(Received for publication, April 14, 1995; and in revised form, June 19, 1995)
From the
We showed previously that replacement of Lys-145 in the IL-1
receptor antagonist (IL-1ra) with Asp resulted in an analog (IL-1ra
K145D) with partial agonist activity. To identify additional amino
acids that affect IL-1 bioactivity, we created second site mutations in
IL-1ra K145D. Substitutions of single amino acids surrounding position
145 were made; none of these substitutions increased the bioactivity of
IL-1ra K145D. However, the insertion of the -bulge (QGEESN) of
IL-1
at the corresponding region of IL-1ra K145D resulted in a
3-4-fold augmentation of bioactivity. An additional increase in
agonist activity was observed when the
-bulge was coexpressed with
a second substitution (His-54
Pro) in IL-1ra K145D. We also show
that the bioactivity of both IL-1ra K145D and the triple mutant IL-1ra
K145D/H54P/QGEESN is dependent on interaction with the newly cloned
IL-1 receptor accessory protein.
The interleukin 1 (IL-1) ()family of cytokines
includes two potent mediators of inflammatory and immune responses,
IL-1
and IL-1
(1) . The third member of this family is
a naturally occurring inhibitor of IL-1 responses termed the IL-1
receptor antagonist (IL-1ra)(2, 3) . IL-1
and
IL-1
mediate cellular signals by binding to an 80-kDa cell surface
receptor, the Type I IL-1 receptor (Type I IL-1R), which is found
mainly, but not exclusively, on T cells and
fibroblasts(4, 5, 6) . IL-1
and
IL-1
also bind to a 68-kDa cell surface receptor found
predominantly on B cells and neutrophils (the Type II IL-1
receptor)(5, 6, 7) . Recent studies indicate
that only the Type I IL-1R is capable of transducing an IL-1 signal,
whereas the Type II IL-1R is dispensable for signaling and may act as a
decoy receptor(8, 9, 10) . IL-1ra binds to
the Type I IL-1R with an affinity equivalent to the IL-1 agonists and
inhibits the binding of both IL-1
and
IL-1
(2, 3, 11, 12) . In
contrast to IL-1
and IL-1
, IL-1ra elicits no discernible IL-1
associated
responses(2, 11, 13, 14) ;
therefore, IL-1ra acts as a pure receptor antagonist.
The mechanism
by which IL-1ra can interact with the Type I IL-1R but not trigger a
biological response is still unclear. The amino acid sequence of IL-1ra
is 19% identical to that of IL-1 and 26% identical to that of
IL-1
(3) . Despite this relatively low sequence
conservation, all three ligands share a common tertiary structure as
determined by x-ray crystallography and
NMR(15, 16, 17, 18, 19, 20) .
Each of these proteins is comprised of 12
-strands, which form a
-barrel structure closed at one end. Thus, amino acid sequence
alignments and structural comparisons among these ligands have not shed
light on the basis for their functional differences.
Because the
Type I IL-1R appears to be essential for IL-1-induced biological
responses, the molecular interactions of the IL-1 ligands with this
receptor have been well characterized. Extensive mutagenesis studies of
both IL-1 and IL-1
have identified the amino acid residues
important for binding of these ligands to the Type I IL-1R (21, 22, 23, 24, 25, 26) .
In these studies, all of the residues involved in binding to the Type I
IL-1R, although not contiguous, are located on one face of the IL-1
structure (the open end of the
-barrel).
Mutagenesis studies
have indicated that residues essential for IL-1 biological activity
could be distinguished from the residues required for receptor binding.
Substitution of Thr-9 (numbering of the mature protein)(27) ,
Arg-11(28) , and Asp-145 (29) in IL-1 each
resulted in significantly reduced bioactivity with little effect on
binding to the receptor. These amino acid residues are located in
regions that are distinct and at a distance from the Type I IL-1R
binding site. To date, the molecular mechanism(s) by which these
residues contribute to IL-1 agonist activity have not been elucidated.
