(Received for publication, January 22, 1996)
From the
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.
Interleukin-2 (IL-2) ()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-2
IL-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,
,
, and
. The
subunit forms a
heteromeric complex with the
subunit that functions to bind IL-2
to the surface of activated T cells(7) . The
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
/
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.
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.
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
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-
-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
PO
, 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
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
PO
, 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
).
Figure 4:
Competitive displacement of I-labeled IL-2.
, rIL-2;
, DAB
IL-2;
, DAB
IL-2(E494K);
, 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.
Figure 5:
Representative dose response analysis of
stimulation of [H]thymidine incorporation by
rIL-2, DA(E149S)B
IL-2, and mutant fusion toxins on
CTLL-2 cells.
, rIL-2;
, DA(E149S)B
IL-2;
, DA(E149S)B
IL-2(T439P);
,
DA(E149S)B
IL-2(E494K);
, DA(E149S)B
IL-2(Q514D). The corresponding mutants with active catalytic
domains did not stimulate [
H]thymidine
incorporation; results are shown in Table 2.
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
10
M 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
values) were 3.6
10
M and
5.7
10
M, respectively (Fig. 4, Table 2). DA(E149S)B
IL-2 was
tested for stimulation of DNA synthesis by a CTLL-2 cell
[
H]thymidine incorporation assay, and a
representative assay is shown in Fig. 5. The stimulation by
10
M DA(E149S)B
IL-2 was 83%
that of the stimulation induced by 10
M IL-2 (Table 2). The stimulation by 10
M DAB
IL-2 was only 4% that of
10
M 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
10
M, approximately 7-fold less than the
wild type cytoxicity (Fig. 3, Table 2). The K
for the high affinity form of the IL-2 receptor
was 2.9
10
M, 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 10
M 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
of 6.6
10
M, and stimulated CTLL-2
[
H]thymidine incorporation by 63% compared to the
same concentration, 10
M, of rIL-2 (Fig. 4, Table 2).
Figure 3:
Dose-response analysis of
DAB IL-2 and mutant fusion toxins on HUT102/6TG cells.
, DAB
IL-2;
, DAB
IL-2(E494K);
, 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
10
M (Fig. 3, Table 2) and a K
of 3.5
10
M (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.
10
M DAB
IL-2(T439P)
stimulated DNA synthesis in CTLL-2 cells to 5% of the level of
10
M rIL-2. The E149S form of DAB
IL-2(T439P) was not cytotoxic and possessed a K
of 1.8
10
M (Table 2).
DA(E149S)B
IL-2(T439P) at 10
M stimulated [
H]thymidine incorporation by 65%
compared to 10
M rIL-2.
DAB IL-2(Q514D) (Fig. 1) possessed an IC
of 4.3
10
M and a K
of 1.9
10
M ( Fig. 3and Fig. 4, Table 2). The cytotoxicity was
therefore, approximately 2000-fold less than that of wild type
DAB
IL-2. The K
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
was 2.0
10
M and, in contrast to the other noncytotoxic forms of
the fusion toxins, at a concentration of 10
M, stimulated CTLL-2 [
H]thymidine
incorporation to only 8% of the level of 10
M 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 results. Also, the Q514D
IL-2 binding domain mutant was less cytotoxic than expected given the K
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.
, DAB
IL-2;
, DAB
IL-2(E494K);
, DAB
IL-2(T439P);
,
DAB
IL-2(Q514D).
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 []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
/
receptor subunit interaction. The authors postulated that
cross-linking of the
/
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 [
H]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
[
H]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
/
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 /
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.
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