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Research Communications |
chemokine binding
Department of
a Pathology,
b Centre for Protein Technology, and
c Centre for HIV Research, University of Edinburgh, U.K.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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. The peptide also inhibits infection of primary
macrophages by M-tropic HIV-1. Thus, 3.7 is a prototype candidate
peptide for a vaccine against HIV-1 and represents a novel approach to
the rational design of peptides that can mimic complex sequence
discontinuous ligand binding sites of clinically relevant
proteins.—Howie, S. E. M., Fernandes, M. L., Heslop,
I., Hewson, T. J., Cotton, G. J., Moore, M. J., Innes,
D., Ramage, R., Harrison, D. J. A discontinuous HIV-1 gp120 C3/C4
domain-derived, branched, synthetic peptide that binds to CD4 and
inhibits MIP-1 chemokine binding.
Key Words: antibody • macrophage • PBMC • monoclonal antibody • CDR2 region
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Based on the peptide sequence of HIV-1 IIIB gp120, we have previously
described the synthesis of a novel 44-mer three-armed, branched peptide
[3.7] 11, 12)
containing four residues necessary for
CD4 binding (Asp-368 and Glu-370 from C3; Trp-427 and Asp-457 from C4),
an oxidized Cys-Cys turn based on the disulfide link between Cys-378
and Cys-445, and two residues, Lys-421 and Gln-422, involved in
M-tropism and the CCR5 binding site 9, 10)
. Peptide 3.7
has a unique structure that could not be reproduced by conventional
genetic engineering. This rationally designed peptide contains both T
and B lymphocyte epitopes, cross reacts with polyclonal anti-gp120
antiserum, binds to the CDR2 region, domain 1 of CD4, and inhibits
macrophage inflammatory protein-1 (MIP-1) chemokine binding and
infection of primary macrophages by M-tropic HIV-1. Apart from the
relevance to HIV-1, this work also represents a generic approach to the
rational design and synthesis of complex peptides with functional
biological properties.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Antibodies
Antipeptide polyclonal mouse serum was raised as described
previously 11, 12)
. The immunoglobulin G (IgG) fraction
was purified using protein G-Sepharose (Pharmacia Biotech Ltd., St.
Alban's, U.K.) in a 0.7 x 10 cm liquid chromatography column
(Sigma, Poole, Dorset, U.K.) according to the manufacturer's protocol.
Anti-CD4 monoclonal antibodies (QS4120 and L120) and sheep anti-gp120
serum (ARP411) were supplied by the NIBSC centralized facility for AIDS
Reagents supported by EU programme EVA (contract BMH4 97/2515) and the
U.K. Medical Research Council; biotinylated-anti-CD4 (MT310), and
rhodamine (TRITC) -labeled antimouse immunoglobulin were obtained from
Dako Ltd., Cambridge, U.K.; phycoerythrin (PE) -labeled goat antimouse
immunoglobulin, horseradish peroxidase-conjugated, and alkaline
phosphatase-conjugated-donkey antisheep serum were purchased from
Sigma.
ELISA
Unless otherwise stated, all reagents were purchased from Sigma.
96-Well ELISA microtiter plates (Corning-Costar Ltd., High Wycombe,
Bucks, U.K.) were coated overnight at 4°C with 3.7 peptide, FMDV
peptide, bovine serum albumin (BSA), or baculovirus expressed
recombinant gp120 derived from the HIV-1 IIIB strain (EVA607 supplied
by the NIBSC centralized facility for AIDS Reagents supported by EU
programme EVA (contract BMH4 97/2515) and the U.K. Medical Research
Council) (100 µl/well) in 0.1 M carbonate/bicarbonate buffer pH 9.6.
The plate was then washed three times with phosphate-buffered saline
(PBS) containing 0.05% Tween 20. Wells were blocked with 1% BSA in
PBS for 1 h at room temperature. After three additional washes, a
1:500 dilution of sheep anti-gp120 serum (100 µl per well, diluted
with 1% BSA in PBS containing 0.05% Tween 20) was added to the wells
and incubated for 2 h at room temperature. The plate was again
washed; optimal dilutions of secondary antibody (horseradish
peroxidase-conjugated or alkaline phosphatase-conjugated donkey
antisheep serum) were added (100 µl per well) and incubated for
1 h at room temperature. Unbound conjugate was removed by washing;
o-phenylenediamine (0.4 mg/ml in phosphate/citrate buffer pH
5.0 containing 0.006% H2O2, 100 µl per well)
or 3 M p-nitrophenyl phosphate (in 0.05 M
Na2CO3, 0.5 mM MgCl2) was added and
the plate was incubated at room temperature. The coloration reaction
was measured at 490 nm for o-phenylenediamine or at 405 nm
for p-nitrophenylphosphate using a Dynatech MR5000
microplate reader.
