(Received for publication, October 11, 1995)
From the
Wild type human (h) interleukin 5 (wt IL5) is composed of two
identical peptide chains linked by disulfide bonds. A gene encoding a
single chain form of hIL5 dimer was constructed by linking the two hIL5
chain coding regions with a Gly-Gly linker. Expression of this gene in
COS cells yielded a single chain IL5 protein (sc IL5) having biological
activity similar to that of wt IL5, as judged by stimulation of human
cell proliferation. Single chain and wt IL5 also had similar binding
affinity for soluble IL5 receptor chain, the specificity subunit
of the IL5 receptor, as measured kinetically with an optical biosensor.
The design of functionally active sc IL5 allowed asymmetric mutagenesis
of the symmetrical IL5 molecule. Such mutagenesis was exemplified by
changes at residues Glu-13, Arg-91, Glu-110, and Trp-111. The receptor
binding and bioactivity data obtained are consistent with a model in
which residues from both IL5 monomers interact with the receptor
chain, while the interaction likely is asymmetric due to the intrinsic
asymmetry of folded receptor. The results demonstrate a general route
to the further mapping of receptor and other binding sites on the
surface of human IL5.
Human interleukin 5 (hIL5) ()is a T cell-derived
cytokine which plays an important role in the differentiation,
proliferation, and activation of eosinophils (Sanderson et
al., 1992; Bentley et al., 1992). Natural hIL5 is a
disulfide-linked, homodimeric glycoprotein with 115 residues per chain.
The high resolution crystal structures of both Escherichia
coli-expressed (Milburn et al., 1993) and Drosophila-expressed hIL5 (Johanson et al., 1995)
have revealed a core of two four-helix bundles. Each four-helix bundle
resembles the four-helix bundle seen in IL2 (Bazan and McKay, 1992),
IL4 (Smith et al., 1992), and GM-CSF (Diederichs et
al., 1991). However, the bundle organization in IL5 is unique in
that helix D of one monomer combines with helices A, B, and C of the
second monomer, and vice versa. In the two-bundle structure, the A and
D helices form one face, the B and C helices a second. The organization
of structural features on each face is palindromic, as observed for
restriction sites in DNA.
Human IL5 receptor is composed of two
different chains, denoted and
(Tavernier et al.,
1991). The
chain is specific for IL5 (Murata et al.,
1992) and has a K
in the 0.1-1
nM range depending on the assays and receptor forms examined
(Li et al., 1996). In contrast, the
chain of hIL5R is
not cytokine-specific but is shared with the receptors for IL3 and
GM-CSF (Tavernier et al., 1991) and appears needed for signal
transduction. A soluble form of hIL5 receptor
chain
(shIL5R
), which has only the extracellular domain of IL5R
,
also has been described. This binds to hIL5 with nanomolar affinity
(Tavernier et al., 1991; Johanson et al., 1995).
Despite the dimeric nature of IL5, a 1:1 binding stoichiometry between
shIL5R
and hIL5 has been reported (Devos et al., 1993;
Johanson et al., 1995).
New insights into the binding
mechanism of IL5 to its receptor are emerging with the availability of
high resolution structure and mutagenesis techniques. Data from hybrid
constructs of mouse/human IL5 suggest that the carboxyl-terminal 36
residues of IL5 interact directly with the IL5R and confer species
specificity (McKenzie et al., 1991). By Ala-scanning
mutagenesis of the carboxyl-terminal region of hIL5, we found
previously that Glu-110 and Trp-111 contribute significantly to
receptor binding (Morton et al., 1995). In addition, mutation
of residues (Glu-89 and Arg-91) in the loop between helices C and D
have been found to affect binding to IL5R
(Tavernier et
al., 1995; Graber et al., 1995). All of these residues
cluster around the interface between the two 4-helix bundles of hIL5
and appear to constitute a central patch for binding to a single
molecule of hIL5R
(Morton et al., 1995). In contrast,
residue Glu-13, which is at the distal ends of the IL5 dimer away from
the helix bundle interface, was suggested to interact with the
chain of IL5R, since mutation at this position resulted in loss of
biological activity but did not affect the binding affinity to the
chain (Tavernier et al., 1995; Graber et al.,
1995).
While emerging data suggest models for the topography of
receptor binding sites in hIL5, a more defined understanding is impeded
by the homodimeric nature of the protein. Because of this, mutagenesis
of wild type hIL5 inevitably has resulted in symmetrical changes in
side chains on both sides of the 4-helix bundle interface. It is thus
unclear whether one or both of the residues found to be important
participate equally in receptor binding and, furthermore, whether the
topology of binding is different for hIL5R versus hIL5R
. One way to overcome this limitation is asymmetric
mutagenesis. Here, we report construction of an active single chain
form of hIL5 dimer, denoted sc IL5, and its use to construct asymmetric
mutations at residues Glu-110, Trp-111, Arg-91, and Glu-13.