In addition to the residues described above, a region of charged
amino acids (a -bulge) positioned between
-strands 4 and 5
has been implicated in the ability of IL-1
to transduce a signal
through the IL-1R(30) . The
-bulge residues (Gln-48 to
Asn-53) form a protrusion on the edge of the open end of the
-barrel adjacent to the receptor binding epitope. Direct evidence
for the importance of these amino acids comes from mutagenesis studies
in which deletions or substitutions of residues in this region reduced
IL-1
agonist activity without affecting receptor
binding(31, 32) . In addition, one group has suggested
that a synthetic peptide containing the
-bulge residues has
immunostimulatory
activity(33, 34, 35, 36) , although
independent confirmation of these results is not available. A similar
-bulge is present at the homologous position in IL-1
, but
absent in IL-1ra.
Previously, we provided evidence for a role for
Asp-145 in IL-1 agonist activity(29) . Whereas the
substitution of Lys for Asp-145 of IL-1
greatly reduced agonist
activity, we showed that the corresponding substitution in IL-1ra
(Lys-145
Asp) conferred partial agonist activity to the receptor
antagonist. In the present study, we extend our examination of the
regions of IL-1 important for agonist activity by creating second site
mutations in IL-1ra K145D that further increase agonist activity. In
addition, we present evidence to suggest that the lack of agonist
activity of IL-1ra is a result of its inability to interact with the
newly cloned and characterized IL-1 receptor accessory protein (IL-1R
AcP)(37) .
Purified human IL-1ra
(huIL-1ra) was supplied by Synergen Inc. Purified human IL-1
(huIL-1
) was the gift of S. Roy, Hoffmann-La Roche Inc.
In a previous study, we showed that the substitution of a
single amino acid (Asp-145 Lys) in huIL-1
reduced
bioactivity by
90%, while retaining 100% binding to the Type I
IL-1R(29) . We noted that this analog of IL-1
, designated
IL-1
D145K, had a phenotype similar to IL-1ra, i.e. binding to the Type I IL-1R, no binding to the Type II IL-1R, and
reduced biological activity compared to wild-type
IL-1
(29) . Alignment of the amino acid residues of
IL-1
and IL-ra indicated that Asp-145 of IL-1
aligned with
Lys-145 of IL-1ra. When the corresponding mutation (K145D) was made in
IL-1ra, this mutant analog (IL-1ra K145D) maintained receptor binding
and gained partial agonist activity(29) . These observations
implicated Asp-145 in IL-1
agonist activity and suggested that
relatively minor alterations of the protein could have marked effects
on biological activity. In this study, we extend our examination of
regions important for IL-1 agonist activity by constructing second site
mutations in the IL-1ra K145D analog using PCR-based site-directed
mutagenesis. Each analog was examined for Type I IL-1R binding on EL-4
cell membranes (as a measure of conformational integrity) and for
agonist activity in the D10 proliferation assay(29) . We were
interested in those analogs that gained significant agonist activity
(>2-fold increase) while maintaining receptor binding.
As shown in Table 1, none of the substitutions at the five positions listed above resulted in the desired effect. The V18S analog and both substitutions at Cys-122 had little or no effect on either the binding or bioactivity of IL-1ra K145D. These results indicate that the identity of these residues is not critical to the structure or activity of the protein. These data also suggest that Cys-122 is not required for disulfide bond formation. The substitution of Thr-108 with Lys resulted in a <2-fold decrease in Type I IL-1R binding and a 3-fold decrease in bioactivity compared to the IL-1ra K145D analog, suggesting either that Thr-108 is moderately important for the structure of the protein or that Lys is not tolerated in this position.
More significant effects were seen
when substitutions were made at Cys-116 in IL-1ra K145D. Replacement of
Cys-116 with Phe resulted in the complete loss of bioactivity, whereas
full receptor binding activity was maintained. The observation that
receptor binding activity is preserved indicates that there are no
gross alterations in the overall structure of the C116F analog. The
complete loss of bioactivity seen with the Cys-116 Phe
substitution is difficult to understand. Cys-116 in IL-1ra overlaps
Phe-117 in IL-1
, and it appears that the phenyl ring in the C116F
analog could be accommodated in the IL-1ra structure.