Cell culture
All tissue culture reagents and plastics were purchased from
Life Technologies Ltd., Paisley, U.K., unless otherwise stated. The
human T lymphocyte-derived cell line H9 was obtained from the European
Collection of Animal Cell Cultures, Porton Down, Salisbury, U.K. The
human monocyte/macrophage-derived cell line MM6 was the kind gift from
Dr. J. A. Ross, Department of Surgery, University of Edinburgh.
Cells were passaged in RPMI 1640 medium containing 10% (vol/vol) fetal
calf serum (FCS), 2 mM L-glutamine, and antibiotics (50 IU/ml of
penicillin, 50 µg/ml of streptomycin) with the addition of 2.5
µg/ml fungizone for H9 cells.
Peripheral blood-derived macrophages were obtained from single donor Buffy-coat preparations obtained from the Scottish National Blood Transfusion Service. Peripheral blood mononuclear cells (PBMC) were isolated by centrifugation over lymphoprep (Nycomed Pharma AS, Oslo, Norway) and washed in PBS. PBMC were plated into a 24-well plate at 5 x 106/well in Iscove's medium containing antibiotics as described above and allowed to adhere to the wells for 1 h. Nonadherent cells were then removed, the wells were washed, and 1 ml Iscove's medium containing antibiotics and 5% heat-inactivated human AB serum (Scottish National Blood Transfusion Service) were added. The cells were cultured overnight and any remaining nonadherent cells were removed. Adherent cells were cultured for another 4 days (at which point they were >95% CD14+ve, MHC II+ve and CD4+ve macrophages by flow cytometry) before infection.
Colocalization of CD4 and 3.7 on the cell surface
MM6 cells were washed three times in PBS, plated in a microtiter
plate at a concentration of 3 x 105 cells/well with
or without 1 µg/well of 3.7 and incubated on ice for 2 h. The
wells were washed with prechilled binding buffer (1 mg/ml GMEM, 10%
FCS, 1 mg/ml HEPES in distilled H2O, pH 7.2) and incubated
with or without 25 µl/well purified mouse antipeptide IgG (2.5 µg)
on ice for 1 h. The wells were washed as above and bound
antipeptide antibody was detected using TRITC-labeled antimouse
immunoglobulin. After further incubation on ice for 30 min and at room
temperature for 15 min, cells were washed with prechilled buffer (PBS,
1% BSA, 0.05% NaN3). Biotinylated mouse antihuman-CD4 mAb
was then added and the plate incubated for an additional 0 min on ice,
washed with flow buffer, and detected with fluorescein isothiocyanate
(FITC) -labeled avidin (Sigma). Cells were fixed in 0.4% formaldehyde
and then transferred to slides with a single drop of glycerol/PBS
before examination under a Zeiss confocal laser scanning microscope.
Flow cytometry
Flow cytometric analysis was carried out using a Coulter EPICS
XL Flow Cytometer (Beckmann-Coulter Electronics, Luton, U.K.) with a 15
mW, single argon ion laser operating at wavelength 488 nm. FITC and PE
fluorescence were detected depending on the individual experiment. The
percentage of positive cells was established relative to background
fluorescence of cells treated with FITC-labeled avidin or PE-labeled
goat antimouse immunoglobulin only. Relative intensities of cell
surface staining were determined by comparing the mean fluorescence
intensity of cell staining within individual experiments.
Anti-CD4 mAb binding
To detect anti-CD4 mAb binding, viable H9 T cells were isolated
by gradient centrifugation on Lymphoprep (Nycomed Pharma AS, Oslo,
Norway) at 1000 x g for 25 min and washed three times
in PBS. Cells were then pelleted in a 96-well microtiter plate at a
concentration of 105 cells/well and 10 µl of 20 nM
peptide was added. After incubation for 2 h on ice, 10 µl
containing 1 µg anti-CD4 mAb was added. After further incubation on
ice for 1 h, wells were washed with prechilled flow buffer (PBS,
1% BSA, 0.05% NaN3) and bound mAb was detected with
PE-labeled goat antimouse immunoglobulin (Dako Ltd.). After incubation
for 1 h on ice, the wells were washed in prechilled flow buffer
and resuspended in 400 µl prechilled flow buffer in scintillation
tubes for analysis.
