(Received for publication, August 22, 1994; and in revised form, December 12, 1994)
From the
Mouse IL-12 acts on both mouse and human cells; human IL-12 acts only on human cells. This species specificity is determined by the p35 subunit of the IL-12 heterodimer. Since mouse and human p35 sequences are 60% identical, the determinants for the species specificity most likely reside in the nonhomologous sequences of mouse p35. To identify the regions on the p35 subunit interacting with the mouse IL-12 receptor, we constructed a series of chimeric mouse-human p35 molecules by replacing mouse sequences with the nonhomologous human counterparts. An IL-12 heterodimer containing a mouse-human p35 chimera with five residues changed in three discontinuous sites had drastically reduced (750-3000-fold) bioactivities on mouse cells. However, the competitive binding activity of the same mutant IL-12 heterodimer on mouse cells was only reduced 30-fold relative to wild-type IL-12. These findings therefore suggest that 1) the mouse p35 subunit participates in both receptor binding and signaling, 2) the mutations introduced into p35 affect signaling to a much greater extent than receptor binding, and 3) the five residues identified on p35 are required for interacting with the mouse, but not with the human IL-12 receptor and as such contribute extensively to the observed species specificity of IL-12.
Interleukin 12 (IL-12) ()is a cytokine produced
primarily by macrophages and B-cells(1) . The cytokine is a
pleiotropic immunologic regulator acting on T- and
NK-cells(2, 3, 4, 5) . Among all the
cytokines characterized thus far, IL-12 is unique because of its
heterodimeric structure. It consists of two disulfide-bonded subunits
with apparent molecular masses of 40 kDa and 35 kDa,
respectively(2, 6) . The cDNAs encoding the two
subunits of IL-12 have been cloned from both human (7, 8) and mouse (9) cells. It was
demonstrated that co-transfection of both p40 and p35 cDNAs is
necessary for secretion of bioactive IL-12. Comparison of the amino
acid sequences of human and mouse IL-12 subunits deduced from the cDNAs
reveals that there is a 60% identity between the p35 subunits and a 70%
identity between the p40 subunits(9) . High affinity receptors
for IL-12 are found mostly on T- and NK-cells(10) . A recent
study (11) described the cloning of a cDNA encoding an IL-12
receptor subunit and showed that this receptor is a member of the
cytokine receptor superfamily, with strong homologies to gp130 and the
receptors for G-CSF and leukemia inhibitory factor. However, this IL-12
receptor subunit (IL-12R
) binds IL-12 only with a low
affinity (K
= 2-5
nM) when expressed in COS cells; thus, an additional receptor
subunit is likely to be required to generate a high-affinity (K
= 50 pM), functional
IL-12 receptor complex (IL-12R)(11) .
Human IL-12 (hIL-12) does not act on mouse cells, whereas mouse IL-12 acts on both human and mouse cells(9) . By creating IL-12 hybrid molecules consisting of human p35 and mouse p40 or mouse p35 and human p40, it was found that the determinants for the species specificity of human IL-12 reside within the human p35 subunit(9) . A recent study using neutralizing monoclonal antibodies specific for the human p40 subunit has demonstrated that these antibodies can block the binding of the IL-12 heterodimer to its receptor(12) . It was also found that the p40 subunit alone is able to form homodimers which bind to IL-12R with affinities similar to the IL-12 heterodimer; however, no signal is transduced when p40 homodimer binds to IL-12R(13, 14) . Thus, the p40 subunit might be primarily involved in receptor binding while p35 may be more critical for signal transduction.
As a first step toward elucidating how the p35 subunit might be involved in receptor binding and/or signaling, we took advantage of the species specificity of hIL-12. A number of human/mouse p35 chimeras were constructed, expressed with wild-type (wt) mouse p40, and evaluated for bioactivity. A minimum of five amino acids unique to mouse p35 and located in three separate sites were found to be necessary for interacting with mIL-12R and transducing a signal in mouse cells. Our data suggest that p35 participates in both receptor binding and signaling, and that the five amino acids identified contribute extensively to the observed species specificity of IL-12.
