HYBRIDOMA Volume 9, Number 6, 1990 Mary Ann Leibert, Inc., Publishers
Monoclonal Antibodies Specific to Human ETS-2 Oncoprotein: Recognition of Epitopes Clustered on the B Domain SHIGEYOSHI FUJIWARA,' SHIGEKI KOIZUMI,' ROBERT J. FISHER,1 NARAYAN K. BHAT,2 and TAKIS S. PAPAS' 'Laboratory of Molecular Oncology, National Cancer Institute 2Program Resources, Inc., Frederick, MD
ABSTRACT Six monoclonal antibodies were prepared from mice immunized with a These antibodies specifically bacterially expressed human ets-2 protein. recognize the two human ets-2-encoded proteins p56 and p54 but failed to react with chicken, mouse, rat, bovine, or monkey proteins, suggesting that the antibodies recognize epitopes specific to the human ets-2 protein. Differential reactivities of these monoclonal antibodies with the peptide fragments generated by partial proteolytic digestion of the bacterially expressed ets-2 protein indicated that the six antibodies recognize at least three distinct epitopes in the B domain of the ets-2 protein. Immunoprecipitation experiments comparing native and denaturing conditions suggested that the ets-2 domain detected by the monoclonal antibodies is masked in the native condition by either protein folding or interacting proteins. The biochemical analysis of the ets-2 protein will be facilitated by the development of these monoclonal antibodies, which may be useful as both domain-specific probes and tools for specifically detecting the human ets-2 protein in heterologous expression systems.
INTRODUCTION The ets gene
family
consists of at least five genes, ets-1. ets-2.
erg,
elk-
1, and el_k-2, which have various degrees of sequence similarity with the ets oncogene carried by the avian acute leukemia virus E26 (1,2,3,4,5,6). A region
of the ets sequence (termed domain C) is evolutionarily extremely well conserved (7) and is similar to the DNA-binding domains of transcription factors in its abundance of basic amino acid residues. In fact, a murine transcription factor, PU.l, was recently shown to have sequence similarity with this domain of the ets family proteins (8). The human ets-2 gene codes for short-lived nuclear proteins of 56 and 54 kilodaltons (KD) whose half-life can be prolonged by the activation of protein kinase C (5,9,10). Thus the steady-state level of the protein can be controlled by protein kinase C activity through a post-translational mechanism. The p56 protein is a phosphorylated form of p54, and this phosphorylation is regulated by Ca2+ signals triggered by the activation of the antigen receptor on T lymphocytes (11). These findings suggest that the ets-2 protein is a transcription factor whose activity is regulated by signal transduction systems, although the biochemical function of the protein is unknown. The expression of this gene is induced in association with cell 559
proliferation (12,13), and its overexpression in mouse NIH/3T3 cells results in neoplastic transformation of the cells (14). The human ets-2 gene is located on a region of chromosome 21 (21q22.3) (15) that has been implicated in Down's syndrome and is involved in the non-random translocations in certain types of leukemias (16). To obtain highly specific immunological reagents for the ets-2 protein and to facilitate its biochemical characterization, we isolated mouse hybridomas producing monoclonal antibodies (MAbs) against the human ets-2 protein. In this paper, we describe the isolation and characterization of MAbs that are specific to the human ets-2 protein and that recognize different epitopes on the protein's B domain.
MATERIALS AND METHODS Purification of the Bacterially Expressed Human ets-2 Protein E. coli cells carrying the ets-2 expression vector pBAN-150 (17) were provided by A. Seth. This vector expresses a truncated form of the human ets2 protein designated p35, containing amino acids 190-469 of the protein (7). Bacterial cells were treated with DNase I and lysozyme and lysed with NP-40, and inclusion bodies containing the p35 protein were isolated by consecutive washes with 1.75 M guanidine hydrochloride, 1 M NaCl, and 1% Triton X-100 (18). The pellet containing p35 was then dissolved in SDS protein sample buffer and subjected to preparatory sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The ets-2 protein band was stained with cold 0.25 M KC1 solution and extracted with phosphate-buffered saline (PBS) containing 0.1% SDS (19).
