells were fixed in 4% (v/v) formaldehyde for 10 min, washed in PBS, stained with 2% (w/v) cresyl
violet acetate and washed in PBS. Migration was assessed by averaging
the count from two high-powered fields (× 400 magnification) per assay
point in each experiment. The data presented are the mean number of
cells/high-powered fields from four independent experiments.
Three-dimensional Structure by Nuclear
Magnetic Resonance
Spectroscopy--
15N/13C-labeled hMIP-1
and hMIP-1
D26A was prepared by established methodology using the
Pichia pastoris expression system (46). Minor modifications
reduced the concentration of [13C]methanol to 4% (w/v)
used in the induction to reduce costs. hMIP-1
D26A was purified from
the culture supernatant by sequential cation exchange and reverse-phase
high-pressure liquid chromatography. Samples for NMR were at ~3
mM concentration, pH 3.5, 10% (v/v) D2O. All
spectra were collected at 318 K.
constraints were obtained from an
heteronuclear single quantum coherence-J experiment (52), constraining
to either
60 (±40)o or
120 (±50)o.
The structure calculation protocol consisted of calculation of
approximate monomer structures, followed by filtering out of probable
dimer NOEs using a filter cutoff of 10 Å. This process was cycled
several times. Dimer NOEs were also checked against the double filtered
NOESY experiment, although this was insufficiently sensitive to detect
all NOEs, and acted mainly as a rigorous check that the correct dimer
interface was present. The monomer structures were oriented as rigid
entities via the dimer NOEs while being maintained at a separation of
100 Å. The monomers were then annealed together and the structures
refined by further annealing cycles. All calculations were performed
using modified variants of the protocol of Nilges et al.
(53), using XPLOR (54). Fig. 4, A and B, were
created using RASMOL (55). An ensemble of 10 structures and a minimized
mean structure of hMIP-1
D26A have been submitted to the Brookhaven
Protein Data Bank, entries 1B50 and 1B53, respectively.
RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
Variant Proteins--
105
single amino acid substituted variants of hMIP-1
were generated by
oligonucleotide site-directed mutagenesis (Fig.
1A). All mutations were
verified at the M13, yeast expression vector, and postexpression stages
by DNA recovery and sequencing (data not shown). Initially, charged
residues implicated in the aggregation process were mutated; however,
the entire molecule was screened such that all hydrophilic residues
were mutated to serine and all hydrophobic residues to alanine.
Sixty-five of the 69 hMIP-1
residues were substituted, and the four
cysteine residues were unmodified to retain structural integrity.
Expression of 75% of the mutants was comparable with that of
hMIP-1
, allowing purification of protein with substitutions at 55 of
the 65 available residues.
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Fig. 1.
Identification of fully active disaggregated
mutants of hMIP-1 . A, the
number of single amino acid substitutions generated at each residue in
hMIP-1
. For a full description of all substitutions see Craig
et al. (41). The number of alternative amino acid
substitutions at each residue, which expressed well (
20% hMIP-1
),
is shown above the origin and those which expressed poorly (
20%
hMIP-1
) below the origin. Cysteine residues at amino acid positions
10, 11, 34, and 50 were not mutated to retain structural integrity.
B and subsequent panels refer to amino acid residues at
which the described properties have been identified are shown with a
tall histogram and variants which were assayed for a property but did
not meet the criteria (e.g. they were not disaggregated or
they were less potent) are shown by short histograms. B,
variants that expressed well and were disaggregated according to native
polyacrylamide gel electrophoresis. Disaggregated variants migrated
substantially further into the gel than hMIP-1
, which remained near
the well. C, variants that were disaggregated according to
sedimentation equilibrium AUC analysis. Disaggregated variants
possessed weight average molecular weights
100,000 Da. D,
disaggregated variants that retained full competitive receptor binding
activity on FDCP-mix A4 cells. Fully active variants were defined as
those with IC50 values
8 nM in this
cellular assay.