Figure 1: Alignment of the amino acid sequences of mouse and human IL-12 p35 subunits. The alignment was obtained using the program GAP in GCG(30) . A vertical line between residues indicates identity, and a colon marks conservative residues. Regions A-D and a-e (see text) are boxed. The vertical double line indicates where N- and C-terminal halves were fused to create the MH and HM chimeras. The vertical single line indicates the boundary between murine and human sequences in the HMMM and MHMM chimeras. The arrow marks the end of the signal peptides.
In a first attempt to determine whether the region responsible for the species specificity is located on the N-terminal or C-terminal half of the p35 molecule, two chimeras were constructed by fusing either the N-terminal half of mouse p35 to the C-terminal half of human p35 (construct MH) or vice versa (construct HM) (Fig. 1). Surprisingly, both these mutants showed full activity when assayed on mouse lymphoblasts (Table 1). In the next set of mutants, using m35 as the template, each of the regions A-D and a-e (Fig. 1) was individually substituted with the corresponding human sequence. All these chimeras showed activities similar to wild-type mouse IL-12 (range for ratio human/mouse = 0.1-2.6; data not shown). These findings therefore suggested that the amino acid residues interacting specifically with the mouse IL-12 receptor may not be located in a single contiguous region.
The strategy subsequently adopted to identify the discontinuous sites is shown in Fig. 2. In contrast to the experiments described above, we now used the cDNAs encoding either the MH or HM variants of p35 as templates for mutagenesis. Within the mouse portion of MH, the four regions A-D were separately changed to the human sequences (Fig. 1). Mutants MH-A, MH-B, and MH-D were active on mouse lymphoblasts (ratio human/mouse = 0.4-0.9; data not shown); Western blot analysis demonstrated that mutant MH-C was not expressed. DNA sequencing revealed that the mutagenesis had introduced a frameshift mutation. Several additional attempts at generating the MH-C mutant also resulted in frameshifts. An alternative method to produce replacements in the C region is described below.
Figure 2: Strategy for the identification of a discontinuous species specificity determinant on the mouse p35 subunit of IL-12. Open boxes A-D and a-e denote human sequences and correspond to the areas shown in Fig. 1. Filled areas indicate mouse sequences. See text for details.
Another set of mutants using the MH template comprised single-residue substitutions in the mouse portion of the sequence other than regions A-D, where each of the nonhomologous single residues at positions 62, 74, 79, 80, 87, 94, 95, 100, 101, and 103 was changed one by one to the human residue (Fig. 1, numbering from the mouse p35 sequence); all of these mutants were active on mouse lymphoblasts (ratio human/mouse = 0.4-2.4; data not shown).
Using the HM template, regions a-e were then substituted with the corresponding human sequences. Three of those five mutants (HM-b, -c, and -e) had activities similar to wt mouse IL-12 (ratio human/mouse = 0.3-0.8; data not shown); only HM-a and HM-d showed significantly reduced levels of bioactivity on mouse lymphoblasts (Table 1). Region a contains 13 amino acids with three identical and one conservative change between human and mouse p35 (Fig. 1). In the next series of experiments, each of the remaining nine residues in region a was therefore changed on the HM-template. Similarly, region d is 11 amino acids long, with three identical residues and four conservative changes, leaving four residues to be changed on the HM-template. This analysis allowed us to identify residue H146 in region a and residue P184 in region d as the only amino acids that could significantly reduce the ability of the chimeras to induce proliferation in mouse cells (mutants HM-146K and HM-184F, see Table 1).
In order to identify residues important for the species specificity within the N-terminal half of mouse p35, two-step polymerase chain reaction mutagenesis was used to replace either the first or second quarter of mouse p35 with the corresponding human sequences (Fig. 1), resulting in chimeras HMMM and MHMM, respectively. The amino acid changes found to be important in the previous step, i.e. 146K and 184F, were also incorporated to yield the mutants HMMM-146K, HMMM-184F, MHMM-146K, and MHMM-184F. For HMMM-146K and HMMM-184F, the levels of bioactivity on mouse lymphoblasts decreased to about 5-10% of wild-type (Table 1), whereas MHMM-146K and MHMM-184F showed wild-type activity on both human and mouse lymphoblasts (ratio human/mouse = 1.1-1.5, data not shown).