MIP-1 binding
Binding of biotinylated-recombinant MIP-1 (R&D Systems Europe
Ltd., Abingdon, U.K.) to MM6 cells was analyzed after the
manufacturer's protocol. Briefly, viable cells were washed three times
in PBS, pelleted in a 96-well microtiter plate at 105
cells/well, and incubated with or without peptide in a total volume of
10 µl PBS with 1% BSA for 2 h on ice. The cells were then
treated with biotinylated MIP-1, which was detected using
FITC-labeled avidin.
Inhibition of HIV-1 BAL infection of primary macrophage cultures
Medium was removed from wells containing adherent macrophages,
spun to remove any nonadherent cells and debris, and reserved. Peptide
or gp120 was added to quadruplicate wells in 100 µl PBS. The
irrelevant random peptide PSS023 and 3.7 were added at 30 µM and
recombinant gp120 from the M-tropic MN strain (supplied by the NIBSC
centralized facility for AIDS reagents supported by EU programme EVA
(contract BMH4 97/2515) and the U.K. Medical Research Council) was
added at 0.3 µM. Only PBS was added to control cells. The cells were
then incubated for 1 h at 37°C. A previously titrated amount of
HIV-1 BAL supernatant (150 µl) was then added to each well such that
virus specific message would be detected 72 h after exposure of
untreated primary macrophages. Cells were then incubated for 30 min at
37°C. After this time, 750 µl of the reserved culture medium was
added to each well and the cells were incubated at 37°C in 5%
CO2 in a humidified incubator.
RT-PCR
Chemokine expression
Total RNA was isolated (Stratagene, Cambridge, U.K.) and 3 µg
of RNA was used for cDNA synthesis using Expand reverse transcriptase,
5x RT buffer, DTT (Boehringer Mannheim, Roche Diagnostics Ltd., Lewes,
U.K.), and oligo (dt) (Oswel Ltd., Southampton, U.K.). Products of this
reaction were used as a template for polymerase chain reaction (PCR)
amplification with Taq DNA polymerase (Promega, Southampton, U.K.) and
primers (Oswel Ltd.): CCR5: antisense - CTC GGA TCC GGT GGA ACA AGA TGG
ATT AT, sense - CTC GTC GAC ATG TGC ACA ACT CTG ACT G; CCR3: antisense
- CCG CTC GAG CAG ACC TAA AAC ACA ATA GAG AGT TCC, sense - CGC GGA TCC
GGG AGA AGT GAA ATG ACA ACC; CXCR4: antisense - CCG CTC GAG CAT CTG TGT
TAG CTG GAG TGA AAA C, sense - CGC GGA TCC GCG GTT ACC ATG GAG GGG ATC;
ß-actin: antisense - CTA GAA GCA TTT GCG GTG GAC GAT GGA GGG,
sense-TGA CGG GGT CAC CCA CAC TGT GCC CAT CTA.
HIV infection
At 72 and 96 h postinfection total RNA was extracted from
duplicate wells using the Qiagen RNeasy spin column kit as per the
manufacturer's instructions (Qiagen Ltd., Crawley, U.K.). The
extracted RNA samples were each treated with 10 units DNaseI (Pharmacia
Biotech Ltd.) for 30 min at room temperature. DNase was inactivated by
addition of EDTA and incubation at 65 °C for 10 min. RNA content of
the samples was measured on a GeneQuant (Pharmacia, Biotech Ltd.). RNA
(0.1 µg) was used for cDNA synthesis using Expand reverse
transcriptase, 5x RT buffer, DTT (Boehringer Mannheim, Roche
Diagnostics Ltd.), and Oligo (dt) (Oswel Ltd.). Products of this
reaction were used as a template for PCR amplification with Taq DNA
polymerase (Helena Bioscience Ltd., Sunderland, U.K.) and primers
(Oswel Ltd.) designed from the HIV-1/HTLVIII reference genome sequence,
Genbank accession number KO3455, - antisense (vpu) – CTA TGA TTA CTA
TGG ACC AC; sense [5'LTR] – CTC TAG CAG TGG CGC CCG AAC AGG G,
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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From sequence data available in the HIV Molecular Immunology Database, this structure contains human cytotoxic T lymphocyte epitopes and human and murine antibody epitopes. The residues Lys-421 and Gln-422 are conserved in T-, M-, and dual tropic isolates but destroy M-tropism when mutated nonconservatively (7) and have recently been shown to be involved in the CCR5 binding site 9, 10) . The peptide 3.7 cross reacts with polyclonal sheep antibody raised against baculovirus expressed gp120 whereas an irrelevant 44 mer peptide with an oxidized Cys-X-Cys turn (FMDV derived from a different organism) does not, indicating that 3.7 contains at least some epitopes present in the native molecule (Table 1 ).