Self-association--
Substantial changes in the
self-association of the hMIP-1
variant proteins were initially
detected by native polyacrylamide gel electrophoresis. Of the 79 variants analyzed, 37, representing 28 residues, migrated more rapidly
through the gel than hMIP-1
(Fig. 1B). Sedimentation
equilibrium AUC analysis was used to provide a quantitative assessment
of the effects of the substitutions on hMIP-1
self-association (36).
Twenty-three of the 37 variants analyzed had substantially reduced
(<100 kDa) weight average molecular weights than hMIP-1
(>100
kDa). These variants represented 16 positions in hMIP-1
at which
substitution leads to significant disaggregation (Fig. 1C).
Native gel electrophoresis appeared to enhance the effects of
substitution on hMIP-1
variants, implying greater effects on
self-association than observed by AUC analysis. The high glycine
concentration in the gel may be responsible for the differences between
the two assays. The AUC analysis is generally accepted to be a
preferable method of analysis of self-association.
Variants--
The
activity of the disaggregated hMIP-1
variants was assessed in a
receptor binding assay using the murine cell line, FDCP-mix A4 (37,
38), which binds and responds to hMIP-1
. Nineteen variants,
representing substitution at 16 residues, were assessed for their
ability to bind the murine MIP-1
receptor expressed by these cells
in a competitive receptor binding assay. The concentration of hMIP-1
or variants required to inhibit iodinated hMIP-1
binding by 50%
(IC50) was estimated. hMIP-1
had an IC50 of
3.27 ± 0.34 ng/ml (mean ± S.E.; n = 81). A
range of hMIP-1
variant receptor binding activities was observed
from wild-type to IC50 values
100 nM.
For the purposes of screening, variants with mean IC50 values
8 nM were considered to be fully active as
they fall within the 99.99% confidence interval for wild-type
hMIP-1
activity (Fig. 1D). The four most active
disaggregated variants representing two residues of hMIP-1
(Asp26 and Glu66) were essentially homogeneous
tetramers at 0.5 mg/ml. Three alternative substitutions D26A, D26S, and
D26Q gave rise to the same properties.
,
hMIP-1
, and RANTES Produces Active Disaggregated
Variants--
Alignment of the protein sequence of hMIP-1
with the
structurally related chemokines hMIP-1
and RANTES reveals homologous charged amino acid residues that may play similar roles in the self-association pathways of these chemokines (Table
I). Substitution of residues D27A and
E67S in hMIP-1
and E26A and E66S in RANTES was performed to
investigate the aggregation and biological properties of the variants.
Aggregation of RANTES was so pronounced that at 0.5 mg/ml the estimate
of weight average molecular weight was unreliable, and therefore a
lower concentration of 0.1 mg/ml was used in these comparisons. AUC
analysis indicated that residues 26 and 66, which are critical for
hMIP-1
self-association, also play a key role in the
self-association of hMIP-1
and RANTES (Fig.
2). Substitution of hMIP-1
at either
residue 26 or 66 resulted in the formation of an essentially
nonassociating protein solution. Substitution at residue 67 in
hMIP-1
was more effective than substitution at residue 27, although
both variants continued to self-associate, reaching a lower weight
average molecular weight than wild-type hMIP-1
. Substitution at
residue 66 in RANTES was more effective than that at residue 26, resulting in the formation of an essentially nonassociating RANTES
solution. The lowest disaggregated chemokine weight average molecular
weights were consistent with dimeric proteins at 0.1 mg/ml.
Amino acid alignment of hMIP-1, hMIP-1
, and RANTES
(38), hMIP-1
, and RANTES is
shown. The positions of the key acidic residues at 26/27 and 66/67 are
boxed.
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Fig. 2.