Figure 1:
Schematic diagram of the
tethered hIL5 dimer with a Gly-Gly linker and its transient COS
expression. A: top, wt IL5; bottom, sc IL5.
The gene of the single chain IL5 was constructed by linking two hIL5
genes in tandem separated by a spacer that encoded the dipeptide
-Gly-Gly-. The amino-terminal half of the molecule is defined as a, the carboxyl-terminal half as b. The wtIL5
contains the full sequence of hIL5 except that the amino-terminal
sequence was NH-GARSEIPTSALVKET (Johanson et
al., 1995). The amino terminus of IL5 (b) of the single chain IL5
is RSEIPTSALVKET. B, expression of single chain hIL5
(Western blot). The supernatant (10 µl) of COS cells transfected
with indicated expression vectors (see ``Materials and
Methods'') was quenched with SDS-PAGE loading buffer (with or
without 100 mM dithiothreitol), run on a 15% SDS-PAGE gel, and
then transferred to Immobilon. The blot was probed with rabbit
anti-hIL5 antibodies.
sc IL5 and wt IL5 were expressed
in COS cells, and the supernatants were analyzed by SDS-PAGE followed
by immunoblotting with a polyclonal anti-hIL5 antiserum (Morton et
al., 1995). As shown in Fig. 1B, sc IL5 had the
same molecular mass (34 kDa) as that of the wt IL5 under
nonreducing conditions. The levels of expression of wt IL5 and sc IL5
were similar. Under reducing conditions, sc IL5 still ran as a dimer
(34 kDa) while wt IL5 was reduced to two monomers (
17 kDa) (Fig. 1B). The fact that hIL5 and sc IL5 had the same
molecular masses under nondenaturing conditions indicated that both
forms were glycosylated similarly and that no intermolecular disulfide
bonds had formed in sc IL5.
Figure 2:
Comparison by biosensor analysis of the
binding kinetics of wt IL5 (top) and sc IL5 (below)
in interacting with shIL5R. A, overlays of sensorgrams
showing binding of various concentrations of shIL5R
to wt IL5 or
sc IL5. The marked numbers indicate the concentrations of shIL5R
.
The increase in response shows the binding of shIL5R
. The decay
represents the dissociation of bound shIL5R
. Data obtained with
the IAsys biosensor. B, calculation of on-rate constant for
the interaction of hIL5 with shIL5R
. The association phases of the
sensorgrams in A were replotted as the slope of the curve at a
given time versus relative response at the time. The straight lines show a line fit to the linear part of the data.
The slopes of these lines give values for k
at each concentration (see Morton et al.(1995)). C, plot of k
versus concentration. The slope of the line to these points gives the
association rate constant. D, determination of dissociation
rate constant. The dissociation phase of the sensorgram at 60 nM shIL5R
in A was replotted as ln(response at time
zero of dissociation/response at time n) versus time.
The straight line shows the line fit to the early part of the
data. The slope of the line gives the dissociation
constant.
The binding of COS-expressed sc IL5 and
wt IL5 to the full length IL5R was also compared by competition
for binding of
I-IL5 to Drosophila cell
membranes containing expressed IL5R
(Johanson et al.,
1995). Single chain IL5 was equally as effective as wt IL5 in
inhibiting the binding of
I-IL5 to the cell membranes
(data not shown), consistent with the biosensor data, suggesting that
sc IL5 and wt IL5 can bind to the same site(s) of full-length IL5R
with similar affinity.
The ability of sc IL5 to induce signal transduction was measured by cell proliferation. Single chain IL5 showed activity comparable to that of wt IL5 in the TF-1 cell assay (Table 1). Overall, there were no major differences in either receptor binding or bioactivity of single chain and wild type IL5.
The effects of asymmetric Ala mutagenesis in sc IL5 for
Glu-110, Trp-111, Arg-91, and Glu-13 are shown in Table 1. In all
cases, with the exception of Glu-13, single site mutations in the b
domain of sc IL5 led to small but finite decreases (50%) in
shIL5R
binding activity compared with wt IL5. However, for each
mutation, the reduction in binding affinity for the asymmetric
construct was 4-19-fold less than that obtained with the
corresponding double mutant (for example, sc IL5 (Glu-110 (b)) versus sc IL5 (Glu-110 (a, b))). Consistent with the binding
affinity data, asymmetric mutagenesis of Glu-110, Trp-111, or Arg-91
all resulted in an increased EC
value compared with that
of sc IL5, but these values were 4-30-fold less than those
obtained with the corresponding double mutants.