Varying results were obtained depending on whether Tyr-147 was substituted with Thr or with Gly in the IL-1ra K145D analog. The Y147T analog lost all detectable activity (both binding and bioactivity), whereas the Y147G analog lost all bioactivity but retained 100% binding. These data suggest that Tyr-147 is important for bioactivity of IL-1ra K145D, either by direct interaction with signaling proteins or for local conformation. These data also indicate that the overall structure of IL-1ra K145D will not tolerate Thr at position 147.
Since none of
the single amino acid substitutions discussed above resulted in
increased bioactivity, the sequence alignments of IL-1ra and IL-1 (3) were examined for regions in which short segments of
IL-1
residues were absent from IL-1ra. One such region includes
the six amino acids comprising the loop between the fourth and fifth
-strands of IL-1
(30, 43) . These six amino
acids (Gln-48 to Asn-53) have been termed a
-bulge and have been
implicated in early IL-1 signaling events and the immunostimulatory
properties of
IL-1(30, 33, 34, 35, 36) .
These six amino acids or a similar
-bulge structure are absent
from IL-1ra. Therefore, the
-bulge region from huIL-1
was
inserted into IL-1ra K145D either after Ile-51 or after Pro-53; the
position of these two amino acids in IL-1ra aligns with the location of
the
-bulge in IL-1
. Two sites were chosen for insertion due
to ambiguities in the structural alignment of IL-1ra and IL-1
in
this region(3) . The insertion of the
-bulge (QGEESN)
after either position 51 or 53 of IL-1ra K145D resulted in analogs that
retained full Type I IL-1R binding and increased bioactivity
3-4-fold (Table 1).
We next tested the ability of the
QGEESN insertion to impart agonist activity to IL-1ra in the absence of
the K145D mutation. The -bulge was inserted either after amino
acid 51 or 53 of IL-1ra. None of the plasmid clones with the insertion
at position 51 of IL-1ra produced the appropriate protein, suggesting
that this analog is extremely unstable in E. coli. We were
able to isolate clones with the insertion at position 53, which
produced protein; however, these analogs retained only 10-20% of
the Type I IL-1R binding activity (data not shown). These data indicate
that the insertion of QGEESN into IL-1ra at either position 51 or 53
results in gross distortion of the structure. This structural
alteration was not seen in the IL-1ra K145D/QGEESN analogs.
Further
analysis of the QGEESN insertion analogs revealed a surprising variant.
In the case of the QGEESN insertion after Ile-51 of IL-1ra K145D, five
individual isolates were analyzed. Four isolates were similar; each
yielded increases in bioactivity in the range of 3-4-fold (Table 1). The fifth isolate had an 8-fold increase in
bioactivity. The plasmid DNA encoding this fifth isolate was isolated
and the nucleotide sequence of the mutated region determined. The DNA
sequence revealed that, in addition to the QGEESN insertion, the His-54
codon was mutated to encode Pro in this clone, probably as a result of
the PCR amplification used to create this analog. We then analyzed the
contribution of the H54P substitution by incorporating this
substitution into the IL-1ra K145D analog. As is shown in Table 1, altering His-54
Pro in IL-1ra K145D results in a
modest 2-fold increase in bioactivity while maintaining receptor
binding. This result indicates that the increased bioactivity of the
triple mutant IL-1ra K145D/H54P/QGEESN is a result of the additive
effects of the individual mutations. Additionally, when the IL-1ra
K145D/H54I analog (replacing His-54 with the aligned IL-1
amino
acid Ile) was made and analyzed, all bioactivity was lost (Table 1). These data indicate that the 2-fold increase in
activity is due to the introduction of Pro and not the removal of His
at position 54 in IL-1ra K145D.
Figure 1:
Competitive binding of
purified IL-1 proteins to EL-4 membranes. Competitive binding to murine
Type I IL-1R was determined by incubating EL-4 membranes with 50 pMI-IL-
and varying concentrations of purified
IL-1 proteins. The percent of radiolabeled ligand bound was determined
as described under ``Materials and Methods.'' IL-
(
), IL-1ra (
), IL-1ra K145D (
), and IL-1ra
K145D/H54P/QGEESN (
).
