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Volume 271, Number 21, Issue of May 24, 1996 pp. 12145-12149
©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
DAB Interleukin-2 Receptor Binding Domain Mutations
CYTOTOXIC PROBES FOR STUDIES OF LIGAND-RECEPTOR INTERACTIONS (*)

(Received for publication, January 22, 1996)

Johanna C. vanderSpek (§),  ,  John A. Sutherland ,  Monthakan Ratnarathorn ,  Kimberly Howland ,  Thomas L. Ciardelli (1),  (2),  John R. Murphy (¶)

From the  (1)Evans Department of Clinical Research and the Department of Medicine, Boston University Medical Center Hospital, Boston, Massachusetts 02118, the Department of Pharmacology and Toxicology, Dartmouth Medical School, Hanover, New Hampshire 03755-3833, and the (2)Veterans Administration Hospital, White River Junction, Vermont 05001

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
REFERENCES

ABSTRACT

Site-directed mutagenesis was used to generate point mutations in the diphtheria toxin-related fusion protein, DAB interleukin-2 (IL-2). Thr-439, in the IL-2 receptor binding domain of the fusion toxin, was changed to a Pro residue. The resultant fusion toxin, DAB IL-2(T439P), was 300-fold less cytotoxic than wild type DAB IL-2, partially as the result of a 100-fold decrease in binding affinity for the high affinity form of the IL-2 receptor. However, DAB IL-2(T439P) stimulated DNA synthesis to a greater extent than expected. Studies of intoxication kinetics indicated that the increased stimulation might result from an increased contact time between the mutated IL-2 receptor binding domain and the receptor, perhaps due to a decreased internalization rate. Another mutant, DAB IL-2(Q514D), in which a Gln residue at position 514 was changed to an Asp, was 2000-fold less cytotoxic than wild type DAB IL-2. This mutant had a 50-fold decrease in binding affinity, did not stimulate DNA synthesis and also had a reduced rate of intoxication. Gln-514 appears to play a role in receptor binding and activation, whereas Thr-439 appears to be involved with receptor binding and signaling internalization of the fusion toxin-receptor complex.

INTRODUCTION

Interleukin-2 (IL-2) (^1)is a 133-amino acid lymphokine protein secreted by activated T-cells. Binding of IL-2 to its high affinity receptor stimulates processes that result in gene activation, DNA synthesis, internalization of the IL-2bulletIL-2 receptor complex, and proliferation of IL-2-dependent T cells (1, 2, 3, 4, 5, 6) . The high affinity form of the IL-2 receptor is composed of at least three subunits, alpha, beta, and . The alpha subunit forms a heteromeric complex with the beta subunit that functions to bind IL-2 to the surface of activated T cells(7) . The beta and subunits associate in a ligand-dependent fashion and appear to be involved with intracellular signaling(8) . Mutational studies have been performed on IL-2 in an attempt to define the structure/function relationships between IL-2 and the receptor subunits(9, 10, 11, 12, 13) . Buchli and Ciardelli (14) identified a Gln residue at position 126 of IL-2 that was involved with binding to the beta/ portion of the high affinity receptor. IL-2 in which Gln-126 was mutated to an Asp residue resulted in an analog with greatly reduced biological activity. Another analog was created in which a Thr residue at position 51 was changed to a Pro residue(15) . This analog, despite a decreased binding affinity, increased DNA synthesis of stimulated human peripheral blood lymphocytes to a much greater extent than expected. The authors postulated that the Thr-51 Pro mutation slowed the internalization rate of the ligand-receptor complex, thereby allowing a greater time interval for signaling activation.

The interleukin-2 diphtheria toxin-related fusion protein, DAB IL-2, is composed of amino acid residues 2-133 of IL-2, genetically fused to the first 386 residues of diphtheria toxin (DT)(16, 17) . The fusion toxin is targeted to cells that express the high affinity form of the IL-2 receptor and is internalized by receptor mediated endocytosis. The fusion toxin is processed and the catalytic domain of DT is translocated across the endocytic membrane, into the cell cytosol, where it ADP-ribosylates elongation factor 2, leading to an irreversible inhibition of protein synthesis and subsequent cell death(18, 19, 20) .