Preparation of Hybridomas and Screening
BALB/c mice (8 week-old females) were immunized intraperitoneally with 50 /ig/mouse of the purified protein as an emulsion with Freund's complete adjuvant. Two and six weeks after the first immunization, mice were given subcutaneous booster injections with 25 /ig/mouse of the antigen. At 7-10 days after each booster shot, serum antibody titers were measured by ELISA (19) and immunoprecipitation (9). Mice with higher titers of antibody against the bacterially expressed ets-2 protein were selected and given a final intraperitoneal antigen injection of 25 /tg/mouse. Four days after the final immunization, spleen cells were isolated and hybridized with the mouse myeloma (NS-1) cells using polyethyleneglycol 4000. Hybridomas were selected by HAT medium (hypoxanthine, aminopterin, thymidine) and screened by ELISA for antibodies specific to the ets-2 protein. Enzyme-Linked Immunosorbent Assay
Polyvinylchloride microtiter plates were activated with 0.25% glutaraldehyde, and the antigen was adsorbed to the plate by incubation with the antigen solution (10 /ig/ml of antigen in PBS containing 0.1% SDS; see ref. 19). After blocking with 20% fetal bovine serum (FBS) in Tris-buffered saline (50 mM Tris-HCl, pH 8.0, 140 mM NaCl; TBS), samples of the supernatant fluid from each hybridoma culture were added to the plate and incubated at room temperature for the microplates were filled with After washing three times by TBS, 2 h. peroxidase-labeled antibodies against mouse IgG and IgM (Boehringer Mannheim Biochemicals, used at a dilution of 1:400 in TBS containing 20% FBS) and incubated at room temperature for 2 h. After three washes with TBS, the plates were developed with 2,2'-azino-di[3-ethyl-benzthiazoline sulfonate(6)] (ABTS).
560
Purification of MAbs
Hybridomas were cultured on a large scale in protein-free medium (Pan-Data Systems, Inc., Rockville, MD) and the MAb in the supernatant was purified by ammonium sulfate precipitation followed by protein A-Sepharose chromatography. The two IgA antibodies, P-96 and 0-55, could not be purified by this method because they do not bind protein A. Therefore, unpurified samples of the culture supernatant containing these antibodies were used in the immunoblot analysis. For immunoprecipitation, ammonium sulfate precipitates of the supernatant samples containing each antibody were coupled with agarose using N-hydroxysuccinimide ester (Affi-Gel 10, BioRad Laboratories). Immunoblot Analysis We also
performed immunoblot analysis of the reactivity of the MAbs and the with the bacterially expressed ets-2 protein, using the method described by Towbin and others (20). Bacterial cells were lysed with SDS protein sample buffer, boiled for 5 minutes, and subjected to SDS-PAGE. The proteins
mouse
were
serum
then electroblotted to
a
nitrocellulose membrane.
The filter
was
blocked
using 3% dry milk in TBS and then incubated at room temperature for 2 h with MAbs or serum diluted appropriately in TBS containing 1% bovine serum albumin and 0.5% Tween-20 (TBS-BT). The filter was washed three times with TBS-BT and further incubated with 12 I-labeled sheep antibody [F(ab')2 fraction] against mouse Ig at room temperature for 2 h. After being washed three times with TBS-BT as above, the filter was exposed to X-ray film. To detect the human ets-2 protein with the MAbs, lysate prepared from Jurkat cells was examined according to the procedures described above, except that biotin-labeled anti-mouse immunoglobulin and streptavidin-labeled alkaline phosphatase were used to develop the filter. Nitro-blue tetrazolium (NBT) and 5-bromo-4-chloro-3-indolyl phosphate (BCIP) were used as substrates. Immunoprecipitation
Labeling of the cells with [35S]methionine, preparation of cell lysate immunoprecipitation of the ets-2 protein were performed as described (9). Summary of MAbs
Antibody
Class/subclass
,
and
TABLE 1 to the Human ets-2 Protein
Light chain
Epitope group
0-55 0-137 P-78 P-96 T-7
K a IgA b \ IgGl k a IgGl k c IgA b k IgGl U-244_IgGl_k_a_
to 'According identical an one
the results of immunopeptide mapping, MAbs reacting with combination of peptide fragments were categorized into of the three groups, a, b, and c (see Results for detail).
Epitope Analysis The epitopes recognized by the MAbs were analyzed by immunopeptide mapping, described by Evan and others (21). One microgram of the bacterially expressed human ets-2 protein p35 was incubated with 100 ng of staphylococcal V8 protease as
561
in a buffer containing 0.125 M Tris-HCl (pH 6.8), 0.1% SDS, 1 mM EDTA, and 10% glycerol for 30 minutes at room temperature. The peptide fragments generated by this partial digestion were fractionated by SDS-PAGE and blotted onto nitrocellulose membrane. The peptides containing the epitopes for the MAbs were detected by immunoblot analysis using 125I-labeled second antibodies, as
described above.