Sedimentation equilibrium AUC analysis of
hMIP-1 , hMIP-1
,
RANTES, and disaggregated chemokine variants. Comparison of the
distribution of wild-type and disaggregated chemokine proteins in the
ultracentrifuge cell at equilibrium. All samples were prepared at 0.1 mg/ml and centrifuged at 15,000 rpm. Absorbance was estimated at three
wavelengths and the most complete data set used for analysis. The
solid line depicts the best fit of the data assuming an
ideal nonassociating protein. The straighter the distribution line and
the more random the scatter of the residuals, the closer the protein
solution is to a nonassociating ideal species. Curvature in the
distribution data or the residuals indicates a self-associating protein
species. Weight average molecular weights for the proteins are
shown.
and its disaggregated mutants are not agonists of CCR1, but
do signal effectively via CCR5. Furthermore, the human mononuclear cell chemotactic activity was not affected by disaggregating substitutions (Fig. 3C).
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Fig. 3.
hMIP-1 ,
hMIP-1
, and RANTES G-protein-coupled receptor
biology is unaffected by the disaggregating substitutions.
A, CCR1 binding. A CCR1-expressing HEK293 cell
membrane-based receptor binding assay was used to evaluate competitive
receptor binding potency. Displacement of 125I-hMIP-1
by
hMIP-1
, hMIP-1
, RANTES, and their disaggregated variants is shown
(mean ± S.E.; n = 3). B, calcium
mobilization via CCR1 and CCR5. HEK293 cells expressing CCR1 or CCR5
were incubated with Fura-2AM and the transient calcium flux induced by
chemokines (100 nM) assessed. Typical data are shown.
C, human mononuclear cell chemotactic activity. Cell
migration in response to chemokines or disaggregated variants was
assessed after 2 h. The mean number of cells per high power field
from four independent experiments were assessed. Data are presented as
migration index, reflecting the number of cells migrating in the
presence of chemokine divided by the number migrating in untreated
controls.
and hMIP-1
D26A by
NMR--
The structures of hMIP-1
and hMIP-1
D26A were
investigated by NMR. In order to assess whether gross structural
changes accompanied the substitution of D26A, backbone 1H
and 15N assignments were also completed for hMIP-1
. The
chemical shift changes between the wild-type and D26A forms are
extremely small, being less than 0.1 ppm for
or amide protons
except adjacent to the site of the mutation. Apart from the mutated
residue itself, the greatest shifts are 0.12 ppm for the amide proton
of the subsequent residue Tyr27 and for the residues across
the
-sheet from residue 26, namely Phe41 (
proton
shift difference of 0.09 ppm) and Leu42 (amide proton shift
difference of 0.12 ppm). These chemical shift changes are much smaller
than would be expected for a major structural disruption where shift
changes of up to 2 ppm would be likely.
D26A was completed, which, in
conjunction with heteronuclear edited NOESY spectra, allowed the
calculation of an ensemble of solution state structures. The
experimental data for the structure calculation included a total of 821 NOE constraints and 29
angle constraints. The NOE constraints break
down into 306 intraresidual, 211 sequential, 98 medium range, and 172 long range monomer NOEs and 34 intermonomer NOEs (these figures count
each NOE only once). A total of 30 structures were calculated, and the
10 with the lowest energy were selected to represent the ensemble of
properly converged structures. The root mean square deviation for the
overlay of individual monomers onto the average structure for residues
15-29 and 39-63 is 0.7 Å for backbone atoms and 1.2 Å for all heavy
atoms. The remaining regions of the protein showed a much higher root
mean square deviation. 15N relaxation data indicated that
significant subnanosecond time scale motions occur for residues 1-4
and 67-69. The higher root mean square deviation for residues 5-15
and the loop from 30-38 are for residues in the dimer interface and
probably reflect the difficulty in properly defining the relative
orientation of the two monomers in the dimer. This is reflected in the
overlay of backbone heavy atoms of the second monomers in the ensemble
of dimers of ~12 Å when only the first monomer in each dimer
structure is overlaid. This introduces a consequent uncertainty in the
dimer interface region. This variability in the dimer structure may represent true flexibility or may simply represent a limitation of
current NMR methodology. That such uncertainty is reasonable is
supported by a comparison of the two published RANTES structures (6,
7). Comparison of the monomers from these structures shows a similar
discrepancy between the average structures that is similar to that
found in our ensemble of structures. In particular, the structures
disagree on the positions of the N-terminal region and the 30-s loop.