The bioactivity of each of these
purified proteins was then tested in the D10 cell proliferation
assay(42) . The dose-response curves for IL-1, IL-1ra,
IL-1ra K145D, and IL-1ra K145D/H54P/QGEESN proteins are shown in Fig. 2. The ED
value for each of these proteins is
listed in Table 2. As reported previously, IL-1ra is completely
devoid of bioactivity(2, 11) , whereas IL-1ra K145D
has
0.6% activity compared to IL-1
(29) . The triple
mutant IL-1ra K145D/H54P/QGEESN has 5.3% bioactivity compared to
IL-1
, confirming that the H54P +
-bulge insertion
mutations result in
9-fold increase compared to the bioactivity of
IL-1ra K145D. As we have noted previously for IL-1
and
IL-1
(23, 25) , these data using purified proteins
confirm the results obtained with crude extracts of the analog proteins (Table 1).
Figure 2:
Proliferative activity of purified IL-1
proteins. The ability to induce the proliferation of D10.N4.M cells was
determined as described under ``Materials and Methods.''
IL-1 (
), IL-1ra (
), IL-1ra K145D (
), and
IL-1ra K145D/H54P/QGEESN (
).
Fig. 3shows
the results of inhibition of the bioactivity of IL-1 and the two
IL-1 analogs by mAb 4C5 (anti-muIL-1R AcP), mAb 35F5 (anti-muType I
IL-1R)(5) , or a control mAb 7B2 (anti-IL-12)(44) . As
described under ``Materials and Methods,'' the amounts of
IL-1
and the IL-1ra analog proteins used in this assay were chosen
to induce equivalent amounts of proliferation (2-3-fold above
ED
). PanelA shows that mAb 35F5
inhibits the bioactivity of IL-1
in a dose-dependent manner,
whereas the control mAb 7B2 has no inhibitory activity. The mAb 4C5
(anti-muIL-1R AcP) also is capable of blocking completely the activity
of IL-1
, although it appears to be less potent than mAb 35F5. The
same pattern of inhibition by these mAbs is seen for both IL-1ra K145D
and IL-1ra K145D/H54P/QGEESN (Fig. 3, B and C,
respectively). These data indicate that, like IL-1
, the
bioactivity of IL-1ra K145D and IL-1ra K145D/H54P/QGEESN requires
interaction with both the Type I IL-1R and IL-1R AcP on D10 cells. One
interesting difference between IL-1
and the IL-1ra analogs was
noted. There was an
25-fold increase in the amount of mAb 4C5
needed to neutralize the activity of IL-1
(IC
2.5 µg/ml), compared to the amount of mAb 4C5 that
inhibited the IL-1ra analogs (IC
0.1 µg/ml).
Figure 3:
Inhibition of bioactivity by mAbs to Type
I IL-1R and IL-1R AcP. D10.N4.M cells were incubated with 5 pg/ml
IL-1 (panelA), 1000 pg/ml IL-1ra K145D (panelB), or 100 pg/ml IL-1ra K145D/H54P/QGEESN (panelC). Serial dilutions of mAb 35F5 (anti-muType
I IL-1R,
), mAb 4C5 (anti-muIL-1R AcP,
), or control mAb
7B2 (
) were added 30-60 min prior to each IL-1 protein.
After 3 days, cells were pulsed with [
H]thymidine
and harvested. The percent activity (proliferation in presence versus absence of each mAb) was determined as described under
``Materials and Methods.''
Amino acid changes that confer agonist activity to IL-1ra
have yielded insight into the regions of the IL-1 ligands that are
important for bioactivity. We have generated second site mutations in
the partial agonist IL-1ra K145D (29) in an attempt to increase
agonist activity while retaining IL-1R binding. We have increased the
bioactivity of IL-1ra K145D 3-fold by the insertion of the IL-1
-bulge into IL-1ra K145D. This activity is further augmented
2-fold by the substitution of His-54 with Pro. Our data suggest that
the agonist activity of IL-1ra K145D and IL-1ra K145D/H54P/QGEESN is
likely due to the ability of these analogs to interact with the newly
cloned IL-1 receptor accessory protein(37) .