Polyclonal Antibodies Against Synthetic Oligopeptides The F-13-W antibody is a rabbit polyclonal antibody directed against the viral ets amino acid sequence FKLSDPDEVARRw, which corresponds to FKLADPDEVARRW in the human ets-2 sequence (amino acids 391-403, see ref. 7). There is only one amino acid substitution (S vs. A at residue 394) between the viral and human sequences in this region. In our previous work (9), this antibody was termed antibody B. The L-13-D antibody is also a rabbit polyclonal antibody and is directed to the carboxy-terminal 13 amino acids (LHAILGVQPDTED) of the human ets2 protein (7). The positions of these oligopeptides in the human ets-2 sequence These polyclonal antibodies have been purified by are shown in Figure 1. affinity chromatography using the corresponding antigen peptides.
L-13-D 469
457
LHAILGVQPDTED
Ulli ¡I FKLADPDEVARRW 403 391
F13W
FIGURE 1. The Bacterially Expressed Human ets-2 Protein, p35: Comparison of Amino Acid Sequence with That of the Mouse ets-2 Protein. The p35 protein starts with and continues to the carboxy terminus. The black areas represent amino acids that are identical in the human and mouse proteins, whereas the white lines indicate individual diverged amino acids. The portion of the ets-2 protein indicated by the broken line is missing from p35. The letters A, B, and C (top) define the hypothetical ets domains. The positions of F-13-W and L-13-D, the oligopeptides used to prepare antibodies, are shown.
190Trp
Preparation of Nuclear Extract Nuclei
were
isolated from
[35S]methionine
labeled CEM cells
as
described
previously (9) and the proteins were extracted with NE buffer (0.42 M KC1, 10 mM HEPES, pH 7.0, 1 mM EDTA, 0.5% Nonidet P-40, 10% glycerol, 3 /ig/ml aprotinin, 0.4 mM phenylmethylsulfonyl fluoride, 0.01 mM Na-p-tosyl-L-lysine chloromethyl ketone, 0.1 mM N-tosyl-L-phenylalanine chloromethyl ketone). The extract was clarified by ultracentrifugation at 25,000 g for 30 minutes and diluted to 0.1 M KC1, with other components of the buffer remaining at the same concentrations. RESULTS Generation of MAbs
The bacterially expressed human ets-2 protein p35 (17) identified as a strong 35 KD band seen only after heat treatment
562
(Figure 1) was (Figure 2A, lane
A
B
C U-244
U-244 Block
0-137
0-137 Block
32* 42" pu. 93 K-
E o
o
68 K43 K-
-p35 p3526 K-
—
•p35 18 K -
2
3
9 10 11 12
13 14 15 16
FIGURE 2. Purification of the Characterization of the MAbs.
Bacterially Expressed Human ets-2 p35 Protein and (A) E. coli cells carrying the ets-2 expression vector pBAN-150 were lysed with SDS protein sample buffer and fractionated by SDS-PAGE, and the gel was stained with Coomassie brilliant blue. Lane 1, protein pattern without heat treatment; lane 2, after heat shock; lane 3, the p35 protein purified by preparative SDS-PAGE. The relative mobilities of the protein standards are shown on the left side of the panel. (B) Immunoblot reaction of purified p35 with the anti-ets antibody. The nitrocellulose membrane harboring p35 fractionated by SDS-PAGE was probed with the F-13-W antibody (lane 1). Lane 2 shows competition with peptide. (C) Specific immunoblot reaction of the MAbs with p35. Total lysate was prepared with SDS protein sample buffer from E. coli carrying no vector (lanes 1, 5, 9, 13); a v-ets expression vector, pTSP-8 (Seth et al., 1989) (lanes 2, 6, 10, and 14); or the ets-2 expression vector pBAN-150 (lanes 3, 4, 7, 8, 11, 12, 15, 16). Bacterial cells were heat treated in lanes 2, 4, 6, 8, 10, 12, 14, and 16. Lanes 1-8 were probed with U-244 MAb and lanes 9-16 with 0-137 antibody. Lanes 5-8 and 13-16 show competition with purified p35. 2) and purified by preparative SDS-PAGE (Figure 2A lane 3). Its identity was confirmed by reactivity with the anti-ets peptide antibody F-13-W (9), which was competed by excess antigen peptide (Figure 2B). The purified protein was injected into BALB/c mice and more than 1000 hybridomas were produced. Initial screening of these hybridomas with an enzyme-linked immunosorbent assay (ELISA) selected out 10 positive clones and an immunoblot analysis confirmed that six of them react specifically with the bacterially expressed human ets-2 protein, p35. The results shown in Figure 2C demonstrate that two of the six MAbs, U244 and 0-137, react with p35 from heat-treated E. coli cells carrying the ets2 expression vector but not from the E. coli carrying the v-ets construct (pTSP8) (17) or from non-heat-treated bacteria containing the e_ts-2 construct. These antibodies did not react with a bacterially expressed human erg protein (data not shown). These reactions were completely abolished by preincubating the MAbs with purified p35 (Figure 2C). The other four clones (T-7, P-78, 0-55, and P-96) showed similar specific reactions with the p35 protein on immunoblot analysis (data not shown). The immunoglobulin classes, subclasses, and light chain types of these MAbs are summarized in Table 1. All antibodies except 055 and P-96 were purified by protein A-Sepharose chromatography and used in the subsequent analyses. 563