The relative orientations of the monomers in the dimer structures
reported for RANTES show a similar corresponding discrepancy. In MCP-1,
a similar variability in dimer structure has been reported between
dimers from two crystal forms crystallized from the same drop (39).
This indicates that in MCP-1 the dimer relationship is not rigidly
fixed. The difficulty of properly defining the dimer led us to
disregard NOEs that could not be clearly ascribed to either inter- or
intramonomer contacts in order not to falsely constrain the ensemble of structures.
D26A (and
of hMIP-1
on the basis on the small chemical shift differences) are
similar to those of hMIP-1
, RANTES, and MCP-1. An example overlay of
the backbone atoms of hMIP-1
and hMIP-1
is shown in Fig.
4A.
View larger version (52K):
[in a new window]
Fig. 4.
Three-dimensional structure of
hMIP-1 D26A and comparative positions of amino
acid residues in hMIP-1
,
hMIP-1
, and RANTES. A, stereo
view of the overlay of the monomer backbone structures of hMIP-1
D26A and hMIP-1
. The regions 1-15, 31-38, and 67-69, which are
discussed in the text, are shown as a thin trace, whereas
the rest of the backbone is shown as a thick trace.
B, from left to right, space-filling
representations of the three-dimensional NMR structures of hMIP-1
D26A, RANTES (6), and hMIP-1
(5) are shown to illustrate the
relative positions of the key acidic amino acid residues involved in
chemokine self-association at positions 26 and 66 (27 and 67 in
hMIP-1
) shaded in dark gray. The positions of
the basic residues in the 44, 45, and 47 positions (positions 45, 46, and 47 in hMIP-1
), which we speculate may be involved in charge
interactions leading to self-association are shaded in
light gray. The terminal residues 1-15 and 67-69 are not
shown in this figure.
, hMIP-1
, and RANTES that are critical for
self-association, and yet mutation of these residues does not affect
the biological activities of these molecules. Substitution at residue
66/67 is more effective at disaggregating the chemokines than are
changes at position 26/27. These key residues must be involved in
interactions that stabilize the multimeric form. Analysis of the
three-dimensional structures of D26A hMIP-1
, hMIP-1
, and RANTES
is complicated by the necessity to generate structures under
nonaggregating acidic conditions, which abolish any interactions that
the 26/27 and 66/67 residues may normally have. Although the structures
show residues 26/27 and 66/67 to be close together in the monomer, it
is not obvious how they interact, if at all (Fig. 4B). We
note that the amide proton of the intervening residue Tyr27
shows protection in hMIP-1
D26A, even though it is apparently solvent exposed and not clearly involved in any hydrogen bonding. Similar protection was observed in RANTES, which was thought to arise
from interaction with the side chain of Asp26 (7). This
cannot be the case with hMIP-1
D26A, and we cannot currently offer an
alternative explanation. The most likely interaction for these acidic
residues would be with basic residues. Substitution of basic residues
at hMIP-1
positions Lys44, Arg45, or
Arg47 effectively inhibited aggregation but also reduced
biological activity (Fig. 1), such that we have been unable to identify
direct contacts for residues 26/27 and 66/67. The interaction of
dimers, in the formation of tetramers, may bring acidic and basic
regions together to stabilize the multimer. Charge removal or reversal would destabilize the tetramer and result in disaggregation.
