(Received for publication, June 6, 1994; and in revised form, November 4, 1994)
From the
Mouse granulocyte-macrophage colony-stimulating factor (mGM-CSF)
proteins with substitutions at residues in the first -helix were
examined for biological activity and receptor binding properties.
Substitution at the buried residue His
affected both
bioactivity and receptor binding. Of the four surface-exposed positions
examined (Arg
, Lys
, Lys
, and
Glu
) only substitutions at Glu
impaired
bioactivity. Proteins with charge reversal substitutions at this
position were partial agonists and weak antagonists of native mGM-CSF
action. All substitutions at Glu
abrogated high affinity
binding. Lys
and Lys
substitution proteins
showed various receptor binding defects. Qualitative and quantitative
measurement of these binding defects identified Lys
as a
residue that interacts specifically with the
subunit of the
mGM-CSF receptor, whereas Lys
appeared to exist at the
GM-R
-subunit/GM-R
-subunit interface as substitutions at
this position produce both high and low affinity binding losses. These
determinations permitted the design of a more potent mGM-CSF
antagonist.
Granulocyte-macrophage colony-stimulating factor (GM-CSF) ()is a pleiotropic cytokine made by various cell types, such
as T cells and monocytes, and is likely to play an important role in
the regulation of hemeatopoiesis(1, 2) . GM-CSF
interacts with specific high affinity cell surface receptors
(GM-R
, K
5
10
M) that are complexes containing at
least two subunits, both of which are members of the cytokine receptor
superfamily(3) . The receptor
chain (GM-R
) binds
GM-CSF specifically with low affinity (K
3
10
M), whereas the
receptor
chain (GM-R
), common to the receptors for GM-CSF,
IL-3, and IL-5, does not bind this protein detectably by itself, but
confers high affinity binding when co-expressed with
GM-R
(4, 5, 6) . Formation of this
complex with GM-CSF is required for receptor activation and cellular
signaling(7) .
Structurally, GM-CSF has been characterized
as a four anti-parallel -helical bundle protein(8) . This
topological fold is shared by many of the known cytokine structures,
including interleukins-2(9) , -4 (10) , and
-5(11) , macrophage colony-stimulating factor(12) , and
growth hormone(13) .
Molecular genetic studies and studies
with neutralizing anti-GM-CSF antibodies have identified regions and
residues in both mouse and human GM-CSF that interact with specific
subunits of
GM-R(14, 15, 16, 17, 18, 19, 20, 21) .
These studies suggest the functional importance of the first and fourth
helix of GM-CSF; in particular, the N-terminal helix of mGM-CSF
interacts directly with mGM-R
in the context of mGM-R
to
form the high affinity ligand-receptor complex(15) . Alanine
scanning mutagenesis of this region identified Glu
as
essential for this interaction in both mouse and human
GM-CSF(16, 17, 18) . Although substitution of
Glu
with alanine in mGM-CSF results in a loss of high
affinity binding, biological activity remains essentially intact. The
biological activity of hGM-CSF Glu
substitution proteins
is reduced, and a basic side chain at this position causes the most
severe defect(18) .
In this report we examine further the
biological and receptor binding properties of mGM-CSF mutant proteins
with substitutions at residues in the first helix of mGM-CSF. We show
that all Glu substitution proteins display only low
affinity binding, but that the biological activity depends on the
nature of the substituted side chain. Substitutions at several other
examined residues lead to a loss in either high or low affinity
binding. We also show that this knowledge can be used to design mGM-CSF
derivatives with antagonistic properties.
Figure 1:
Biological activity and competition
binding of wild-type and mutant proteins. A, proliferation of
NFS60 cells was measured as a function of protein concentration using
the MTT assay(32) . Assays were performed in quadruplicate with symbol error bars indicating standard deviation. Curves were
generated using a 4-parameter logistical fit and all correlation
coefficients exceeded 0.990. B, competition of I-mGM-CSF on NFS60 cells were performed with a constant
concentration of 3.0
10
M
I-mGM-CSF in triplicate with symbol error bars indicating standard deviation. Under these conditions both high
and low affinity sites were detected using the Ligand program (33) . Each pair of A and B panels represents
multiple amino acid substitutions for a specific residue as indicated
Arg
(1), Lys
(2),
His
(3), Lys
(4); and
Glu
(5). Substitutions are represented by each
symbol as follows:
, wild-type;
, Gly;
, Met;
Pro;
, Ala;
, Leu;
, Val; &cjs0513;, His;
, Phe;
., Trp;
., Tyr;
, Gln;
, Ser;
&cjs1730;., Arg;
, Glu; &cjs1730;,
Lys.