In the present study, we introduced the Q126D and T51P mutations described above, as well as an E106K mutation, into the IL-2 receptor binding domain of DAB IL-2. We studied the effects of these mutations on cytotoxicity, binding affinity, and kinetics of cytotoxicity. We also created analogous mutations, in which the catalytic domain of DT was mutated to a nontoxic form, so we could study the effects of the IL-2 receptor binding domain on stimulation of DNA synthesis. Our results indicate that the Gln residue is involved with binding affinity and activation of DNA synthesis, and that activation may affect cytotoxicity. The Thr residue appears to affect receptor binding and signaling internalization of the fusion toxin-receptor complex. The Glu residue that was mutated does not appear to play a critical role in the IL-2 binding domain of the fusion toxin.

EXPERIMENTAL PROCEDURES

Plasmid, Bacterial Strains, and Fusion Toxin Products

A schematic representation of the gene encoding DAB IL-2, the restriction digest sites used, and the single amino acid residue changes are shown in Fig. 1. The plasmid encoding DAB IL-2(T439P) was created by a 306-nucleotide MluI to HindIII exchange from the plasmid encoding the T51P mutation in IL-2(15) . This exchange encompassed the region of IL-2 containing the Thr-51 Pro mutation. The plasmids encoding DAB IL-2(Q514D) and DAB IL-2(E494K) were created by PCR mutagenesis of the wild type, DAB IL-2 gene, and cassette exchange encompassing the mutated site(21) . In the case of the plasmid encoding DAB IL-2(Q514D), a 229-nucleotide XbaI to SalI exchange was performed, and for DAB IL-2(E494K), a 241-nucleotide XbaI to HindIII cassette exchange was performed. The E149S mutation in the DT catalytic domain was introduced into each of the above mutants by an NcoI to SphI exchange from the gene encoding DA(E149S)B IL-2. In all cases the constructs were sequenced by the dideoxy chain termination method (22) using a Sequenase reagent kit (U. S. Biochemical Corp.) The mutant fusion toxins, and the corresponding mutations in IL-2, are listed in Table 1.

Figure 1: Schematic representation of the gene encoding DAB IL-2. PCR mutagenesis and cassette exchange were used to create the indicated amino acid changes in the catalytic and IL-2 receptor binding domains.


Escherichia coli JM101 was the host strain for plasmid propagation, and the HMS174 or HMS174 DE3 strain (Novagen, Madison, WI) was used as host for the expression of DAB IL-2 and all the mutants.

Oligonucleotide Synthesis

Oligonucleotides were synthesized on an Applied Biosystems model 391 PCR Mate DNA synthesizer. The oligonucleotides were removed from the columns and deprotected as recommended by Applied Biosystems. The oligonucleotides were vacuum-dried, resuspended in TE buffer, and the concentration determined using absorbance A readings (23) .

Polymerase Chain Reaction (PCR)

PCR was performed using a PCR reagent kit (Perkin Elmer Corp.). The conditions for PCR were 1 min at 95 °C for strand separation, 1 min at 37 °C for primer hybridization, and 1 min at 72 °C for the polymerase activity. The cycle was repeated 25 times.

Expression and Purification of Diphtheria Toxin-related Fusion Proteins

Expression of DAB IL-2, DAB IL-2(T439P), DAB IL-2(E494K), DAB IL-2(Q514D), and their corresponding E149S catalytic domain mutations, were all under control of the T7 polymerase promoter in derivatives of pet11d (Novagen, Madison WI).

The plasmids encoding DAB IL-2, DA(E149S)B IL-2, DAB IL-2(E494K), and DA(E149S)B IL-2(E494K) were transformed into HMS174 for expression. The bacteria were propagated to an A = 0.8 in LB/ampicillin/maltose, and protein expression was induced by the addition of the coliphage derivative, CE6. The expressed proteins formed inclusion bodies under these conditions, and these inclusion bodies were isolated, denatured, and refolded as described previously(24) . After refolding overnight at 4 °C, the proteins were concentrated using a Filtron concentrator and an M(r) 10,000 cut-off filter (Filtron, Northborough, MA). The proteins were then purified further by ion exchange chromatography on DEAE-Sepharose (Pharmacia Biotech Inc.). The fusion toxins were applied to the column and washed extensively with 10 mM phosphate buffer, pH 7.2. The proteins were eluted using a linear 0-0.8 M KCl gradient.

