©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Structure-Function Analysis of the p35 Subunit of Mouse Interleukin 12 (*)

(Received for publication, August 22, 1994; and in revised form, December 12, 1994)

Jun J. Zou David S. Schoenhaut (§) Daisy M. Carvajal Rajeev R. Warrier David H. Presky Maurice K. Gately Ueli Gubler (¶)

From the Department of Inflammation/Autoimmune Diseases, Hoffmann-La Roche Inc., Nutley, New Jersey 07110-1199

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

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.


INTRODUCTION

Interleukin 12 (IL-12) (^1)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.


MATERIALS AND METHODS

Plasmids, Recombinant IL-12 Proteins and Antibodies

The expression plasmid pEF-BOS (15) was obtained from Dr. S. Nagata at the Osaka Bioscience Institute. Purified recombinant human and mouse IL-12 proteins were obtained from F. Podlaski and corresponding polyclonal anti-IL-12 antisera from Dr. J. Hakimi (Dept. of Inflammation/Autoimmune Diseases, Hoffmann-La Roche). The rat monoclonal antibody 2B5 was raised against purified recombinant mouse IL-12 and is specific for the IL-12 heterodimer. (^2)

Mutagenesis of the p35 Subunit cDNAs

Chimeric p35 cDNAs (constructs HM, MH, HMMM, or MHMM, see text) were generated using the polymerase chain reaction-based overlap extension technique (16) and Pfu polymerase (Stratagene). The other mutants involving replacements of smaller sequence blocks were generated by Kunkel mutagenesis(17) , using synthetic mutagenic oligonucleotides and single-stranded pEF-BOS containing p35 cDNA as the template. For confirmation, all the constructs were sequenced using an automated 373A DNA sequencer (Applied Biosystems Inc.).

Expression and Quantitation of Chimeric IL-12 Proteins in COS Supernatants

Mutant and wild-type IL-12 subunit cDNAs were subcloned into the mammalian expression vector pEF-BOS (15) using the XbaI sites. Plasmids encoding p35 and p40 subunits were co-transfected into COS cells by the DEAE/dextran method(18) . Three days after transfection, COS media were collected, and levels of secreted IL-12 proteins were estimated by Western blot analysis, using the Enhanced Chemiluminescence (ECL) detection system (Amersham) and laser scanning densitometry(19, 20) . Various amounts of purified wt mIL-12 or hIL-12 run on the same blot were used to generate a standard curve for quantitation of unknown amounts of sample IL-12.

Lymphoblast Proliferation Assay

The proliferative response of phytohemagglutinin-activated human lymphoblasts to hIL-12, mIL-12, or mutant IL-12 was measured in a 48-h assay as described(21) . The proliferative response of concanavalin A-activated mouse lymphoblasts to wt and chimeric mIL-12 was measured as described(9) . All samples were assayed using triplicate dilution points.

Production and Assay of IFN-

IFN- production by C57BL/6 mouse splenocytes was induced by culture with IL-12 as described previously(9) , and murine IFN- was measured by use of an enzyme-linked immunosorbent assay(22) . For production of human IFN-, 2.5 times 10^6 human peripheral blood mononuclear cells/0.5 ml of culture were incubated for 48 h at 37 °C in culture medium (9) containing 5% human AB serum (Irvine Scientific), 200 units/ml human rIL-2 (from Dr. F. Khan, Hoffmann-La Roche), and the indicated amounts of IL-12. Human IFN- was measured in an enzyme-linked immunosorbent assay similar to that for mouse IFN- (22) but using mouse anti-human IFN- monoclonal antibody clone 69 (from Dr. H. Gallati, F. Hoffmann-La Roche Ltd., Basel, Switzerland), 2.5 µg/ml, to coat plates and peroxidase-conjugated anti-human IFN- monoclonal antibody clone 69, 300 ng/ml, for detection of captured IFN-. Cultures for production of IFN- were set up in duplicate, and supernatant fluids were assayed for IFN- in triplicate.

Purification of an IL-12 Protein Containing a p35 Subunit Triple Mutant

cDNAs encoding the wt mouse p40 (mp40) and the mouse p35 (mp35) triple mutant (mC(2)146K184F) (see text) were co-transfected into COS cells by electroporation(23) . 20-24 h after transfection, the standard growth medium containing fetal calf serum was replaced with serum-free medium. Secreted mutant IL-12 protein was collected after 3 days. The mutant mIL-12 protein was then purified by an immunoaffinity column constructed with the monoclonal antibody 2B5 that is specific for the IL-12 heterodimer. Purified mutant IL-12 protein was quantitated by the fluorescamine method(24) .

IL-12 Receptor Binding Assays

I-mIL-12 proteins were labeled by the method of Bolton-Hunter(25) . Binding studies using I-mIL-12 and phytohemagglutinin-activated human lymphoblasts or concanavalin A-activated mouse lymphoblasts were performed as described (26) with the following modifications. Triplicate samples of cells (2 times 10^6) were incubated with I-mIL-12 (1 times 10^4 cpm) in the presence of increasing amounts of mutant or wt IL-12 protein for 90 min at room temperature. Receptor binding data were analyzed using the nonlinear regression programs EBDA and Ligand as described previously(26) .

Molecular Modeling

The secondary structure of the mouse p35 subunit was predicted using the method of Chou and Fasman (27) and by alignment with the sequence of IL-6. IL-6 structure predictions have been published by Bazan (28) and Savino et al.(29) . A three-dimensional model for the structure of p35 was then generated as follows. Helices within p35 were located by identifying segments with a heptad repeat consisting of hydrophobic residues. These hydrophobic residues were aligned with the corresponding patterns of hydrophobic residues in G-CSF. The loops within p35 were defined based on comparison to the beta-strand loops found in growth hormone. The computer program SYBYL (Tripos Associates Inc., St. Louis, MO) was used to assist modeling. Surface probability analysis for amino acid residues was performed using the program PeptideStructure from the Genetics Computer Group (GCG)(30) .


