(Received for publication, September 20, 1995; and in revised form, November 7, 1995)
From the
Granulocyte-macrophage colony-stimulating factor (GM-CSF) is a member of the four-helix bundle family of cytokines/growth factors which exhibit several activities. It is a hematopoietic growth factor, a cytokine involved in inflammatory and immune processes, an adjunct for cancer therapy, and an anti-tumor immunomodulator. Studies of interactions between GM-CSF and its receptor and identification of small peptides presenting binding capacity to the receptor are important goals for the development of GM-CSF analogs. Here we describe the study of two cyclic peptides, 1785 and 1786, developed based on structural analysis of the GM-CSF region mimicked by anti-anti-GM-CSF recombinant antibody 23.2. These peptides were designed to structurally mimic the positions of specific residues on the B and C helices of human GM-CSF implicated in receptor binding and bioactivity. Both 1785 and 1786 were specifically recognized by polyclonal anti-GM-CSF antibody (stronger for 1786 than 1785). 1786 also competitively inhibited binding of GM-CSF to the GM-CSF receptor on HL-60 cells and demonstrated antagonist bioactivity, as shown by its reversal of GM-CSF's ability to inhibit apoptosis of the GM-CSF-dependent cell line MO7E. These studies support the role of residues on the GM-CSF B and C helices in receptor binding and bioactivity and suggest strategies for mimicking binding sites on four-helix bundle proteins with cyclic peptides.
Granulocyte-macrophage colony-stimulating factor (GM-CSF) ()is a hematopoietic growth factor and a cytokine involved
in many inflammatory and immune processes. GM-CSF activates
antigen-presenting cells (monocytes, macrophages, and dendritic cells),
increases major histocompatibility complex class II
expression-enhancing antigen presentation, and increases macrophage
anti-tumor activity(1) . Recently it has been used as an
important adjunct in cancer therapy for bone marrow recovery following
chemotherapy and transplantation(2) . Moreover, GM-CSF induces
protective immune responses against lymphoma cells if fused with a
tumor-derived idiotype, eliciting tumor-specific immunity(3) .
GM-CSF also enhances the immunogenicity of tumor cells when expressed
by them, resulting in induction of protective anti-tumor immunity,
while other cytokines such as IL-2, IL-4, IL-5, IL-6,
-interferon,
or tumor necrosis factor-
are less effective(4) .
The
crystal structure of human GM-CSF (5, 6, 7, 8) reveals a four-helix
bundle organization similar in some respects to that described for
growth hormone(9) , IL-2(10) , and
IL-4(11, 12, 13, 14) . The related
cytokines macrophage colony stimulating factor (15) and IL-5
are organized as dimers of four-helix bundles(16) . GM-CSF
activity is mediated by binding to specific cellular receptors
(GM-CSFR) which belong to a recently described supergene
family(17, 18, 19, 20, 21, 22, 23) .
The high affinity GM-CSFR is comprised of an chain (GM-CSFR
)
specific for GM-CSF(20) , and a
chain
(
), which can also associate with the IL-3 and IL-5
receptor
chains(21) . The GM-CSFR
imparts
specificity to the interaction with GM-CSF, and when expressed without
is able to bind GM-CSF, albeit with lower affinity
than the heterodimeric receptor(24) . The high affinity
receptor (GM-CSFR
and
) appears to be the
signal-transducing unit(25, 26) , with a sequential
binding of GM-CSF to GM-CSFR
followed by binding to
postulated.
GM-CSF and the related four-helix bundle cytokines are important targets for drug design and production of low molecular weight analogs which mimic the native ligand. Studies of ligand-receptor intermolecular interactions which help delineate their active sites should allow the development of small molecules able to mimic the larger polypeptide ligands. Such small drugs, created based on analysis of the most important binding interactions, could circumvent problems of immunogenicity, antigenicity, rapid proteolysis by serum proteolytic enzymes, short serum half-life, and low oral bioavailability, commonly presented by large polypeptides.
In prior
studies, linear peptide analogs of GM-CSF were produced by dividing the
human GM-CSF sequence into six peptides(27) . This strategy led
to the identification of two peptides with receptor binding and
antagonist activity. One peptide corresponding to residues 17-31
(the A helix) inhibited high affinity receptor binding, while a second
peptide corresponding to residues 54-78 (the B and C helices)
inhibited low affinity receptor binding(27) . This implicates
these sites in intermolecular interactions with the GM-CSFR. We also
have used a recombinant antibody (rAb) as a GM-CSF mimic(28) .