The fusion toxins DAB IL-2(T439P), DA(E149S)B IL-2(T439P), DAB IL-2(Q514D), and DA(E149S)B IL-2(Q514D) were expressed from HMS174 DE3. One liter of bacterial cultures were incubated in Luria broth containing 100 µg/ml ampicillin and 25 µg/ml chloramphenicol to A = 0.8. Expression of the fusion toxins was induced by addition of isopropyl-beta-D-thiogalactopyranoside to a final concentration of 1.0 mM. The bacteria were incubated an additional 2-3 h, with shaking, at 37 °C and then harvested by centrifugation. The bacterial pellets were resuspended in 50.0 ml of buffer 101 (50 mM KH(2)PO(4), 10 mM EDTA, 750 mM NaCl, 0.1% Tween 20, pH 8.0) and sonicated on ice for 15 min (dial 6, 40% duty cycle, pulsed microtip, Branson Cell Disruptor). The lysates were centrifuged at 600 times g for 20 min at 4 °C, and the resulting supernatants were 0.4-µm filtered. The filtrates were loaded onto an anti-diphtheria immunoaffinity column and washed with several column volumes of buffer 101. The fusion toxins were eluted in buffer 104 (100 mM KH(2)PO(4), 4 M guanidine-HCl, 0.1% Tween 20, pH 7.2) in final volumes of approximately 50.0 ml. The proteins were next dialyzed overnight, at 4 °C, against refolding buffer (50 mM Tris-Cl, pH 8.0, 50 mM NaCl, 5 mM reduced glutathione, 1 mM oxidized glutathione). The proteins were further purified by ion exchange chromatography on DEAE-Sepharose, as described above.

All protein concentrations were determined using Pierce protein assay reagent. The proteins were analyzed for purity by electrophoresis on a 12% SDS-polyacrylamide gel, stained with Coomassie Blue (Fig. 2).

Figure 2: SDS-polyacrylamide gel electrophoresis of purified proteins. Lane 1, DAB IL-2; lane 2, DA(E149S)B IL-2; lane 3, DAB IL-2(T439P); lane 4, DA(E149S)B IL-2(T439P); lane 5, DAB IL-2(Q514D); lane 6, DA(E149S)B IL-2(Q514D); lane 7, DAB IL-2(E494K), lane 8, DA(E149S)B IL-2(E494K). Molecular weight standards are indicated (10).


Cytotoxicity Assays

HUT 102/6TG cells were maintained in complete RPMI 1640 medium (Life Technologies, Inc.) (supplemented with 10% fetal bovine serum (HyClone Laboratories, Logan, UT): 2 mM glutamine, 50 IU/ml penicillin, and 50 µg/ml streptomycin). For the cytotoxicity assays, 5 times 10^4 cells in 100 µl of complete medium were seeded into each well of a 96-well, V-bottomed microtiter dish (Linbro). The fusion toxins were added so the final volume was 200 µl/well, and the fusion toxin concentrations ranged from 10M down to 10M. The plates were incubated for 18 h at 37 °C in a 5% CO(2) atmosphere and then centrifuged at 170 times g for 5 min. The medium was aspirated and replaced with 200 µl of leucine-free minimal essential medium (Life Technologies, Inc.) containing 1.0 µCi/ml [^14C]leucine (280 mCi/mmol, DuPont NEN), 2 mML-glutamine, 50 µg/ml streptomycin, and 50 IU/ml penicillin. The cells were pulsed for 90 min and then centrifuged at 170 times g for 5 min. The medium was removed, and the cells were lysed by addition of 60 µl/well 0.4 M KOH and incubated at room temperature for 10 min. The proteins were precipitated out by addition of 140 µl/well 10% trichloroacetic acid and incubating for another 10 min at room temperature. The insoluble proteins were collected on glass fiber filters using a PhD cell harvester (Cambridge Technology, Inc., Watertown, MA). Radioactivity was determined according to standard methods. All assays were performed in quadruplicate, and medium alone served as a control.