To further define the
critical region(s) within the first quarter of the mutants HMMM-146K or
HMMM-184F, several additional p35 chimeras were constructed. Blocks A,
B, or C within the first quarter of mp35 ( Fig. 1and Fig. 2) were changed one by one; residue changes H146K or P184F
were also incorporated, resulting in mutants M-A-146K, M-B-146K,
M-C-146K, M-A-184F, M-B-184F, and M-C-184F. Only mutants M-C-146K and
M-C-184F showed a significant decrease in bioactivity on mouse
lymphoblasts (Table 1). Subsequently, the crucial residues in
region C were located to region C (Fig. 1) by
generating and analyzing mutants M-C
-146K and
M-C
-184F (see Table 1). The corresponding mutants
M-C
-146K and M-C
-184F showed no decrease in
bioactivity on mouse cells (ratio human/mouse = 0.5-0.7,
not shown). Within the C
region, three out of four residues
differ between human and mouse p35. Replacing each one of these three
amino acids together with 146K or 184F resulted in chimeras with
wild-type activities (human/mouse ratios = 0.3-1.7; data
not shown). Thus, the residues in the C
region are
important as one block for the bioactivity of mouse p35 on mouse cells.
Finally, generation of the triple mutants M-C-146K/184F and
M-C
-146K/184F demonstrated that changing a minimum of five
amino acids in three discontinuous sites on mouse p35 drastically
reduces the ability of the resulting mutant IL-12 to act on mouse cells (Table 1). The results thus demonstrated that residues located at
these three sites on mouse p35 are crucial for bioactivity on mouse
cells but not on human cells and suggested that these residues are
important elements in determining the species specificity of IL-12.
Figure 3:
SDS-polyacrylamide gel electrophoresis
analysis of purified mutant mouse IL-12 composed of wt mouse p40
subunit and the triple mutant p35 subunit mC146K184F. The
proteins were analyzed on a 10% polyacrylamide gel and silver-stained. Lane 1, crude, serum-free COS cell medium containing the IL-12
mutant. Lane 2, 100 ng of the purified mutant. Lane
3, 200 ng of purified wt mIL-12. Molecular mass standards
(Amersham) are indicated in kilodaltons. The arrow indicates
the position of mouse IL-12.
Because of the availability of
purified mutant IL-12, its bioactivity could be assessed at protein
concentrations much higher than what was possible with crude COS
supernatants. As shown in Fig. 4A, mouse IL-12
containing the p35 triple mutant mC146K184F was inactive at
concentrations below 10 pM in a mouse lymphoblast assay. At a
concentration of approximately 300 pM, the specific activity
of the mutant when compared to wt mouse IL-12 was reduced by about
750-fold (2.6
10
versus 2
10
units/mg). In contrast, the mutant showed only a slightly reduced
specific activity relative to wt IL-12 when assayed on human
lymphoblasts (4.4
10
versus 2
10
units/mg, 4-fold decrease, Fig. 4B). The
ratios of the specific activities as determined in human and mouse
lymphoblast assays are 1 for wt mouse IL-12 and 170 for the purified
mutant IL-12. These results are thus consistent with the earlier
results obtained with crude material from COS supernatants (Table 1).
Figure 4:
Comparison in the lymphoblast
proliferation assay of purified wt human and mouse IL-12 and purified
mouse IL-12 containing the p35 subunit triple mutant
mC146K184F. A, mouse lymphoblast assay; B, human lymphoblast assay. Incorporation of tritiated
thymidine was plotted as a function of IL-12 concentration. The results
from one of three essentially identical experiments are shown.
--
, wt human IL-12;
- -
- - -
, wt mouse IL-12;
- - -
, mouse
IL-12 mutant. Each point represents triplicate
samples.
The ability to induce IFN- production by T-
and NK-cells has been recognized as one of the key bioactivities of
IL-12 both in vitro and in
vivo(2, 3, 4, 5, 22) .
The mutant was therefore tested in assays measuring the induction of
IFN-
production by mouse and human cells. Fig. 5A shows
that on mouse cells, this activity of the mutant was drastically
reduced when compared to wt mouse IL-12. As demonstrated before, human
IL-12 was unable to stimulate mouse cells to produce
IFN-
(9) . In contrast, the production of IFN-
from
human cells was induced by hIL-12, mIL-12, and the mutant (Fig. 5B), although the mutant was 4-10-fold less
active than wt mIL-12. Thus, the IL-12 mutant is similarly defective in
its ability to induce mouse lymphoblast proliferation or production of
IFN-
.