Based on homology to
hGM-CSF, His of mGM-CSF appears to occupy a buried
position in the first
-helix. This buried position is less
tolerant to substitution than the surface exposed residues (with the
exception of Glu
). Substitution at His
with
charged or polar residues resulted in a reduced biological activity,
whereas hydrophobic or aromatic substitutions are well tolerated (Fig. 1, panel 3A). All His
mutants
reached full plateau.
For most substitution mutants at Arg, Lys
,
His
, and Lys
, a two-site fit of the high
affinity receptor binding data was statistically significant (data
shown for Lys
and Lys
; Table 1and Table 2). Substitution at Arg
with either a
negatively charged (R11E) or positively charged (R11K) residue, neutral
(R11G), hydrophobic (R11L), or aromatic (R11W) residue had no effect on
either high or low affinity binding constants (data not shown). This
residue was not studied further.
Mutant proteins with charged and
polar substitutions at position His showed a reduced
ability to compete for
I-mGM-CSF (Fig. 1, panel 3B), in agreement with the observed loss in biological
activity (Fig. 1, panel 3A). Since this is a buried
position in the protein, losses in activity and binding are probably
due to structural perturbations of the protein core. Proteins mutated
at this position were also not studied further.
Mutations at
position Lys did not alter noticeably the biological
activity of the resulting mutant protein. However, significant binding
defects were observed (Fig. 1, panels 2A and 2B, Table 1). For some Lys
mutants, only
one affinity site is detected (Glu, Gly, Ser, Val, Trp, and Tyr). For
others, two sites were still detected but both were reduced in affinity (e.g. Phe and Gln). In general, reduction of the high affinity
binding constant was less than 10-fold, while values for the low
affinity K
showed wider variation with a
concomitant increase in the %CV, indicating that the calculated value
for the low affinity binding constant was less reliable under these
conditions. Several Lys
mutants displaying a range of high
affinity defects were selected and analyzed for their low affinity
binding to mGM-R
expressed alone on stably transfected L-cells (Fig. 2A). Of the five mutant proteins tested, four had
a low affinity K
similar to mGM-CSF (K
= 7.2
10
M; Table 4). From this analysis we conclude that
the reduction seen in the high affinity binding constant for Lys
substitution proteins results from losses in binding to the
mGM-R
in the mGM-R
complex. Protein K14P is the
exception. A proline substitution at this position affected the low as
well as the high affinity binding constant (Table 1).
Figure 2:
Competition binding of mutant and
wild-type mGM-CSF proteins. A, competition of I-mGM-CSF on L cells transfected with mGM-R
by
, mGM-CSF;
, K14A;
, K14E;
, K14G;
,
K14P; and
, K14S. B, competition of
I-mGM-CSF on L cells transfected with mGM-R-
by
, mGM-CSF;
, K20G;
, K20M;
, K20P;
,
K20A;
, K20L;
, K20V;
+, K20F;
+,
K20H;
+, K20W;
+, K20Y;
, K20Q;
,
K20S; &cjs1730;+, K20R;
, K20E. Assays were performed in
triplicate using a constant concentration of 2.0
10
M
I-mGM-CSF with symbol error bars indicating standard
deviation.
Mutant
proteins with Lys substitutions also showed losses in both
high and low affinity binding as measured on NSF60 cells (Fig. 1, panel 4B, Fig. 2B, Table 2and Table 4). To distinguish between losses in
binding to the mGM-R
and/or mGM-R
, Lys
substitution proteins were analyzed on mGM-R
-expressing
L-cells. Whereas most substitutions at Lys
did not affect
the low affinity binding constant as measured on L-cells, small amino
acid substitutions (Ala, Gly, and Ser) at this position led to a
10-fold reduction in mGM-R
binding (Fig. 2B, Table 4). The introduction of proline at this position profoundly
disturbed the binding of the resulting protein to both high and low
affinity receptor.