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To determine whether there was any basis for potential binding of the
sequence Lys-421–Gly-431 to beta-chemokine receptor, the
sequence was compared against that of binding sites on the
beta-chemokines MIP1, MIP1ß, and regulated on
activation, normal T expressed and secreted (RANTES), the 9-10
NH2-terminal amino acid residues proximal to the first
Cys-Cys residues (15)
. There was no sequence homology, but
Hopp and Woods (16)
hydropathy and molecular weight plots
showed that Lys-424–Gly-432 was similar to the chemokines in terms of
charge and size (Fig. 2
), such that it might fit within a receptor for these chemokines.
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Detection of peptide with mouse immune IgG
BALB/C mice were immunized with four doses of 3.7, as previously
described (12)
, and the IgG fraction of serum purified by
protein-G-Sepharose column affinity purification. The peptide induced a
specific class-switched IgG antibody response without coupling to a
carrier molecule, indicating the presence of both helper T and B
lymphocyte epitopes (Fig. 3
). The purified IgG did not bind to an irrelevant peptide, FMDV, of
similar size.
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3.7 colocalizes with CD4
Dual immunofluorescence studies with CD4-positive MM6 cells showed
that 3.7 bound by antipeptide antibody and detected with TRITC-labeled
goat antimouse immunoglobulin colocalized with biotinylated anti-CD4
monoclonal antibody, detected using FITC labeled avidin (Fig. 4
).
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3.7 binds to the CDR2 region, domain 1 of CD4
To confirm that 3.7 binds to CD4 and to determine to which region
it binds, the interaction of different anti-CD4 mAb's with H9 T cells
was assessed in the presence of gp120 or 3.7 or an irrelevant peptide
of similar size, FMDV. The mAb's used were Q4120 and L120. Q4120 binds
to the CDR2 region, domain 1 of CD4, and inhibits gp120 binding to CD4;
L120 binds to domain 4 of CD4 and does not inhibit binding of gp120
(12)
. The mAb's were used at pretitrated concentrations,
which gave 30–50% maximal binding to allow inhibition to be detected.
Both gp120 and 3.7 inhibited the binding of Q4120 mAb, but not L120
mAb, to H9 cells, although gp120 was more efficient on a molar basis
(Table 2
).
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3.7 inhibits the binding of MIP-1 to MM6 cells
To investigate the possibility that, like gp120, 3.7, may
also interact with chemokine receptors, the ability of 3.7 to inhibit
MIP-1 binding to MM6 cells was studied. The macrophage-derived MM6
cell line was selected because these cells are CD4 positive, express
CCR3 and CCR5 mRNA (Fig. 5a
), and strongly bind recombinant human MIP-1. Both 3.7
and gp120 significantly inhibited the binding of MIP-1 to MM6 cells
whereas the irrelevant peptide FMDV did not inhibit binding (Fig. 5b,c
)
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3.7 inhibits HIV-1 infection of primary macrophages
Since 3.7 bound to both CD4 and chemokine receptors, its effect on
the infectivity of the M-tropic HIV-1 BAL strain in primary peripheral
blood-derived macrophages was investigated (see Fig. 6
). Using a semiquantitative reverse transcription (RT) -PCR with
ß-actin as a reference housekeeping gene, recombinant gp120 blocked
infection 72 and 96 h after infection; the irrelevant peptide
PSS023 did not block at either time point. The 3.7 peptide markedly
inhibited infection on day 3 and to a lesser extent on day 4.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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binding to H9 cells. The peptide
was designed to include four of five residues in the native molecule
known to be critical for CD4 binding 7, 8)
and the
results suggest that the peptide is capable of adopting a structure
that allows it to bind to the same region of CD4 as gp120 does. The
peptide is less efficient on a molar basis than recombinant gp120,
which is not surprising since the percentage of peptide molecules
folded in any one particular configuration will be relatively small.