Human ets-2 Protein Recognized by the MAbs In our previous work we identified two proteins, p56 and p54, as the products of the human ets-2 gene (11); p56 is a phosphorylated form of p54. To determine whether the MAbs and the polyclonal antiserum show the same specific reactivity with the human ets-2 protein, human T-lymphocytes of the CEM cell line were labeled with [35S]methionine and the cell lysate was tested by immunoprecipitation. The results (Figure 3A) show that all six MAbs react with p56 and p54, as does the polyclonal antiserum. Although the p54 species was only weakly detected by the P-96 antibody in the experiment shown, this protein was consistently detected in the subsequent experiments. These reactivities were completely blocked by adding the bacterially expressed ets-2 protein p35 (Figure 3A, lanes 2, 4, 6, and 12). The strong background reactions observed with the P-96 and 0-55 antibodies (Figure 3A, lanes 8 and 9) are due to the impurity of the antibody preparations, because ammonium sulfate precipitates from the culture supernatant were directly conjugated with agarose beads and used for immunoprecipitation. The p56 proteins detected by the six MAbs and the polyclonal antiserum showed identical peptide maps after partial digestion with staphylococcal V8 protease (22), indicating that all of these antibodies recognize the same p56, the product of the human ets-2 gene (Figure 3B).
A
B O
O Q-
-
O
CL
-
Ki
mi
« I
O
O
o.
¡ïi
iii
O O
O-
O
O 3
5 6
7 8
Q.
200 K
-93 K -69 Kets-2' e,S
' P56" |p54-
:$ K8 -46 K-
12 3
4
-30 K 12 3
4
5
6
7
8
9
10 11
12
3. (A) Reactivity of MAbs with the Human ets-2 Protein. Immunoprecipitation of the human ets-2 protein by the MAbs. Human T-lymphocytes of CEM line were labeled with [35S]methionine and the cell lysate was immunoprecipitated with polyclonal anti-ets-2 serum (lanes 1 and 2), MAb U-244 (lanes 3, 4, and 7), MAb T-7 (lanes 5 and 6), MAb P-96 (lane 8), MAb 0-55 (lane 9), MAb P-78 (lane 10), and MAb 0-137 (lanes 11 and 12). Immunocompetition with purified p35 is shown in lanes 2, 4, 6, and 12. Lanes 1-6 and lanes 7-12 were The relative mobilities of the protein standards are run on separate gels. shown. (B) Peptide mapping by partial digestion with staphylococcal V8 protease. The ets-2 protein p56 detected by the six MAbs was excised from the gel and analyzed as described (22). Lanes 1-3 and lanes 4-8 were analyzed on separate gels. A 30 ng sample of the enzyme was added to each lane.
FIGURE
564
N. CO
i-
97 K 68 K ets-2
43 K-
12
3
4
Immunoblot Detection of the Human ets-2 Protein by MAbs. Jurkat FIGURE 4. cells were treated with TPA (10 nM) for 6 h (lanes 2 and 4) and analyzed as described in Materials and Methods. Same number of the cells were processed similarly but without TPA treatment (lanes 1 and 3). The filter was probed with the 0-137 antibody (lanes 3 and 4) or purified murine IgGl, used as a negative control (lanes 1 and 2). The ets-2 protein is indicated by an arrow. The p56 and p54 proteins were not well separated in this experiment. The reactivity of these MAbs on immunoblot analysis was tested using the human T-lymphocyte line Jurkat, as described in Materials and Methods. The T7 antibody detected the ets-2 protein from Jurkat cells treated with the tumorpromoting phorbol ester 12-0-tetradecanoylphorbol-13-acetate (TPA), while the protein was below the detectable level in the control cells (Figure 4). This is consistent with our previous finding that protein kinase C activation increases the half-life of ets-2 protein and thereby elevates its steady state level. The results of this study also indicate that the steady-state level of the ets-2 protein can be measured by immunoblot analysis using the MAb. Our quantitative study showed that control Jurkat cells have approximately 1500 molecules of the ets-2 protein, which is close to the threshold level observed in immunoblot analysis using the avidin-biotin system (S. Koizumi, manuscript in preparation). The U-244 antibody gave a similar result (data not shown).