Molecular modeling of the rAb 23.2 allowed the identification of
complementarity determining regions (CDRs) as sites of structural
mimicry of GM-CSF, focusing attention on the CDRI region mimicking
residues on the B and C helices of GM-CSF. After synthesis and
characterization of CDRI, CDRII, and CDRII peptides, the CDRI peptide
exhibited specific GM-CSF receptor binding and antagonist
bioactivity(28) . Thus, these studies suggest that residues on
the B and C helices of GM-CSF mediate binding to the low affinity
receptor (GM-CSFR alone).
Here we describe the development of two cyclic peptide GM-CSF mimics (1785 and 1786) obtained from structural analysis of the GM-CSF region mimicked by rAb 23.2(28) . Cysteines were introduced in the peptide structures at the amino and carboxyl termini to allow cyclization. The cyclized peptides were specifically bound by polyclonal anti-GM-CSF antibody (stronger for 1786 than for 1785). Moreover, 1786 competes with GM-CSF for binding to the GM-CSF receptor present on HL-60 cells and reverses GM-CSF's prevention of apoptosis of MO7E cells. Thus, 1786 represents a structurally designed biological and receptor antagonist of GM-CSF.
Figure 1: Development of 1785 and 1786 peptides. The structure of GM-CSF (left ) was determined from coordinates derived from the crystal structure (J. M. LaLonde, K. Swaminathan and D. Voet, manuscript in preparation), displayed on the MacImdad program (Molecular Applications Group, Palo Alto, CA) on a Macintosh Quadra 950 computer. The critical residues of 54-61 region of B helix and of 77-83 region on C helix are reported. These residues are introduced in 1785 (upper right) and 1786 (lower right) sequences together with glycines, alanines, and cysteines (for peptide cyclization). Peptide tridimensional structures are also reported.
For the agarose evaluation, 350 µl of 5 10
cells/ml were washed, added to 20 µl of lysis buffer (10
mM EDTA, 50 mM Tris HCl pH 8.0, 0.5% N-lauroylsarcosine sodium salt (Sarkosyl), 0.5 mg/ml
proteinase K) and incubated for 1 h at 50 °C. After addition of 10
µl of 0.45 mg/ml RNase and incubation at 50 °C for 1 h, the
samples were mixed with 10 µl of 10 mM EDTA, pH 8.0, 0.03%
bromphenol blue, 1% Nue Sieve GTG agarose (FMC BioProducts, Rockland,
ME), heated at 70 °C for 10 min, loaded into a 1.2% agarose gel and
run for 1 h at 100 V using TBE buffer (89 mM Tris base, 89
mM boric acid, 2 mM EDTA pH 8.0). The gel was stained
with ethidium bromide (Sigma) and photographed under ultraviolet light.
Mono- and oligonucleosome fragments present in the cytoplasmic
fraction of cell lysates were detected following the protocol for
``Cell Death Detection ELISA'' kit. Briefly, the microtiter
plate was coated with anti-histone solution and, after incubation with
a 1:10 dilution of the lysate derived from 2.5 10
cells, DNA was detected by the anti-DNA-peroxidase system
according to the kit instructions, with color development read at 405
nm.
Mass spectrometry analysis was performed on the oxidized peptides to confirm that oxidation had resulted in intrachain disulfide bond formation, as opposed to formation of oligomers. The mass spectrometry study showed that >90% of the oxidized 1785 peptide was represented by a peak at molecular mass 1514, with the theoretical molecular mass for 1785 being 1511 daltons. Similarly, >90% of the 1786 peptide was seen as a peak at molecular mass 1637, the theoretical molecular mass being 1639. Thus, both of the oxidized peptides were >90% in the monomeric form, with only trace contamination by oligomers (dimers and trimers).