Figure 5:
IL-12 induced IFN- production by
mouse splenocytes (A) or by human peripheral blood mononuclear
cells (B). Cells were incubated with the indicated
concentrations of purified human IL-12 (
), wt mouse IL-12
(
), and mouse IL-12 mutant (
) (see ``Materials and
Methods'' for details). Similar results were obtained in one
additional assay on mouse splenocytes and two additional assays on
human peripheral blood mononuclear cells.
In order to analyze whether the failure of the mutant to
activate mouse lymphocytes was due to the inability to bind to mouse
IL-12 receptors or inability to transduce a signal, we performed
competitive binding studies using both mouse and human lymphoblasts. As
shown in Fig. 6A, binding of labeled mouse IL-12 (41
pM) to mouse IL-12 receptors in the presence of increasing
amounts of either the mutant or wt human or mouse IL-12 showed that
human IL-12 has a very low affinity for mouse IL-12R (IC = 130 nM). The affinity of the mutant IL-12 for
the receptor is reduced about 30-fold when compared to wt mouse IL-12
(IC
values of 1.5 nMversus 45
pM). In contrast, the affinity of the mutant IL-12 for the
human IL-12 receptor is only reduced about 5-fold (IC
values of 270 pMversus 60 pM, Fig. 6B). The data thus suggest that the changes
introduced into the p35 triple mutant affect both receptor binding and
signaling; however, the signaling process appears to be affected to a
much greater degree.
Figure 6:
Analysis of competitive binding to mouse
and human cells by purified wt and mutant mouse IL-12. A,
inhibition of binding of I-labeled mouse IL-12 (41
pM) to mouse lymphoblasts by increasing amounts of wt human,
wt mouse, or mutant IL-12. B, inhibition of binding of
I-labeled mouse IL-12 (39 pM) to human
lymphoblasts by increasing amounts of wt human, wt mouse, or mutant
IL-12.
- - -
, wt human IL-12;
-
, wt mouse IL-12;
- - - - -
,
mouse IL-12 mutant. Each point is the average of triplicate samples;
one of two experiments with similar results is
shown.
By generating mouse-human chimeras of the p35 subunit, which were co-expressed with wt mouse p40 subunit, we were able to identify the major residues involved in the specific interactions with mIL-12R. The advantage of using mouse/human chimeras instead of deletions or insertional mutations was to avoid dramatic conformational changes in the resulting p35 subunits. All of the IL-12 chimeras described in this study were active on human lymphoblasts, indicating that the mutations had no significant detrimental effects on the conformation of the resulting IL-12 proteins.
One of the mutants,
mC146K184F, incorporated only five amino acid changes in
three different areas (K57E, H58F, S60P, H146K, and P184F). The ability
of this mutant to induce either cell proliferation or production of
IFN-
in mouse cells when compared to wt mIL-12 was dramatically
(750-3000-fold) reduced. On the other hand, the same activities
when measured on human cells were only slightly impaired
(4-10-fold). The data thus indicate that these five amino acid
residues are required for signaling on mouse cells only and as such
constitute the basis for the observed species specificity of IL-12. The
slightly reduced bioactivities of the mutant IL-12 on human cells could
conceivably be caused by minor conformational changes; such changes
could affect either the p35 subunit alone or could also have an impact
on the dimerization of the mutant p35 with the wt p40 subunit.
Competitive binding experiments using mouse cells showed that the
IC of the purified mutant was reduced about 30-fold
relative to wt mIL-12, indicating that the p35 subunit of IL-12 is
indeed involved in binding to the mouse IL-12 receptor. A similar
discrepancy between binding and signaling properties of specific
mutants was also observed for IL-6(32, 33) . Such a
discrepancy could be due to the fact that in order to signal, the five
residues that we have identified on mouse p35 have to interact with
specific residues on mouse IL-12 receptors. These interactions could
contribute partially to the ligand-receptor binding; however, the major
contributions to binding would come from the p40 subunit and from the
residues common to human and mouse p35 sequences. It has been
demonstrated that the p40 subunit alone is able to form homodimers
which bind to IL-12R with affinities similar to the IL-12 heterodimer;
however, no signal is transduced(13, 14) . Therefore,
the results of our mutagenesis studies of the p35 subunit support the
hypothesis that the p40 subunit is primarily involved in receptor
binding while p35 may play more of a role in signal transduction, with
only a partial contribution to receptor binding.