Immunopeptide Mapping To address the question of whether these six MAbs recognize the same epitope different epitopes on the ets-2 protein, we tested the reactivity of each antibody with a mixture of peptide fragments generated by partial digestion of p35 with staphylococcal V8 protease. The MAbs could be divided into three groups, according to their patterns of reactivity with the peptides (Figure 5). The U-244 and 0-55 antibodies recognize a common set of peptides, while the 0137 and T-7 antibodies reacted with a different set of fragments. The P-96 antibody detected still another set of peptides. Results not shown in Figure 5 indicate that the P-78 antibody recognized the same combination of peptides or
565
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11
ÏIL
U-244 V8 0,g) O i-; O O O O 1-
¿
I HP
1
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ïI
123456789
1234
FIGURE 5. Epitope Mapping by the ets-2 MAbs. (A) A 1 /tg sample of purified p35 protein was partially digested with 100 ng of staphylococcal V8 protease. The peptides were fractionated by SDS-PAGE and electroblotted on nitrocellulose membrane. The filter was then cut into strips and probed with the MAbs, as indicated at the top of the panel. Epitopes were visualized with 125I-labeled second antibodies. (B) A 1 /ig sample of p35 protein was digested with the amount of the protease indicated and the epitope was detected by the MAb U-244 as in
(A).
detected
by U-244 and 0-55. These results are summarized in Table 1. As included two anti-peptide antibodies, F-13-W and L-13-D, directed to known amino acid sequences in the ets-2 primary protein structure (Figure 1). The F-13-W antibody recognizes the amino acid sequence FKLSDPDEVARRW (amino acids 391-403) and L-13-D is directed to the carboxy-terminal thirteen amino acids LHAILGVQPDTED (7). These two reference antibodies recognized almost identical combinations of peptides, that are different from any of the combinations observed with the MAbs (Figure 5). Thus these results indicate that the six MAbs recognize at least three different epitopes on the ets-2 protein. These studies also suggest that the epitopes detected by the MAbs are located somewhere between the Trp residue (amino acid 190, see ref. 7) which starts the p35 protein and the F-13-W sequence, because the F-13-W and L-13-D (carboxy terminal) antibodies recognized almost identical combinations of peptide fragments and these as
standards,
we
combinations are different from any of those observed with the six MAbs. It remains to be seen whether the MAbs within each group recognize an identical epitope or closely spaced epitopes that remained joined on the smallest peptide of several thousand daltons generated by the protease digestion.
Specificity of the MAbs for Human ets-2 Protein To test whether the MAbs recognize ets-2 proteins from other species, we prepared [35S]methionine-labeled lysates of chicken embryo fibroblasts, rat embryo fibroblasts, mouse thymocytes, and African green monkey Vero cells. In the immunoprecipitation using these lysates (Figure 6), the L-13-D antibody
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Monoclonal Antibodies Specific to Human ETS-2 Oncoprotein: Recognition of Epitopes Clustered on the B Domain SHIGEYOSHI FUJIWARA,' SHIGEKI KOIZUMI,' ROBERT J. FISHER,1 NARAYAN K. BHAT,2 and TAKIS S. PAPAS' 'Laboratory of Molecular Oncology, National Cancer Institute 2Program Resources, Inc., Frederick, MD
ABSTRACT Six monoclonal antibodies were prepared from mice immunized with a These antibodies specifically bacterially expressed human ets-2 protein. recognize the two human ets-2-encoded proteins p56 and p54 but failed to react with chicken, mouse, rat, bovine, or monkey proteins, suggesting that the antibodies recognize epitopes specific to the human ets-2 protein. Differential reactivities of these monoclonal antibodies with the peptide fragments generated by partial proteolytic digestion of the bacterially expressed ets-2 protein indicated that the six antibodies recognize at least three distinct epitopes in the B domain of the ets-2 protein. Immunoprecipitation experiments comparing native and denaturing conditions suggested that the ets-2 domain detected by the monoclonal antibodies is masked in the native condition by either protein folding or interacting proteins. The biochemical analysis of the ets-2 protein will be facilitated by the development of these monoclonal antibodies, which may be useful as both domain-specific probes and tools for specifically detecting the human ets-2 protein in heterologous expression systems.