RESULTS

Construction, Expression, and Bioactivity of Mouse/Human IL-12 Chimeras

Mouse IL-12 acts on both human and mouse cells, whereas human IL-12 acts only on human cells. This one-way species specificity is determined by sequences within the p35 subunit of IL-12(9) . Since mouse and human p35 subunit sequences are about 60% identical, the determinants for the species specificity are likely to reside in regions with lower sequence conservation. Fig. 1illustrates the alignment of the mouse and human p35 subunit sequences and how these regions were assigned. The amino acid sequences were broken up into nonoverlapping blocks of 9-12 residues; this length allowed the generation of a single mutagenic oligonucleotide for use in the Kunkel-mutagenesis procedure. Nine of these blocks (marked A-D and a-e) with a sequence identity of 50% or less were chosen for initial mutagenesis. The strategy was to (i) replace these sequence blocks in mouse p35 with the corresponding human sequences, either singly or in combination, (ii) express these mouse p35 mutants in COS cells together with wt mouse p40, and (iii) determine the proliferative activity of the resulting IL-12 chimeras on both human and mouse lymphoblasts. A mutant with the desired phenotype would show reduced levels of activity on mouse lymphoblasts but full activity on human lymphoblasts. In addition to evaluation in the two proliferation assays, expression levels of some of the IL-12 chimeric proteins in the COS supernatants were also monitored by quantitative Western blot analysis using polyclonal antibodies against either human or mouse IL-12 (see ``Materials and Methods''); these protein estimations were used to calculate the specific activities listed in Table 1. Activities of the IL-12 chimeras on human and mouse lymphoblasts were compared as follows. The ratio of the activities determined in the two bioassays was calculated for each mutant (Table 1). Use of this ratio allowed rapid identification of chimeras that were defective on mouse lymphoblasts only; additionally, this initial comparison did not rely on the protein quantitation by Western blot. For wt mouse IL-12 expressed in COS cells, this ratio was calculated to be in the range of 0.5 to 1.9 (n = 12, independent transfections); the corresponding value for wt human IL-12 was >24. In addition, the specific activities of the IL-12 mutants on mouse lymphoblasts were also expressed as a percentage of the specific activity of wt mIL-12, the latter being defined as 100%.


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(2) (Fig. 1) by generating and analyzing mutants M-C(2)-146K and M-C(2)-184F (see Table 1). The corresponding mutants M-C(1)-146K and M-C(1)-184F showed no decrease in bioactivity on mouse cells (ratio human/mouse = 0.5-0.7, not shown). Within the C(2) 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(2) 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(2)-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.

Purification and Characterization of IL-12 Containing the p35 Triple Mutant C(2)146K184F

In order to characterize the IL-12 heterodimer containing this triple mutant in detail, including testing its binding affinity for the IL-12 receptor, it was necessary to purify the mutant from conditioned COS cell medium. The excess free p40 subunit that is always present in such medium would interfere with the binding of IL-12 heterodimers to the IL-12 receptor(31) . An affinity column using the rat monoclonal antibody 2B5 that is specific for the mouse IL-12 heterodimer was constructed, and the mutant IL-12 was purified from serum-free COS medium by this affinity column. SDS-polyacrylamide gel electrophoresis and silver staining showed that the protein preparation contained only the 75-kDa doublet bands characteristic of wt mouse IL-12 (the appearance of the doublet is probably due to differential glycosylation) (Fig. 3).


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 mC(2)146K184F. 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 mC(2)146K184F 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 times 10^5versus 2 times 10^8 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 times 10^7versus 2 times 10^8 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 mC(2)146K184F. 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. box--box, wt human IL-12; - - - - -, wt mouse IL-12; circle- - -circle, 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 (box), wt mouse IL-12 (), and mouse IL-12 mutant (circle) (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. box- - -box, wt human IL-12; -, wt mouse IL-12; circle- - - - -circle, mouse IL-12 mutant. Each point is the average of triplicate samples; one of two experiments with similar results is shown.




DISCUSSION

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, mC(2)146K184F, 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(2) 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 alpha-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(2), 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, (^3)again indicating a direct interaction between this receptor beta-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.


FOOTNOTES

*
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
Present address: CADUS Pharmaceuticals, 777 Old Sawmill River Rd., Tarrytown, NY 10591.

To whom correspondence and reprint requests should be addressed: Dept. of Inflammation/Autoimmune Diseases, Hoffmann-La Roche Inc., 340 Kingsland St., Nutley, NJ 07110-1199. Tel.: 201-235-7481; Fax: 201-235-5046.

(^1)
The abbreviations used are: IL, interleukin; IL-12R, interleukin 12 receptor; mp35, mouse p35 subunit; hp35, human p35 subunit; G-CSF, granulocyte-colony-stimulating factor; wt, wild type; IFN-, interferon-.

(^2)
D. Presky, manuscript in preparation.

(^3)
R. Chizzonite, personal communication.


ACKNOWLEDGEMENTS

We thank Vincent Madison for construction of the p35 model, Victoria Wilkinson for providing monoclonal antibody 2B5, Frank Podlaski for help with the purification of the mutant, Alvin Stern for advice on the fluorescamine method, Lucy Foppiani and Rongshen Hsiao for oligonucleotide synthesis, Joe Levine and John Duker for DNA sequencing, Richard Chizzonite and Grace Ju for critical comments on the manuscript, and Anne Chua and Amy Herbitter for their enthusiastic support.


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