Although the p35 mutant shows significantly reduced bioactivities (750-3000-fold) in two different assay systems on mouse lymphocytes and a moderately reduced binding affinity for the receptor (30-fold), it could not inhibit the proliferation of mouse lymphoblasts induced by 3 pM wt IL-12, even when present at a concentration of 333 pM (data not shown). This finding may be explained by the fact that the mutant needs to be present in an at least 30-fold excess over wt IL-12 in order to compete with wt IL-12 for binding to the receptor; however, when assayed by itself at concentrations above 10 pM, the mutant has slight agonistic properties and stimulates the proliferation of mouse lymphoblasts (Fig. 4A). Thus, because of these weak agonistic properties, the mutant cannot function as an antagonist.
Little is known about the secondary and tertiary
structure of IL-12 besides the fact that the p35 subunit shares a
limited sequence homology with IL-6 and G-CSF(34) , and that
p40 resembles the extracellular domain of the IL-6
receptor(9, 35) . Since the three-dimensional
structure of G-CSF has been determined by x-ray
crystallography(36) , a structural model for p35 was built
based on its homology with G-CSF (Fig. 7). The positions of the
four antiparallel helices in this model are similar to the predictions
based on the methods of Chou and Fasman(27) ,
Bazan(28) , and Savino et al.(29) , the latter
two being based on an alignment of p35 with IL-6. As shown in Fig. 7, the five amino acid residues identified in this study
form a cluster on one side of the molecule. Region C is
located at the C terminus of helix A close to the beginning of the A-B
loop, residue His-146 maps to the beginning of helix C and residue
Pro-184 is located on the C-D loop. Computer analysis using the program
PeptideStructure in GCG (30) indicates that the residues in
these three regions have a high probability for being located on the
surface of the protein, supporting the model that these residues may
directly interact with the receptor.
Figure 7:
Predicted tertiary structure of the p35
subunit of mouse IL-12. The model was built based on the limited
homology between the p35 subunit and G-CSF and the known crystal
structure of G-CSF(36) . The -helices are shown as cylinders. In the model shown, helix A extends from amino acid
32 to 57, helix B from residue 117 to 136, helix C from residue 145 to
168, and helix D from residue 188 to 215 (see Fig. 1for amino
acid numbering). The locations of the three sites C
,
His-146, and Pro-184 are indicated. See text for more details.
Based on our results, it is tempting to speculate about the potential differences between human and mouse IL-12 receptors. One could postulate that there must be at least two sets of amino acid residues on mouse p35 that can interact with IL-12 receptors. One set of amino acids might be common to both human and mouse p35, necessary for the interactions with the mouse receptor. The same set would be necessary and sufficient for interaction and signaling on the human receptor. However, in order to activate mouse cells, an additional set of amino acid residues unique to mouse p35 might be required. This set of residues is responsible for the observed species specificity of IL-12.
The IL-12/IL-12 receptor system is
more complex when compared to some of the other cytokine systems
because both IL-12 ligand and IL-12 receptor are heterodimeric in
nature(11) . What role does each of the IL-12 subunits play in
interacting with the subunits of the receptor? It was concluded that
the p40 subunit is directly involved in receptor binding because
monoclonal antibodies specific for the p40 subunit blocked receptor
binding and bioactivity on human lymphoblasts(12) . The same
antibodies also inhibit IL-12 binding to the recently cloned human low
affinity IL-12R, (
)again indicating a direct
interaction between this receptor
-subunit and p40. On the other
hand, the contribution of the p35 subunit to receptor binding and
signaling has been less clear. The difficulty in assessing the role of
p35 is at least partly due to the fact that antibodies to native p35
are difficult to generate. Most of the monoclonal antibodies that have
resulted from immunizing animals with native IL-12 have shown
specificity for p40, and only a few antibodies were specific for the
IL-12 heterodimer(1, 12) . Antibodies raised against Escherichia coli-expressed p35 do not recognize native IL-12
efficiently and do not neutralize IL-12(1) . An initial
characterization of the p35 subunit that does not rely on specific
antibodies has now shown that mouse p35 participates both in receptor
binding and signaling. The knowledge obtained from this study will be
helpful for further dissecting the mechanism of interactions between
the IL-12 heterodimers with the IL-12R complex.