Mutagenesis of mMIP-1
, substituting up to three C-terminal acidic
residues, also resulted in substantial disaggregation implicating the
C-terminal
-helix in multimer formation (18). It is possible that
the position and/or flexibility of the C-terminal
-helix is critical to self-association and that substitution at residues 26/27 or 66/67
subtly alters its position or flexibility, thereby decreasing the
stability of multimers.
or hMIP-1
. The eight-fold increase in p24 antigen release
was accompanied by a 10-fold increase in infectious virus titer, as
determined by the titration of extracellular virus on U87.CD4 cells
expressing either CCR5 or CXCR4 co-receptors (data not shown). It is
striking that stimulation was not observed in cultures treated with the disaggregated variants of RANTES.
View larger version (51K):
[in a new window]
Fig. 5.
Chemokine-mediated neutralization of HIV
infectivity. A, PHA-stimulated peripheral blood
mononuclear cells were incubated with 0.1 µM wild-type
and dissagregated chemokine variants for 2 h prior to infection
with a 100 TCID50 of HIV isolates SF-2 (T-tropic) or SF-162
(M-tropic). Cultures were incubated for 18 h, washed, and
replenished with fresh medium containing the relevant chemokine. Five
days postinfection, the extracellular supernatant was harvested and the
levels of HIV-1 p24 core antigen present determined. B,
PHA-stimulated peripheral blood mononuclear cells were incubated with
increasing concentrations of hMIP-1 , hMIP-1
, RANTES, and
disaggregated RANTES mutants E26A and E66S and infected with 100 TCID50 of SF-2 and SF-162. Cultures were incubated for
18 h, washed, and replenished with fresh medium containing the
relevant chemokine. Five days postinfection, the extracellular
supernatant was harvested and the levels of HIV-1 p24 core antigen
present determined.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
, hMIP-1
, and RANTES are CC chemokines that tend to
self-associate, forming high molecular mass aggregates. This
self-association is a dynamic, reversible process, and it is generally
accepted that chemokine concentrations in vivo may be too
low to encourage such aggregation. However, there are natural
circumstances that may cause high local concentrations of chemokines.
These include platelet degranulation, in inflammatory disease (40), and
accumulation on cell membranes via receptors or glycosaminoglycans. In
addition, should chemokines be used clinically, their concentration
will be high at the injection site, encouraging self-association and precipitation which may reduce tissue penetration and induce
inflammation. We believe that CC chemokine self-association must be
fully characterized and its in vivo relevance determined if
the immunomodulatory properties of these chemokines are to be fully understood.
as a model system, and have also investigated the aggregation
of hMIP-1
and RANTES. Extensive mutagenesis and biophysical
characterization, comprising the production of 105 single amino acid
substituted variants of hMIP-1
identified two residues,
Asp26 and Glu66, as the key elements to the
self-association process. Identical or analogous residues were found in
homologous positions in both hMIP-1
and RANTES, suggesting that
their substitution might affect self-association. Substitution of
Asp27 and Glu67 in hMIP-1
and
Glu26 and Glu66 in RANTES had similar effects,
substantially disaggregating the chemokines and resulting in the
production of proteins with greatly improved solution properties. Our
analysis suggests that a single charged residue at either 26/27 or
66/67 is insufficient to support extensive self-association and that
two charged residues must be present. Data presented here and elsewhere
demonstrate that both aggregating and disaggregated forms of hMIP-1
generally have equivalent biological potencies (18, 22, 38).
Disaggregated hMIP-1
and RANTES variants retain the biological
activities and receptor selectivities associated with the aggregating
forms. We have therefore generated novel reagents to evaluate the role of hMIP-1
, hMIP-1
, and RANTES self-association in
vitro and in vivo. Since the GPCR-mediated activities
of these chemokines-receptor binding, signal transduction, and
chemotaxis are unaffected by the substitutions which disaggregate the
chemokines, self-association is probably irrelevant to these
activities. If this proposition is true, any biological differences
observed between the wild-type chemokines and their disaggregated
variants are probably due to their differences in their
self-association and probably act via non-GPCR-mediated events.