In contrast to the other mutant proteins
examined, all Glu substitution proteins competed
I-mGM-CSF for only a single class of binding sites on
NSF60 cells (Fig. 1, panel 5B, Table 3). The
affinity of Glu
substitution proteins (K
= 1-4
10
M) for
these sites suggested that binding is to mGM-R
. Scatchard analysis
of one of the Glu
substitution proteins, E21A, has shown
that both affinity and number of binding sites were consistent with
binding of this mutant protein to mGMR
only(16) . Again,
the exception was E21P. This protein had a low affinity binding
constant that was at least three orders of magnitude higher than the
other Glu
proteins (Fig. 5B, Table 3), probably caused by severe structural
changes(36) .
Figure 5:
Two views of the mGM-CSF C backbone
indicating important structural features and identifying specific amino
acid side chains. Helix A (red) and helix D (blue)
form an anti-parallel helix pair and comprise the receptor binding
site(38) . Disulfide bonds are indicated in yellow.
Amino acid residues significant to this study are colored according to
the properties of their side chains, negatively charged (E), red; positively charged (K and R), blue; and aromatic (H), green.
Figure 3:
Biological activity of wild-type and
mutant mGM-CSF proteins. Proliferation of NFS60 cells in response to
mGM-CSF (), E21K (
), and K14E/E21K (
) was measured
as a function of protein concentration using the MTT
assay(32) . Assays were performed in quadruplicate with symbol error bars indicating standard
deviation.
Figure 4:
Antagonism of the mGM-CSF response by
mutant mGM-CSF proteins. Proliferation of NFS60 cells in response to
E21K () and K14E/E21K (
) in the presence of 3.5
10
M mGM-CSF using the MTT
assay(32) . Assays were performed in quadriplicate with the symbol error bars indicating standard
deviation.
The involvement of the amino-terminal region of mGM-CSF in
receptor binding and biological activity is well
documented(16, 17) . In particular, it is now clear
that residues in the first -helix of mGM-CSF directly contact the
mGM-CSF receptors. The data presented here identify in detail the
qualitative and quantitative contribution of several residues in this
region to this interaction. This knowledge was essential to our
designing a mGM-CSF receptor antagonist.
We have previously shown
that Glu plays a pivital role in binding to the mGM-R
in the high affinity complex. Substitution of Glu
with Ala
abrogates high affinity binding(16) . The data presented here
show that none of the 14 amino acid substitutions are tolerated at this
position. Previous work concludes that Glu
can be replaced
with Asp without serious consequences to bioactivity(36) .
Analysis of the equivalent Glu
position in hGM-CSF shows
that Glu
can be replaced with an Asp without noticeable
loss in either bioactivity or binding(18) . Taken together,
these data show the absolute requirement of an acidic residue at this
position. All other substitutions we tested resulted in a complete loss
of high affinity binding, while low affinity binding remained
unaffected. These data identify Glu
unequivocally as a
residue that interacts with mGM-R
only. Even though Glu
mediates high affinity binding, only charge reversal
substitutions cause a noticeable loss in biological activity.
Maintaining biological activity in the absence of high affinity binding
seemingly contradicts the observation that signaling occurs through a
complex of the mGM-R
and mGM-R
subunits in the presence of
ligand. However, we have previously shown for one of the Glu
substitution proteins, E21A, that this protein still can be
cross-linked to the mGM-R
complex, even in the absence of any
detectable high affinity binding(16) . Taken together these
data indicate that the interaction of Glu
with mGM-R
,
although pivital for the measured high affinity binding of mGM-CSF to
its receptor, is not necessary for receptor activation. The absence of
a Glu residue at position 21 apparently does not prevent mGM-R
from participating in complex formation and signaling and suggests that
other mGM-CSF residues contact mGM-R
.