Binding Assays

The displacement of I-labeled recombinant IL-2 from the high affinity form of the IL-2 receptor was measured following the method of Wang and Smith(25) . HUT102/6TG cells were harvested and washed thoroughly in complete RPMI 1640 medium. The cells (1 times 10^6/point) were incubated with 200 pMI-labeled recombinant IL-2 (0.7 µCi/pmol, DuPont NEN) in the presence or absence of increasing concentrations of unlabeled recombinant IL-2, DAB IL-2, or the related mutant fusion toxins. The cells were overlaid on a mixture of 80% 550 fluid (Accumetric Inc., Elizabethtown, KY) and 20% paraffin oil (density = 1.03 g/ml) and incubated at 37 °C for 20 min. The reactions were centrifuged in a microcentrifuge for 2 min. The cell pellet, representing bound ligand, and the aqueous phase, representing unbound ligand, were counted in a Beckman Gamma 5500 counter. The apparent dissociation constants (K(i) values) were determined based on the concentration of unlabeled ligand required to displace 50% I-labeled recombinant IL-2 binding. (The K(i) for DAB IL-2(T439P) is an estimate determined from Fig. 4, as an IC was not reached, even at the highest concentration of toxin tested).

Figure 4: Competitive displacement of I-labeled IL-2. times, rIL-2; bullet, DAB IL-2; circle, DAB IL-2(E494K); up triangle, DAB IL-2(Q514D); , DAB IL-2(T439P). The results for the corresponding catalytic domain mutations are not shown here, but are reported in Table 2.


Activation Assay

CTLL-2 cells were maintained in complete RPMI, as above, that was further supplemented with 5 µM 2-mercaptoethanol and 1 nM recombinant IL-2. For the assays, the CTLL-2 cells were washed twice in the above medium, without IL-2, and seeded in the medium, again without IL-2, at 1.5 times 10^4 cells/well in a 96-well, V-bottomed microtiter plate. The catalytic domain mutants of the fusion toxins, or recombinant IL-2, were added to final concentrations ranging from 10M down to 10M. The cells were incubated at 37 °C for 24 h and then 1.0 µCi of [^3H]thymidine (6.7 Ci/mmol, DuPont NEN) was added to each well. The cells were pulsed for 6 h at 37 °C, harvested on glass fiber filters, and radioactivity was determined using standard methods. Each assay point was performed in triplicate or quadruplicate, and the assays were performed several times for each fusion toxin (Fig. 5). The results for activation reported in Table 2are a comparison of activation by 10M concentrations of the fusion toxins to 10M IL-2.

Figure 5: Representative dose response analysis of stimulation of [^3H]thymidine incorporation by rIL-2, DA(E149S)B IL-2, and mutant fusion toxins on CTLL-2 cells. , rIL-2; bullet, DA(E149S)B IL-2; circle, DA(E149S)B IL-2(T439P); , DA(E149S)B IL-2(E494K); up triangle, DA(E149S)B IL-2(Q514D). The corresponding mutants with active catalytic domains did not stimulate [^3H]thymidine incorporation; results are shown in Table 2.


Kinetic Assays

HUT 102/6TG cells were seeded in 100 µl of complete medium, at a concentration of 5 times 10^4 cells/well, as described above for the cytotoxicity assays. The cytotoxic forms of the fusion toxins were added to a final concentration of 10M, and the cells were incubated for 1, 3, 5, 9, 12, 15, or 18 h before pulsing and harvesting, as described above.

RESULTS

A schematic representation of the gene encoding DAB IL-2 is shown in Fig. 1. The mutations in the IL-2 receptor binding domain of the gene, where nucleotides were changed to encode different amino acid residues, are indicated. The Glu Ser mutation in the catalytic domain of DT is also indicated. The mutations in IL-2, corresponding to the mutations in the IL-2 receptor binding domain of the fusion toxins, are shown in Table 1.