INTRODUCTION The ets gene
family
consists of at least five genes, ets-1. ets-2.
erg,
elk-
1, and el_k-2, which have various degrees of sequence similarity with the ets oncogene carried by the avian acute leukemia virus E26 (1,2,3,4,5,6). A region
of the ets sequence (termed domain C) is evolutionarily extremely well conserved (7) and is similar to the DNA-binding domains of transcription factors in its abundance of basic amino acid residues. In fact, a murine transcription factor, PU.l, was recently shown to have sequence similarity with this domain of the ets family proteins (8). The human ets-2 gene codes for short-lived nuclear proteins of 56 and 54 kilodaltons (KD) whose half-life can be prolonged by the activation of protein kinase C (5,9,10). Thus the steady-state level of the protein can be controlled by protein kinase C activity through a post-translational mechanism. The p56 protein is a phosphorylated form of p54, and this phosphorylation is regulated by Ca2+ signals triggered by the activation of the antigen receptor on T lymphocytes (11). These findings suggest that the ets-2 protein is a transcription factor whose activity is regulated by signal transduction systems, although the biochemical function of the protein is unknown. The expression of this gene is induced in association with cell 559
proliferation (12,13), and its overexpression in mouse NIH/3T3 cells results in neoplastic transformation of the cells (14). The human ets-2 gene is located on a region of chromosome 21 (21q22.3) (15) that has been implicated in Down's syndrome and is involved in the non-random translocations in certain types of leukemias (16). To obtain highly specific immunological reagents for the ets-2 protein and to facilitate its biochemical characterization, we isolated mouse hybridomas producing monoclonal antibodies (MAbs) against the human ets-2 protein. In this paper, we describe the isolation and characterization of MAbs that are specific to the human ets-2 protein and that recognize different epitopes on the protein's B domain.
MATERIALS AND METHODS Purification of the Bacterially Expressed Human ets-2 Protein E. coli cells carrying the ets-2 expression vector pBAN-150 (17) were provided by A. Seth. This vector expresses a truncated form of the human ets2 protein designated p35, containing amino acids 190-469 of the protein (7). Bacterial cells were treated with DNase I and lysozyme and lysed with NP-40, and inclusion bodies containing the p35 protein were isolated by consecutive washes with 1.75 M guanidine hydrochloride, 1 M NaCl, and 1% Triton X-100 (18). The pellet containing p35 was then dissolved in SDS protein sample buffer and subjected to preparatory sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The ets-2 protein band was stained with cold 0.25 M KC1 solution and extracted with phosphate-buffered saline (PBS) containing 0.1% SDS (19).
Preparation of Hybridomas and Screening
BALB/c mice (8 week-old females) were immunized intraperitoneally with 50 /ig/mouse of the purified protein as an emulsion with Freund's complete adjuvant. Two and six weeks after the first immunization, mice were given subcutaneous booster injections with 25 /ig/mouse of the antigen. At 7-10 days after each booster shot, serum antibody titers were measured by ELISA (19) and immunoprecipitation (9). Mice with higher titers of antibody against the bacterially expressed ets-2 protein were selected and given a final intraperitoneal antigen injection of 25 /tg/mouse. Four days after the final immunization, spleen cells were isolated and hybridized with the mouse myeloma (NS-1) cells using polyethyleneglycol 4000. Hybridomas were selected by HAT medium (hypoxanthine, aminopterin, thymidine) and screened by ELISA for antibodies specific to the ets-2 protein. Enzyme-Linked Immunosorbent Assay
Polyvinylchloride microtiter plates were activated with 0.25% glutaraldehyde, and the antigen was adsorbed to the plate by incubation with the antigen solution (10 /ig/ml of antigen in PBS containing 0.1% SDS; see ref. 19). After blocking with 20% fetal bovine serum (FBS) in Tris-buffered saline (50 mM Tris-HCl, pH 8.0, 140 mM NaCl; TBS), samples of the supernatant fluid from each hybridoma culture were added to the plate and incubated at room temperature for the microplates were filled with After washing three times by TBS, 2 h. peroxidase-labeled antibodies against mouse IgG and IgM (Boehringer Mannheim Biochemicals, used at a dilution of 1:400 in TBS containing 20% FBS) and incubated at room temperature for 2 h. After three washes with TBS, the plates were developed with 2,2'-azino-di[3-ethyl-benzthiazoline sulfonate(6)] (ABTS).
560
Purification of MAbs
Hybridomas were cultured on a large scale in protein-free medium (Pan-Data Systems, Inc., Rockville, MD) and the MAb in the supernatant was purified by ammonium sulfate precipitation followed by protein A-Sepharose chromatography. The two IgA antibodies, P-96 and 0-55, could not be purified by this method because they do not bind protein A. Therefore, unpurified samples of the culture supernatant containing these antibodies were used in the immunoblot analysis. For immunoprecipitation, ammonium sulfate precipitates of the supernatant samples containing each antibody were coupled with agarose using N-hydroxysuccinimide ester (Affi-Gel 10, BioRad Laboratories). Immunoblot Analysis We also
performed immunoblot analysis of the reactivity of the MAbs and the with the bacterially expressed ets-2 protein, using the method described by Towbin and others (20). Bacterial cells were lysed with SDS protein sample buffer, boiled for 5 minutes, and subjected to SDS-PAGE. The proteins
mouse
were
serum
then electroblotted to
a
nitrocellulose membrane.