The chemokine receptors CCR3 and CCR5 that bind MIP-1 18, 19)
have been shown to be coreceptors for macrophage tropic
HIV-1 gp120 binding 3, 4, 20-22)
. However, the nature of
the chemokine receptor binding site on gp120 is not yet fully
understood, although it is known to involve conformational determinants
9, 10)
and the V3 loop 23, 24)
. M-tropism
has been shown to involve residues Lys-421 and Gln-422 of the C4 region
(7)
, which were incorporated into the design of 3.7
(12)
and have since been shown to be involved in the CCR5
binding site 9, 10)
. The Hopp and Woods hydropathy values
and the molecular weights of residues 424-432 of 3.7 suggested that it
might be capable of low affinity binding to receptors for MIP-1,
MIP-1ß, and RANTES in addition to CD4. Like gp120, 3.7 did inhibit
binding of MIP-1 to MM6 cells, suggesting that the peptide may adopt
a structure that allows it to bind to beta-chemokine
receptors as well as CD4 or that its binding to CD4 causes either a
steric alteration or a down-regulation of MIP-1 receptors. We
believe it is unlikely that 3.7 signals through CD4 to cause chemokine
receptor down-regulation or cytoskeletal changes that render the
receptor less accessible to MIP-1, because all the experiments were
conducted on ice. The binding inhibition is not a nonspecific peptide
interaction as control irrelevant peptide FMDV had no effect. A number
of mechanisms exist by which 3.7 may be inhibiting MIP-1. First, 3.7
may induce a conformational change in CD4 that causes CD4 to associate
with the MIP-1 receptor and, hence, allosterically occlude the
MIP-1 binding site. Second, a single molecule of 3.7 may bind to
both CD4 and the MIP-1 receptor simultaneously. Third, separate
molecules of 3.7 may be binding to MIP-1 receptor and CD4.
Because of the ability of 3.7 to bind to both CD4 and chemokine receptors, we tested its ability to inhibit infection with an M-tropic virus. Using primary, peripheral blood-derived macrophages, we found that 3.7 could indeed inhibit infection with the HIV-1 BAL strain whereas the irrelevant peptide PSS023 had no effect. It may appear paradoxical that a sequence derived from a T cell tropic isolate is able to inhibit ligand binding normally associated with macrophage tropic isolates, but the sequence used is conserved in both T and M tropic isolates. Whereas the V3 loop has been described as necessary for binding to chemokine receptors 6, 24) , other regions of gp120 have also been implicated 10, 25-27) . It has been reported that the V3 loop interacts with residues from the C4 region 28, 29) . Hence, changes in the conformation of the V3 loop may determine whether the residues involved in coreceptor binding from conserved regions of gp120 are in a position that allows interaction of the native molecule with particular coreceptors.
That the synthetic peptide 3.7 derived from three discontinuous sequence stretches of conserved regions can adopt a structure that allows it to interact with cell surface ligands of native gp120 and partially inhibit infection of primary macrophages has implications for the development of both therapeutic intervention and a synthetic vaccine. This approach also has more general implications for the synthesis of novel peptides representing complex, sequence discontinuous ligand binding sites of important biological proteins.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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2 Abbreviations: BSA, bovine serum albumin; FCS, fetal
calf serum; FITC, fluorescein isothiocyanate; Ig, immunoglobulin; M,
macrophage; MIP-1, macrophage inflammatory protein-1; PBMC,
peripheral blood mononuclear cells; PBS, phosphate-buffered saline;
PCR, polymerase chain reaction; PE, phycoerythrin; RANTES, regulated on
activation, normal T expressed and secreted; T, T lymphocyte; TRITC,
rhodamine.
Received for publication January 21, 1998.
Revision received October 26, 1998.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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This article has been cited by other articles:
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  T. J. Hewson, J. J. Logie, P. Simmonds, and S. E. M. Howie A CCR5-Dependent Novel Mechanism for Type 1 HIV gp120 Induced Loss of Macrophage Cell Surface CD4 J. Immunol., April 15, 2001; 166(8): 4835 - 4842. [Abstract] [Full Text] [PDF]
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| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
), MIP-1ß (
), and
RANTES (
) with 3.7 (424-432, x). c) Molecular weight
comparison of the receptor binding sites of MIP-1