Figure 2:
Binding of polyclonal Ab against GM-CSF to
1786 and 1785 peptides. Binding was performed by ELISA assay as
described under ``Material and Methods.'' The graphs shown
are referred to the case of peptides at 1.5 µg/well. Similar
results were obtained with 3, 4.5, and 6 µg/well. Binding of 1786,
1785, and control peptides both to anti-GM-CSF polyclonal antibody and
to preimmunization serum (normal mouse serum, NMS) are
reported. The values are obtained subtracting the A of wells without peptides from the A
of
wells with peptides at different concentrations. The mean ± S.D.
of duplicate wells is shown for decreasing amounts of polyclonal
anti-GM-CSF antibody.
Figure 3:
Inhibition of I-GM-CSF
binding to HL-60 cells by peptides. The radioreceptor assay was
performed as reported under ``Materials and Methods,'' using
10
cells/test. The specific proportion of count/min bound
was determined subtracting the proportion of counts/min bound under
identical conditions in the presence of saturating amounts of unlabeled
GM-CSF (50 nM). The percent inhibition of binding of 1786,
1785, and control peptide is reported versus increasing
amounts of peptides together with the S.D. of duplicate
tests.
Both the agarose gel and the ELISA results (Fig. 4) indicated clear antagonist activity for the 1786
peptide, with reversal of GM-CSF's prevention of apoptosis.
Increasing the amount of 1786 in presence of GM-CSF increased the
amount of apoptosis seen (IC of
85 µM).
When incubated with the cells in medium alone, the 1786 peptide did not
prevent DNA degradation, excluding any agonist activity by the peptide.
The same peptide, in the presence of U87 cell supernatant, presented
the same type of dose-dependent behavior in increasing apoptosis as
shown in presence of GM-CSF (IC
of
65
µM). The 1786 effect was not seen in the presence of TPA
which prevents apoptosis in a receptor independent fashion, indicating
that the antagonist activity was GM-CSF receptor-dependent. In
contrast, the 1785 peptide did not demonstrate agonist or antagonist
activity in these apoptosis assays. This indicates that 1786, which
inhibits GM-CSF receptor binding, has a similarly specific GM-CSF
receptor-dependent antagonist bioactivity.
Figure 4: Inhibition of GM-CSF's prevention of apoptosis by peptides. Apoptosis was evaluated both by running cell lysate in an agarose gel (left, reported only for the case of peptides at 160 µg/ml) and by determining mono- and oligonucleosomes with an ELISA kit (right, reported only for the significant 1786 peptide). The assays were performed as indicated under ``Materials and Methods.'' Lysates from cells incubated with or without peptides in presence or absence of factors preventing apoptosis (GM-CSF, TPA, U87 supernatant) were loaded into gel (left) or analyzed by ELISA reporting the percentage of maximal apoptosis, as referred to the absence of any reagent preventing apoptosis (right).
The interaction of GM-CSF with its receptor has been the
subject of intense investigation. Prior studies with GM-CSF mutants
indicated that residues on the first (A) helix of GM-CSF are involved
in the binding to high affinity receptor (the
GM-CSFR complex) but not to low affinity
receptor (GM-CSFR
alone)(24, 39, 40) .
This is illustrated most strikingly by studies using mutants of residue
Glu-21 of GM-CSF, which inhibit binding of GM-CSF to the low affinity
receptor, but display little activity in inhibiting binding to the high
affinity receptor(39, 41, 42) . Based on
these experiments, it has been proposed that the first
helix of
GM-CSF is responsible for binding to
(40) .
Murine and human GM-CSF display species specificity and are not
cross-reactive. As substitutions are scattered throughout these
molecules, it was possible to swap regions of murine and human GM-CSF
to locate sites critical for receptor interaction(35) . These
studies indicated a critical role for amino acids 21-31 (A helix)
and 77-94 (including the C helix) in mediating the activity of
human GM-CSF, suggesting that the second site may be involved in
binding to the GM-CSFR. Additional mutagenesis
studies(42, 43, 44, 45) , mapping of
neutralizing monoclonal
antibodies(46, 47, 48, 49, 50) ,
and synthetic peptide studies (47, 51, 52) suggest other potential
interaction sites. Thus, in spite of considerable study, the
GM-CSFR
interaction site(s) on GM-CSF remain incompletely
characterized.