The fusion toxins DAB IL-2, DA(E149S)B IL-2, DAB IL-2(E494K), DA(E149S)B IL-2(E494K), DAB IL-2(T439P), DA(E149S)B IL-2(T439P), DAB IL-2(Q514D), and DA(E149S)B IL-2(Q514D) were expressed and purified as described under ``Experimental Procedures,'' Following purification, the proteins were separated by electrophoresis on 12% SDS-polyacrylamide gels and stained with Coomassie Blue (Fig. 2). The full-length forms of the proteins all migrated at approximately 57.7 kDa, in agreement with their calculated molecular weights. Western blot analysis was also performed and indicated the proteins were all immunoreactive with DT antibody (data not shown).

DAB IL-2 possessed an IC of 2.2 times 10M in the cytotoxicity assay (Fig. 3, Table 2). The corresponding catalytic domain mutation, DA(E149S)B IL-2, in which a Glu residue was changed to a Ser residue, was not cytotoxic (Table 2). The apparent binding affinities (K(i) values) were 3.6 times 10M and 5.7 times 10M, respectively (Fig. 4, Table 2). DA(E149S)B IL-2 was tested for stimulation of DNA synthesis by a CTLL-2 cell [^3H]thymidine incorporation assay, and a representative assay is shown in Fig. 5. The stimulation by 10M DA(E149S)B IL-2 was 83% that of the stimulation induced by 10M IL-2 (Table 2). The stimulation by 10M DAB IL-2 was only 4% that of 10M IL-2. DAB IL-2(E494K), in which a Glu residue in the IL-2 binding domain was changed to a Lys residue (Fig. 1), possessed an IC of 1.6 times 10M, approximately 7-fold less than the wild type cytoxicity (Fig. 3, Table 2). The K(i) for the high affinity form of the IL-2 receptor was 2.9 times 10M, approximately 8-fold less than the binding affinity of the wild type (Fig. 4, Table 2). DAB IL-2(E494K) stimulated DNA synthesis to 3% of the level of 10M rIL-2 (Table 2). The corresponding fusion toxin with the E149S catalytic domain mutation, DA(E149S)B IL-2(E494K), was not cytotoxic, possessed a K(i) of 6.6 times 10M, and stimulated CTLL-2 [^3H]thymidine incorporation by 63% compared to the same concentration, 10M, of rIL-2 (Fig. 4, Table 2).

Figure 3: Dose-response analysis of DAB IL-2 and mutant fusion toxins on HUT102/6TG cells. bullet, DAB IL-2; circle, DAB IL-2(E494K); up triangle, DAB IL-2(Q514D); , DAB IL-2(T439P). The corresponding catalytic domain mutations were not cytotoxic, and the results are reported in Table 2.


DAB IL-2(T439P) (Fig. 1) possessed an IC of 6.5 times 10M (Fig. 3, Table 2) and a K(i) of 3.5 times 10M (Fig. 4, Table 2). This represents approximately a 300-fold decrease in cytotoxicity and approximately 100-fold less binding affinity when compared to wild type fusion toxin. 10M DAB IL-2(T439P) stimulated DNA synthesis in CTLL-2 cells to 5% of the level of 10M rIL-2. The E149S form of DAB IL-2(T439P) was not cytotoxic and possessed a K(i) of 1.8 times 10M (Table 2). DA(E149S)B IL-2(T439P) at 10M stimulated [^3H]thymidine incorporation by 65% compared to 10M rIL-2.

DAB IL-2(Q514D) (Fig. 1) possessed an IC of 4.3 times 10M and a K(i) of 1.9 times 10M ( Fig. 3and Fig. 4, Table 2). The cytotoxicity was therefore, approximately 2000-fold less than that of wild type DAB IL-2. The K(i) was approximately 50-fold less than that of wild type. DAB IL-2(Q514D) stimulated CTLL-2 cell DNA synthesis by 2% compared to rIL-2 (Table 2). DA(E149S)B IL-2(Q514D) was not cytotoxic, the K(i) was 2.0 times 10M and, in contrast to the other noncytotoxic forms of the fusion toxins, at a concentration of 10M, stimulated CTLL-2 [^3H]thymidine incorporation to only 8% of the level of 10M rIL-2 stimulation (Fig. 5, Table 2).