Structural
alignment of IL-1, IL-1
, and IL-1ra reveals that both
IL-1
and IL-1
have a
-bulge structure between the fourth
and fifth
-strands, whereas no such structure is found in IL-1ra.
We therefore inserted the six amino acids comprising the IL-1
-bulge into IL-1ra K145D in an attempt to augment agonist
activity. There have been suggestions from previous studies that the
-bulge region of IL-1
(QGEESN, residues 48-53) may be
important for bioactivity. Simoncsits et al.(32) have
recently shown that deletion of amino acids 52-54 (SND) in
IL-1
reduces Type I IL-1R binding by 10-fold and biological
activity by 1000-fold. In addition, the substitution of the
-bulge
with loops from various protease inhibitors led to a reduction in
bioactivity without a significant effect on binding to EL-4
cells(31) . One group has also suggested that a synthetic
peptide derived from IL-1
(VQGEESNDK), which contains the six
-bulge amino acids, has immunostimulatory but no inflammatory
effects normally associated with
IL-1(33, 34, 35) . Additionally, this same
group has shown that the insertion of VQGEESNDK into recombinant human
ferritin H chain and recombinant flagellin from Salmonella muenchen increased the immunogenicity of these antigens in
mice(36) .
We compared the activities of purified IL-1,
IL-1ra K145D, and IL-1ra K145D/H54P/QGEESN to further characterize the
changes that lead to agonist activity. Since we have postulated that
the IL-1R AcP is involved in IL-1 signal transduction, we tested the
ability of mAb 4C5 (anti-muIL-1R AcP) to inhibit the bioactivity of the
purified IL-1ra mutants. As expected, the bioactivity of IL-1
was
fully inhibited by mAb 4C5 (Fig. 3A). This mAb was also
able to inhibit completely the activity of both IL-1ra K145D and IL-1ra
K145D/H54P/QGEESN (Fig. 3, B and C).
The observation that 4C5 inhibits the activity of the IL-1ra analogs
confirms that the agonist activity of IL-1ra K145D and IL-1ra
K145D/H54P/QGEESN involves an association with the IL-1R AcP. These
data would suggest that IL-1ra fails to elicit biological responses
because it is unable to interact productively with the IL-1R AcP.
Consistent with this hypothesis, we have shown previously, using
protein cross-linking, that both IL-1
and IL-1
associate with
a complex of the Type I IL-1R and IL-1R AcP on the cell surface,
whereas IL-1ra associates only with the Type I IL-1R(37) .
Cross-linking studies to demonstrate direct interactions between the
IL-1ra analogs and the IL-1R AcP are in progress.
Since the
bioactivity of IL-1ra K145D is completely inhibited by mAb 4C5, it
appears that this single amino acid substitution is sufficient to allow
IL-1ra to establish a productive interaction with the IL-1R AcP. It is
not clear whether this association is through the direct binding of
Asp-145 to the IL-1R AcP or if the K145D substitution affects local
secondary structure, allowing other amino acids to interact. The latter
possibility is unlikely, since the crystal structure of the IL-1
D145K analog is identical to wild-type IL-1
(23) . The
mechanisms by which the insertion of QGEESN and the H54P substitution
increase agonist activity of IL-ra are also not known. Interestingly,
the
8-9-fold increase in bioactivity of IL-1ra
K145D/H54P/QGEESN compared to IL-1ra K145D appears to be the cumulative
effect of all three mutations, suggesting that each mutation can
contribute in an additive manner to enhance agonist activity.
The
cumulative effects of these three mutations are also interesting since
their positions on the IL-1ra protein appear to be spatially separated (Fig. 4). Ile-51 and His-54 are located on the open face of the
-barrel of IL-1ra, whereas Lys-145 is located away from the open
barrel end(20) . In IL-1
, the same relative positions of
the
-bulge and Asp-145 are observed (15, 16, 18, 30) with the two
regions separated by the known Type I IL-1R binding site (Fig. 4). As discussed above, it is difficult to assess whether
the
-bulge or residue 145 is important for direct intermolecular
interaction (e.g. with the IL-1R AcP) or to maintain the local
conformation.