, hMIP-1
, and
RANTES we have shown that the effects of chemokines on HIV-1 infection
are concentration-dependent. At lower concentrations (
100
nM) hMIP-1
, hMIP-1
, and RANTES inhibit infection of
PBMC of SF-162 virus, but at higher concentrations (
1000
nM) RANTES enhances infection by both SF-2 and SF-162
viruses. Interestingly, disaggregated RANTES variants failed to enhance
HIV-1 infection, instead acting as effective inhibitors of M-tropic
HIV-1 infection at all concentrations tested. Since all of the other
biological activities tested were normal, we conclude that RANTES
aggregation is directly responsible for this stimulation of HIV-1
infection. We speculate that this stimulation is not mediated by GPCRs
and currently consider interactions with glycosaminoglycans to be a
potential mechanism of RANTES-mediated viral stimulation.
and hMIP-1
aggregation that will be
revealed only by use of the relevant assay system. Nonaggregating
variants of hMIP-1
, hMIP-1
, and RANTES may be essential for the
safe clinical evaluation of these chemokines. hMIP-1
D26A, also
known as BB-10010, has proven to be safe and well tolerated in phase I
and phase II clinical trials even after large doses (300 µg/kg) have
been administered by subcutaneous injection (25-27, 38).
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ACKNOWLEDGEMENTS |
---|
We thank our many departmental colleagues for support and help, especially the technical expertise of Julie Lewis, Sue Hodgson, Richard Marcus, and Ingrid Holme. We thank the Biotechnology and Biological Sciences Research Council and the Welcome Trust for instrumentation in Sheffield and Micron Separations for provision of FELIX software.
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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.
The atomic coordinates and structure factors (codes 1B50 and 1B53) have been deposited in the Protein Data Bank, Brookhaven National Laboratory, Upton, NY.
a To whom correspondence should be addressed. Tel.: 44-1865-748747 (ext. 2240); Fax: 44-1865-781034; E-mail: czaplewski{at}britbio.co.uk.
c Supported by the Lister Institute for Preventive Medicine and the Medical Research Council.
e Supported by the Wellcome Trust.
g Current address: Dept. of Biochemistry, 80 Tennis Court Rd., Cambridge CD2 1GA, UK.
h Current address: Zeneca Pharmaceuticals, Mereside, Alderley Park, Macclesfield SK10 4TG, UK.
i Current address: OSIRIS Therapeutics Inc. 2001 Aliceanna St., Baltimore, MD 21231-2001.
j Current address: The Beatson Institute for Cancer Research, Switchback Rd., Bearsden, Glasgow G61 1BD, UK.
k Current address: Oxagen, 91 Milton Park, Abingdon OX14 4RX, UK.
l Current address: Dept. of Biochemistry and Genetics, University of Newcastle, Newcastle NE2 4HH, UK.
m Current address: Murex Biotech Ltd., Central Rd., Dartford, DA1 5LR, UK.
n Current address: Prolifix Ltd., 91 Milton Park, Abingdon OX14 4RY. UK.
o Current address: School of Biological Science, University of Manchester M13 9BT, UK.
p Current address: Cambridge Antibody Technology, The Science Park, Melbourn SG8 6JJ, UK.
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ABBREVIATIONS |
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The abbreviations used are: GPCR, G-protein-coupled receptor; MIP, macrophage inflammatory protein; RANTES, regulated on activation normal T-cell expressed; IL, interleukin; HIV, human immunodeficiency virus; HPLC, high performance liquid chromatography; AUC, analytical ultracentrifugation; PBS, phosphate-buffered saline; FDCP, Factor-dependent cell lines, Paterson; BSA, bovine serum albumin; NOE, nuclear Overhauser effect; NOESY, nuclear Overhauser effect spectroscopy; PBMC, peripheral blood mononuclear cell.
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REFERENCES |
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