The above results were of interest as the T439P IL-2 binding domain mutant stimulated DNA synthesis by CTLL-2 cells to a greater extent than one would expect, given the K(i) results. Also, the Q514D IL-2 binding domain mutant was less cytotoxic than expected given the K(i) results. For this reason, kinetic assays of cytotoxicity were performed. DAB IL-2 and DAB IL-2(E494K) possessed the same rates of intoxication. DAB IL-2(T439P) and DAB IL-2(Q514D) exhibited reduced rates of intoxication (Fig. 6).

Figure 6: Inhibition of protein synthesis in HUT102/6TG cells by 10M DAB IL-2 and related mutant fusion toxins after incubation for the indicated times. bullet, DAB IL-2; circle, DAB IL-2(E494K); , DAB IL-2(T439P); up triangle, DAB IL-2(Q514D).


DISCUSSION

Studies of the relationships between IL-2 and its receptors are important to identify the various functions of IL-2, as well as the structural elements involved with these functions. This information is required for the design of analogs with expanded therapeutic applications. In the present work we use the IL-2-directed fusion toxin, DAB IL-2, to further study some amino acid residues previously identified as involved in the processing of IL-2 and its receptors. The purpose of this work was to expand on the knowledge already obtained for these residues, to ascertain how these residues affect the function of the fusion toxin, and to demonstrate the potential of DAB IL-2 as a useful agent for the study of ligand-receptor interactions.

Buchli and Ciardelli (14) created an IL-2 analog in which a Gln residue at position 126 of IL-2 was changed to an Asp residue. Their results indicated that the Asp-126 mutant stimulated [^3]thymidine incorporation in human T-lymphocytes and CTLL-2 cells to a lesser degree than wild type IL-2. The binding affinity of the D126 mutant was greatly decreased, and the loss was due to a disruption of the beta/ receptor subunit interaction. The authors postulated that cross-linking of the beta/ receptor subunits is the likely signaling event for activity of IL-2, and that Gln-126 is involved with binding and cross-linking the subunits, either as a contact position or allosterically.

We constructed and studied DAB IL-2(Q514D), which contains the analogous mutation in the IL-2 receptor binding domain of DAB IL-2. For comparison, we also studied DAB IL-2(E494K), a form of DAB IL-2 containing a mutation in the IL-2 binding domain that we already knew exerted minimal effects on cytotoxicity. DAB IL-2(Q514D) was 2000-fold less cytotoxic than wild type DAB IL-2 and possessed a decreased binding affinity, and the corresponding protein with the catalytic domain mutation did not stimulate [^3H]thymidine incorporation. The cytotoxicity kinetic assays indicate that DAB IL-2(Q514D) inhibited protein synthesis at a slower rate than wild type DAB IL-2. The rate of inhibition reflects the rate of binding and toxin entry into the cell cytosol. These results indicate that Gln-514, in the receptor binding domain of DAB IL-2, is involved with binding affinity, stimulation of DNA synthesis and rate of toxin internalization. These results are all consistent with the findings for the Gln-126 residue of IL-2. DAB IL-2(E494K) was 7-fold less cytotoxic than DAB IL-2, probably as a direct result of the 8-fold decrease in binding affinity. The rate of protein synthesis inhibition was the same as for DAB IL-2. DA(E149S)B IL-2(E494K) stimulated [^3H]thymidine incorporation, although not as much as wild type, DA(E149S)B IL-2. The effects imposed by the Glu-494 mutation to a Lys probably result from structural changes in the IL-2 binding domain of the fusion toxin, leading to the decrease in binding affinity.

Chang et al.(15) discovered that mutating Thr-51 of IL-2 to a Pro residue resulted in an IL-2 analog with a decreased binding affinity, but the corresponding loss in stimulation of DNA synthesis was much lower than expected. They postulated that either the Thr-51 Pro mutation resulted in a conformational change that partially mimicked a change required for IL-2 to facilitate the beta/ subunit cross-linking necessary for signaling activation, or the Thr-51 Pro mutation slowed the internalization rate for the ligand-bound receptor complex, thereby allowing a greater time for the signaling interval. The corresponding residue in the IL-2 receptor binding domain of DAB IL-2 was mutated to create DAB IL-2(T439P). DAB IL-2(T439P) was 300-fold less cytotoxic than wild type and possessed a decreased binding affinity, but still stimulated DNA synthesis as well as the control mutation, DAB IL-2(E494K). SDS-polyacrylamide gel electrophoresis of DAB IL-2(T439P) (Fig. 2, lane 3) shows this protein was subject to breakdown after freezing and thawing, probably due to conformational effects the Thr-439 Pro mutation had on the protein. (DAB IL-2(T439P) was originally isolated as full-length protein, as shown by gel electrophoretic analysis performed during the purification procedures; data not shown.)