In our group use of synthetic peptides, anti-peptide
antisera, and neutralizing monoclonal antibody to map epitopes on
GM-CSF important for bioactivity have led to several conclusions: a
peptide corresponding to residues 17-31 of the A helix, as well
as antibodies against this peptide, are able to inhibit GM-CSF
dependent cellular proliferation; the 17-31 peptide also inhibits
GM-CSF binding to the high affinity receptor but not to the low
affinity receptor; a peptide corresponding to residues 54-78
overlapping the B and C helices is recognized by two neutralizing
monoclonal antibodies to GM-CSF and exhibits antagonist
bioactivity(27) . This suggests a model of receptor interaction
where residues on the B and C helices of GM-CSF, the opposite face of
the A helix, are involved in interactions with GM-CSFR, while
residues on the A helix mediate binding to
(27) . This model is supported by analysis of
a rAb mimic of GM-CSF (23.2) as well as a peptide derived from the CDRI
sequence of the rAb 23.2. The CDRI peptide and the rAb were shown to
exhibit structural similarity to residues on the GM-CSF B and C
helices; both the peptide and the rAb mimic were bound by neutralizing
anti-GM-CSF monoclonal antibody 126.213 and exhibited biological and/or
receptor antagonist activity(28, 33) .
The purpose of this study was to further test this model by developing additional peptides which mimic the position of specific residues on the GM-CSF B and C helices, and evaluating them for receptor binding and biological activity. The structure of the two peptides discussed in this report derived from our prior studies, with 1785 and 1786 designed to structurally mimic potential contact residues on the GM-CSF B (residues 54-61) and C (residues 77-83) helices. The two peptides were synthesized with cysteines at both the amino and carboxyl termini in order to develop cyclic forms, thereby constraining the conformations of the peptides and providing more accurate mimicry of the B and C helical face of GM-CSF. These peptides also allowed us to evaluate whether reverse turn peptide mimics, such as those developed from the rAb 23.2 CDRI sequence, could be developed from simple structural considerations, obviating the need to develop them by library screening or antibody mimicry.
The cyclic peptides were easily prepared by oxidation overnight, reaching almost 100% oxidation and 90% yield (only traces of dimer and trimer were detected by mass spectrometry analysis). The cyclized monomer peptides were therefore used in binding tests to polyclonal antibody against GM-CSF and to GM-CSF receptor present on HL60 cells. In both cases peptide 1786 showed good binding capacity, displaying competitive behavior toward GM-CSF in the radioreceptor assay. On the other hand, peptide 1785 demonstrated a lower binding affinity to polyclonal anti-GM-CSF antiserum and complete lack of interaction with the GM-CSF receptor. In order to establish their bioactivity, the peptides were assayed in an apoptosis assay. GM-CSF is known to prevent apoptosis of MO7E cells(37, 38) . These cells were incubated, in presence or absence of different concentrations of peptides, with fixed amounts of GM-CSF or U87 supernatant (a source of GM-CSF). TPA, an agent which also prevents apoptosis but via a different mechanism not involving the GM-CSF receptor, allowed the specificity of the reaction to be evaluated(37) . Peptide 1786, but not 1785 or control peptides, displayed biological antagonist activity: increasing the amount of peptide 1786 resulted in an increase in apoptosis in response to GM-CSF or U87 supernatant, while no effect was seen in the presence of TPA.
The IC for peptide 1786 in the apoptosis assay
was similar for both GM-CSF and U87 supernatant (65-85
µM). This is somewhat smaller than the calculated K
for peptide inhibition of binding to low
affinity receptor sites on HL-60 cells (270 µM). However,
the low affinity sites do not appear to mediate bioactivity, while the
high affinity sites do(21, 39) . Interestingly, if a
similar EC
is assumed for the high affinity sites, the
calculated K
for peptide 1786 in the binding assay
is 59 µM(36) , much closer to the IC
observed in the apoptosis assay. This supports the role of high
affinity sites in mediating bioactivity.
Based on these studies, peptide 1786 represents a receptor antagonist of GM-CSF, supporting our conclusions from molecular-structural analysis utilizing recombinant antibodies (28) for the identification of residues critical for bioactivity. Moreover, these studies suggest that similar peptide mimics can be designed based on structural information derived from knowledge of potential contact residues. The ability to design such mimics may be readily extended to other systems where sufficient structural and biological information is available to delineate potential contact residues. This should allow for the analysis of potential contact residues on novel backbones as well as the rational design of receptor antagonists with potential clinical utility.