The filter
was
blocked
using 3% dry milk in TBS and then incubated at room temperature for 2 h with MAbs or serum diluted appropriately in TBS containing 1% bovine serum albumin and 0.5% Tween-20 (TBS-BT). The filter was washed three times with TBS-BT and further incubated with 12 I-labeled sheep antibody [F(ab')2 fraction] against mouse Ig at room temperature for 2 h. After being washed three times with TBS-BT as above, the filter was exposed to X-ray film. To detect the human ets-2 protein with the MAbs, lysate prepared from Jurkat cells was examined according to the procedures described above, except that biotin-labeled anti-mouse immunoglobulin and streptavidin-labeled alkaline phosphatase were used to develop the filter. Nitro-blue tetrazolium (NBT) and 5-bromo-4-chloro-3-indolyl phosphate (BCIP) were used as substrates. Immunoprecipitation
Labeling of the cells with [35S]methionine, preparation of cell lysate immunoprecipitation of the ets-2 protein were performed as described (9). Summary of MAbs
Antibody
Class/subclass
,
and
TABLE 1 to the Human ets-2 Protein
Light chain
Epitope group
0-55 0-137 P-78 P-96 T-7
K a IgA b \ IgGl k a IgGl k c IgA b k IgGl U-244_IgGl_k_a_
to 'According identical an one
the results of immunopeptide mapping, MAbs reacting with combination of peptide fragments were categorized into of the three groups, a, b, and c (see Results for detail).
Epitope Analysis The epitopes recognized by the MAbs were analyzed by immunopeptide mapping, described by Evan and others (21). One microgram of the bacterially expressed human ets-2 protein p35 was incubated with 100 ng of staphylococcal V8 protease as
561
in a buffer containing 0.125 M Tris-HCl (pH 6.8), 0.1% SDS, 1 mM EDTA, and 10% glycerol for 30 minutes at room temperature. The peptide fragments generated by this partial digestion were fractionated by SDS-PAGE and blotted onto nitrocellulose membrane. The peptides containing the epitopes for the MAbs were detected by immunoblot analysis using 125I-labeled second antibodies, as
described above.
Polyclonal Antibodies Against Synthetic Oligopeptides The F-13-W antibody is a rabbit polyclonal antibody directed against the viral ets amino acid sequence FKLSDPDEVARRw, which corresponds to FKLADPDEVARRW in the human ets-2 sequence (amino acids 391-403, see ref. 7). There is only one amino acid substitution (S vs. A at residue 394) between the viral and human sequences in this region. In our previous work (9), this antibody was termed antibody B. The L-13-D antibody is also a rabbit polyclonal antibody and is directed to the carboxy-terminal 13 amino acids (LHAILGVQPDTED) of the human ets2 protein (7). The positions of these oligopeptides in the human ets-2 sequence These polyclonal antibodies have been purified by are shown in Figure 1. affinity chromatography using the corresponding antigen peptides.
L-13-D 469
457
LHAILGVQPDTED
Ulli ¡I FKLADPDEVARRW 403 391
F13W
FIGURE 1. The Bacterially Expressed Human ets-2 Protein, p35: Comparison of Amino Acid Sequence with That of the Mouse ets-2 Protein. The p35 protein starts with and continues to the carboxy terminus. The black areas represent amino acids that are identical in the human and mouse proteins, whereas the white lines indicate individual diverged amino acids. The portion of the ets-2 protein indicated by the broken line is missing from p35. The letters A, B, and C (top) define the hypothetical ets domains. The positions of F-13-W and L-13-D, the oligopeptides used to prepare antibodies, are shown.
190Trp
Preparation of Nuclear Extract Nuclei
were
isolated from
[35S]methionine
labeled CEM cells
as
described
previously (9) and the proteins were extracted with NE buffer (0.42 M KC1, 10 mM HEPES, pH 7.0, 1 mM EDTA, 0.5% Nonidet P-40, 10% glycerol, 3 /ig/ml aprotinin, 0.4 mM phenylmethylsulfonyl fluoride, 0.01 mM Na-p-tosyl-L-lysine chloromethyl ketone, 0.1 mM N-tosyl-L-phenylalanine chloromethyl ketone). The extract was clarified by ultracentrifugation at 25,000 g for 30 minutes and diluted to 0.1 M KC1, with other components of the buffer remaining at the same concentrations. RESULTS Generation of MAbs
The bacterially expressed human ets-2 protein p35 (17) identified as a strong 35 KD band seen only after heat treatment
562
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U-244 Block
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0-137 Block
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FIGURE 2. Purification of the Characterization of the MAbs.