DAB IL-2(T439P) and DAB IL-2(Q514D) both possessed poor binding affinities, but DAB IL-2(T439P) was 10-fold more cytotoxic. The fact that DAB IL-2(T439P) stimulated DNA synthesis and DAB IL-2(Q514D) did not lead us to speculate that the difference in stimulatory effect may account for the difference in cytotoxicity, i.e. stimulation of DNA synthesis leads to enhancement of cytotoxicity. The cytotoxicity kinetics assay for DAB IL-2(T439P) showed that this mutant fusion toxin, like DAB IL-2(Q514D), possessed a decreased rate of cytotoxicity compared to the wild type and DAB IL-2(E494K). This leads to the conclusion that the Thr-439 Pro mutation in the IL-2 receptor binding domain of DAB IL-2, and the corresponding Thr51 to Pro mutation in IL-2, exerted greater than expected stimulatory effects at least partially due to a decreased rate of internalization.

The results from these studies confirm and expand on the previous findings for studies performed with IL-2. The Asp residue at position 126 of IL-2 is involved with binding of IL-2 to its receptors. The rate of internalization of the IL-2-receptor complex is decreased and, possibly due to a decrease in beta/ cross-linking, signaling stimulation of DNA synthesis is decreased. Additionally, mutation of this residue in the IL-2 binding domain of the fusion toxin DAB IL-2 decreases cytotoxicity to a greater degree than expected, indicating that the loss in stimulation of DNA synthesis, affects cytotoxicity. It appears that the stimulatory effect of the IL-2 binding domain on DNA synthesis enhances cytotoxicity. The Thr residue at position 51 of IL-2 is involved with binding, and this effect may be conformational. The rate of ligand-receptor internalization is decreased when this residue is changed to a Pro, and an increase in DNA stimulation occurs. This effect results in a greater than expected cytotoxicity when the corresponding residue in the IL-2 binding domain of DAB IL-2 is mutated.

FOOTNOTES

*
This work was supported in part by Public Health Service Grant CA-60934 from the NCI, National Institutes of Health (to J. R. M.). 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.

§
Special fellow of the Leukemia Society of America. To whom correspondence should be addressed. Tel.: 617-638-6010; Fax: 617-638-6009.

Supported by National Institutes of Health Grant R01 GM 52858-01, American Cancer Society Grant FRA 385, and the Norris Cotton Cancer Center.

(^1)
The abbreviations used are: IL-2, interleukin-2; DT, diphtheria toxin; PCR, polymerase chain reaction.