Figure 4:
Location of amino acids important for
agonist activity. The structures of huIL-1 (left; (15) ) and IL-1ra (right; Footnote 2) are shown. The
backbone of IL-1
is indicated by the ribbon in blue. The
backbone of IL-1ra is indicated by the ribbon in red. Residues
in magenta were previously identified as essential for
IL-1
binding to the murine Type I IL-1R(23) . Residues in yellow in IL-1ra denote amino acids where substitution
(His-54, Lys-145) or insertion (Ile-51) resulted in an increase in
agonist activity. The location of Asp-145 and the
-bulge residues
in the IL-1
structure are shown in cyan.
A direct measurement of binding of the IL-1ra analogs
to the IL-1R AcP is not possible because the accessory protein does not
bind IL-1 ligands except in the presence of the Type I
IL-1R(37) . However, the observation that 25-fold more mAb
4C5 is needed to inhibit IL-1
bioactivity on D10 cells compared to
the amount of mAb 4C5 necessary to block the IL-1ra analogs (Fig. 3) suggests that the interaction of the analogs with the
IL-1R AcP may be of lower avidity than that of IL-1
. The
relatively weak interaction of the IL-1ra analogs with the accessory
protein would then account for their lower agonist activity compared to
IL-1
.
We attempted to assess the ability of the QGEESN
insertion to enhance agonist activity of IL-1ra in the absence of the
K145D substitution. Whereas the insertion of the -bulge into
IL-1ra K145D augmented agonist activity, the insertion of the
-bulge into IL-1ra appeared to reduce receptor binding as well as
bioactivity. We also attempted to remove the six
-bulge amino
acids from IL-1
. This deletion led to very poor protein production
and poor receptor binding, (
)suggesting that this region
plays a role in the structural stability of IL-1
.
In addition
to the -bulge insertion, an alternative strategy was the
substitution of amino acids in IL-1ra that were predicted to be in
close proximity to Lys-145 with the corresponding aligned residues in
IL-1
. None of the single amino acid substitutions that we created
yielded a significant increase in the biological activity of IL-1ra
K145D (Table 1). The Tyr at position 147 appears to be the most
important of the amino acids we tested since substitution of this
residue abolished the biological activity of IL-1ra K145D. Substitution
at this position with Thr also abolished binding, indicating that the
structure of this analog was drastically altered. Residue 116 also
appears to be important since substitution at this site with Phe
abolished the biological activity of IL-1ra K145D. It is unclear what
role(s) Tyr-147 and Cys-116 play in agonist activity since neither
residue is conserved in IL-1
or IL-1
.
The observation that
the His-54 Pro substitution is able to augment agonist activity
of both IL-1ra K145D and IL-1ra K145D/QGEESN (Table 1) indicates
that the residue at position 54 plays an important role independent of
the
-bulge insertion. Although it is offset by one residue in the
structural alignment, the mutant Pro-54 in IL-1ra may mimic effects of
Pro-57 in IL-1
. We have not determined whether the His-54
Pro substitution enhances activity in the absence of the K145D
substitution. The analogous substitution in IL-1
(Ile-56
Pro) led to a complete loss of binding and bioactivity. These data are
not surprising, since Ile-56 has been identified as a critical residue
in the binding site in IL-1
for the Type I IL-1R(23) .
These data suggest that His-54 in IL-1ra may not play the same role as
Ile-56 in IL-1
.
The effect of insertion of the -bulge into
IL-1ra K145D confirms the importance of this region for IL-1 biological
activity. The inhibition of both IL-1ra K145D and IL-1ra
K145D/H54P/QGEESN by mAb 4C5 (anti-muIL-1R AcP) supports the hypothesis
that IL-1R AcP is involved in IL-1 agonist activity. Our results also
begin to elucidate the mode of action of IL-1ra. The antagonist
function of IL-1ra is most likely the result of two characteristics: 1)
the ability to compete with IL-1
and IL-1
for binding to the
Type I IL-1R, and 2) the inability to form a productive complex with
the Type I IL-1R and IL-1R AcP. Since IL-1ra K145D/H54P/QGEESN only
regains
5% of the agonist activity of IL-1
, insertion or
substitution of other amino acid residues may lead to additional
increases in biological activity.