Studies on hGM-CSF and
hIL-3, cytokines which share the receptor, show similar results.
Substitution of the homologous acidic residues in these two cytokines
also leads to a loss of high affinity
binding(18, 37) . However, there is a closer
correlation between loss of high affinity binding and loss of activity
for both hGM-CSF and hIL-3. Taken together, these data suggest that
Glu
in GM-CSF and the homologous acidic residue in IL-3
and presumably also IL-5 are functionally identical; this residue
determines high affinity binding. However, the extent to which its
absence affects the biological activity varies and presumably depends
on how many other residues contribute to the interaction with
GM-R
.
Our data revealed that at least 2 other residues in the
first -helix of mGM-CSF interact with mGM-R
. Substitutions at
Lys
and Lys
both disrupted high affinity
binding as indicated by decreased high affinity K
values. Lys
substitutions exclusively reduced high
affinity binding, whereas some Lys
substitutions affected
high affinity binding and others low affinity binding. However, none of
the substitutions at either position reduced the biological activity.
The binding defects of Lys
mutant proteins are generally
somewhat smaller than Lys
defects and fell into two
categories, those that were caused by defects in binding to mGM-R
and those caused by defects in binding to mGM-R
. Substitutions
that reduced the binding to mGM-R
had small side chains (Ala, Ser,
and Gly), suggesting that size plays a critical role at this position.
Homology modeling based on the high resolution crystal structure of
hGM-CSF (8) indicates the most likely location of these
residues on the
-helix 1 mGM-CSF backbone (Fig. 5). Both
Lys
and Glu
are located on the exterior of
-helix 1 toward the groove formed by the
-helix 1 and
-helix 3 and well situated for possible direct interaction with
mGM-R
. It has been suggested that the groove between the first and
third helix provides most of the GM-R
contact(38, 39) . Our data on Lys
and
Glu
support this hypothesis. On the other hand, Lys
is at the interface of
-helix 1 and
-helix 4, which is
the region thought to provide mGM-R
specific contacts. Our data
support a dual role for Lys
in binding mGM-R
and
mGM-R
. It is somewhat surprising that Glu
does not
play a more important role in mGM-R
binding considering its
location between Lys
and
Glu
(15, 16, 17) . Recent
mutagenesis of the hGM-CSF equivalent position, Asn
, has
confirmed that this residue contributes little to binding and
bioactivity(38) . Fig. 5shows that the Glu
side chain is somewhat removed from the groove between helix 1
and helix 3 and apparently does not contribute to binding of GM-R
.
Based on functional and structural comparison of hGM-CSF and GH two
potential receptor-binding sites on hGM-CSF have been
suggested(39) . Analogous to site I of GH(40) , it was
proposed that GM-CSF binds to its primary binding receptor, GM-R,
through
-helix 4. The interaction of the first
-helix of
GM-CSF with the GM-R
in the high affinity complex appears to be
similar to the site II interactions of GH and GH-R. Receptor
antagonists of GH were made by mutations of site II residues that
prevent the second GH-R from participating in the formation of the GH-R
dimer(41) . We have extrapolated that finding to mGM-CSF and
tested the partial agonist E21K as a receptor antagonist. E21K weakly
antagonized mGM-CSF. By eliminating other mGM-R
-specific contact
residues in mGM-CSF, we anticipated improved antagonistic mGM-CSF
derivatives. The K14E, E21K double mutant was indeed a better
antagonist than E21K. These experiments show that detailed studies
which not only identify key residues involved in protein-protein
interactions, but also characterize the nature of that interaction,
make it possible to rationally design cytokine derivatives with altered
properties.
GM-CSF, IL-3, and IL-5 all bind specific Rs but
share a common R
. Identification of two R
-specific contact
residues in mGM-CSF led to derivatives with antagonistic properties.
This finding may have direct implications for the design of IL-3 and
IL-5 antagonists. A strategy to find such molecules should focus on
elimination of R
-specific contacts, in particular, the conserved
acidic residue in the first
-helix of these cytokines and a second
residue corresponding to mGM-CSF Lys
.