REFERENCES

  1. Gillis, S., Ferm, M. M., Ou, W., and Smith, K. A. (1978) Immunology 120, 2027
  2. Beckner, S. K., and Farrar, W. L. (1987) Biochem. Biophys. Res. Commun. 145, 176-182 [CrossRef][Medline] [Order article via Infotrieve]
  3. Mills, G. B., Cragoe, E. J., Jr., Gelfand, E. W., and Grinstein, S. (1985) J. Biol. Chem. 260, 12500-12507 [Abstract/Free Full Text]
  4. Ishii, T., Takeshita, T., Numata, J., and Sugamura, K. (1988) J. Immunol. 141, 174-179 [Abstract]
  5. Rayhel, E. J., Fields, T. J., Albright, J. W., Diamantstein, T., and Hughes, J. P. (1988) Biochem. J. 249, 333-338 [Medline] [Order article via Infotrieve]
  6. Farrar, W. L., and Anderson, W. B. (1985) Nature 315, 233-235 [CrossRef][Medline] [Order article via Infotrieve]
  7. Landgraf, B. E., Goldstein, B., Williams, D. P., Murphy, J. R., Sana, T. R., Smith, K. A., and Ciardelli, T. L. (1992) J. Biol. Chem. 267, 18511-18519 [Abstract/Free Full Text]
  8. Takeshita, T., Asao, H., Ohtani, K., Ishii, N., Kumaki, S., Tanaka, N., Munakata, H., Nakamura, M., and Sugamura, K. (1992) Science 257, 379-382 [Abstract/Free Full Text]
  9. Berndt, W. G., Chang, D. Z., Smith, K. A., and Ciardelli, T. L. (1994) Biochemistry 33, 6571-6577 [CrossRef][Medline] [Order article via Infotrieve]
  10. Berndt, W. G., and Ciardelli, T. L. (1992) Interleukin-2 , pp. 12-28, Blackwell Scientific Publications, Oxford
  11. Zurawski, S. M., and Zurawski, G. (1992) EMBO J. 11, 3905-3910 [Medline] [Order article via Infotrieve]
  12. Collins, L., Tsien, W. H., Seals, C., Hakimi, J., Weber, D., Bailon, P., Hoskins, J., Greene, W. C., Toome, V., and Ju, G. (1988) Proc. Natl. Acad. Sci. U. S. A. 85, 7709-7713 [Abstract/Free Full Text]
  13. Suave, K., Nachman, M., Spence, C., Bailon, P., Campbell, E., Tsien, W. H., Kondas, J. A., Hakimi, J., and Ju, G. (1991) Proc. Natl. Acad. Sci. U. S. A. 88, 4636-4640 [Abstract/Free Full Text]
  14. Buchli, P., and Ciardelli, T. (1993) Arch. Biochem. Biophys. 207, 411-415
  15. Chang, D. Z., Tasayco, M. L., and Ciardelli, T. L. (1995) Mol. Pharmacol. 47, 206-211 [Abstract]
  16. Williams, D. P., Parker, K., Bacha, P., Bishai, B., Borowski, M., Genbauffe, F., Strom, T. B., and Murphy, J. R. (1987) Protein Eng. 1, 493-498 [Abstract/Free Full Text]
  17. Williams, D. P., Snider, C. E., Strom, T. B., and Murphy, J. R. (1990) J. Biol. Chem. 265, 11885-11889 [Abstract/Free Full Text]
  18. Bacha, P., Williams, D. P., Waters, C., Williams, J. M., Murphy, J. R., and Strom, T. B. (1988) J. Exp. Med. 167, 612-622 [Abstract/Free Full Text]
  19. Waters, C. A., Schmike, P. A., Snider, C. E., Itoh, K., Smith, K. A., Nichols, J. C., Strom, T. B., and Murphy, J. R. (1990) Eur. J. Immunol. 20, 785-791 [Medline] [Order article via Infotrieve]
  20. Williams, D. P., Wen, Z., Watson, R. S., Boyd, J., Strom, T. B., and Murphy, J. R. (1990) J. Biol. Chem. 265, 20673-20677 [Abstract/Free Full Text]
  21. Ausubel, F. M., Brent, R., Kingston, R. E., Moore, D. D., Seidman, J. G., Smith, J. A., and Struhl, K. (1991) Current Protocols in Molecular Biology , Vol. 1, pp. 8.5.7-8.5.9, Suppl. 15, Greene Publishing Associates/John Wiley & Sons, Inc., New York
  22. Sanger, F., Nicklen, S., and Coulsen, A. R. (1977) Proc. Natl. Acad. Sci. U. S. A. 74, 5463-5467 [Abstract/Free Full Text]
  23. Maniatis, T., Fritsch, E. F., and Sambrook, J. (1982) Molecular Cloning: A Laboratory Manual, pp. 90-91, 468, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY
  24. vanderSpek, J. C., Mindell, J. A., Finkelstein, A., and Murphy, J. R. (1993) J. Biol. Chem. 268, 12077-12082 [Abstract/Free Full Text]
  25. Wang, H. M., and Smith, K. A. (1987) J. Exp. Med. 166, 1055-1069 [Abstract/Free Full Text]
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