Mutagenesis of
Glu-13 in sc IL5 did not affect IL5R binding activity, as expected
from earlier-reported results (Tavernier et al., 1995; Graber et al., 1995), but did cause a marked decrease in biological
activity. The effect on activity of the single site E13A mutant in sc
IL5 was nearly 10-fold less than that with the corresponding double
mutant in wt IL5.
Finally, we also formed two asymmetric double
mutants in the sc IL5 system, namely (E13A (a), G110A (b)) and (E13A
(b), G110A (b)). In the former, the mutations were in one 4-helix
bundle, while in the latter, the two mutations were in different
bundles. In both cases, the effects on IL5R binding were similar
to that seen in the asymmetric E110A-alone mutants, while the decrease
in bioactivity was far greater when each bundle contained one mutation
than when one bundle had both while the other had none.
In this study, we constructed a tethered dimer of hIL5 with a
two-Gly linker. This single chain hIL5 has properties very similar to
native hIL5 dimer in both receptor chain binding and biological
activity. Single chain hIL5 offers an opportunity to study the effect
of changes in one of the two monomers, and hence one of the two 4-helix
bundles, on properties of hIL5.
Since hIL5 has a 2-fold palindromic
symmetry and binds to shIL5R with a 1:1 stoichiometry, we were
interested in investigating whether the IL5R
binding site is
formed by one or both monomers and how the topography of binding sites
for receptor
and
chains could lead to signal transduction.
Previous mutagenesis studies have mapped the IL5R
binding site to
residues near the central symmetry axis of the dimer, with residues
Glu-110, Trp-111, and Arg-91 being the most important (Tavernier et
al., 1995; Graber et al., 1995; Morton et al.,
1995). Accordingly, we made asymmetric mutations at these positions on
sc IL5. Our data do not fit well to a simple half-site reactivity
model, since mutants with residues changed only on one monomer did not
have complete wt activity (all showed
50% lower affinity). Also,
the increased K
values caused by these mutations
were mainly due to an increase in the dissociation rate k
(Table 1). This is not expected for a
half-site reactivity model, since the interaction face between ligand
and receptor should remain the same for an ideal half-site model.
However, each of the single-site mutants showed a much weaker decrease
in IL5R
binding than the double-site mutants. This result argues
against the possibility that residues on both monomers are equally
involved in IL5R
binding. Our data are consistent with a model in
which residues from both monomers form a central shared patch to
interact with IL5R
while the asymmetric receptor
chain uses
structural elements from this patch asymmetrically to stabilize the
IL5-IL5R
complex. However, we do not believe it possible to
conclusively define the detailed way in which
chain binding
occurs from this first asymmetric mutagenesis study. Even less can be
ventured for
chain binding from the E13A mutants, since we do not
yet know the stoichiometry of the IL5-IL5R
interaction.
A significant caveat in any interpretation of our asymmetric mutagenesis data is that we cannot be certain to what extent mutations may affect the folded structure of sc IL5. At least for the E110A and W111A mutants, several observations argue that gross conformational changes are unlikely. 1) Symmetrical mutants for these residues in wt IL5 have stabilities similar to that of native sequence wt IL5 (Morton et al., 1995). 2) Single chain IL5 had receptor binding and signal transduction activities equivalent to those of native sequence wt IL5. 3) The asymmetric mutants in sc IL5 for E110A and W111A were likely no more perturbed structurally than the double mutants. Furthermore, all of the mutants reported here were found to bind to the monoclonal antibody 24G9; this antibody appears to be conformationally dependent since it does not bind to reduced and alkylated hIL5 (data not shown). Hence, it is likely that the sc mutants we have made so far are not grossly unfolded. Nonetheless, we cannot rule out more subtle conformational changes as a factor in altered functional properties. This must await more deliberate structural analysis of purified mutants, a planned objective of future work.
In conclusion, we have
designed an IL5 system for asymmetric mutagenesis and have exemplified
the approach of asymmetric mutagenesis with sc IL5. The results
obtained suggest a model for IL5-IL5R receptor recognition in
which residues from both 4-helix bundle domains contribute to the
binding of a single molecule of IL5R
, possibly by formation of a
central patch as suggested previously (Morton et al., 1995).
However, the receptor may use this symmetrical patch asymmetrically to
stabilize the IL5-IL5R
complex due to the asymmetry of the
receptor molecule. It remains for further mutagenesis and, importantly,
structural analysis of key mutants, to deduce a more refined and
certain understanding of binding site topography for IL5R
and
ultimately the way this topography leads to signal transduction through
IL5R
.