Bacterially Expressed Human ets-2 p35 Protein and (A) E. coli cells carrying the ets-2 expression vector pBAN-150 were lysed with SDS protein sample buffer and fractionated by SDS-PAGE, and the gel was stained with Coomassie brilliant blue. Lane 1, protein pattern without heat treatment; lane 2, after heat shock; lane 3, the p35 protein purified by preparative SDS-PAGE. The relative mobilities of the protein standards are shown on the left side of the panel. (B) Immunoblot reaction of purified p35 with the anti-ets antibody. The nitrocellulose membrane harboring p35 fractionated by SDS-PAGE was probed with the F-13-W antibody (lane 1). Lane 2 shows competition with peptide. (C) Specific immunoblot reaction of the MAbs with p35. Total lysate was prepared with SDS protein sample buffer from E. coli carrying no vector (lanes 1, 5, 9, 13); a v-ets expression vector, pTSP-8 (Seth et al., 1989) (lanes 2, 6, 10, and 14); or the ets-2 expression vector pBAN-150 (lanes 3, 4, 7, 8, 11, 12, 15, 16). Bacterial cells were heat treated in lanes 2, 4, 6, 8, 10, 12, 14, and 16. Lanes 1-8 were probed with U-244 MAb and lanes 9-16 with 0-137 antibody. Lanes 5-8 and 13-16 show competition with purified p35. 2) and purified by preparative SDS-PAGE (Figure 2A lane 3). Its identity was confirmed by reactivity with the anti-ets peptide antibody F-13-W (9), which was competed by excess antigen peptide (Figure 2B). The purified protein was injected into BALB/c mice and more than 1000 hybridomas were produced. Initial screening of these hybridomas with an enzyme-linked immunosorbent assay (ELISA) selected out 10 positive clones and an immunoblot analysis confirmed that six of them react specifically with the bacterially expressed human ets-2 protein, p35. The results shown in Figure 2C demonstrate that two of the six MAbs, U244 and 0-137, react with p35 from heat-treated E. coli cells carrying the ets2 expression vector but not from the E. coli carrying the v-ets construct (pTSP8) (17) or from non-heat-treated bacteria containing the e_ts-2 construct. These antibodies did not react with a bacterially expressed human erg protein (data not shown). These reactions were completely abolished by preincubating the MAbs with purified p35 (Figure 2C). The other four clones (T-7, P-78, 0-55, and P-96) showed similar specific reactions with the p35 protein on immunoblot analysis (data not shown). The immunoglobulin classes, subclasses, and light chain types of these MAbs are summarized in Table 1. All antibodies except 055 and P-96 were purified by protein A-Sepharose chromatography and used in the subsequent analyses. 563
Human ets-2 Protein Recognized by the MAbs In our previous work we identified two proteins, p56 and p54, as the products of the human ets-2 gene (11); p56 is a phosphorylated form of p54. To determine whether the MAbs and the polyclonal antiserum show the same specific reactivity with the human ets-2 protein, human T-lymphocytes of the CEM cell line were labeled with [35S]methionine and the cell lysate was tested by immunoprecipitation. The results (Figure 3A) show that all six MAbs react with p56 and p54, as does the polyclonal antiserum. Although the p54 species was only weakly detected by the P-96 antibody in the experiment shown, this protein was consistently detected in the subsequent experiments. These reactivities were completely blocked by adding the bacterially expressed ets-2 protein p35 (Figure 3A, lanes 2, 4, 6, and 12). The strong background reactions observed with the P-96 and 0-55 antibodies (Figure 3A, lanes 8 and 9) are due to the impurity of the antibody preparations, because ammonium sulfate precipitates from the culture supernatant were directly conjugated with agarose beads and used for immunoprecipitation. The p56 proteins detected by the six MAbs and the polyclonal antiserum showed identical peptide maps after partial digestion with staphylococcal V8 protease (22), indicating that all of these antibodies recognize the same p56, the product of the human ets-2 gene (Figure 3B).
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3. (A) Reactivity of MAbs with the Human ets-2 Protein. Immunoprecipitation of the human ets-2 protein by the MAbs. Human T-lymphocytes of CEM line were labeled with [35S]methionine and the cell lysate was immunoprecipitated with polyclonal anti-ets-2 serum (lanes 1 and 2), MAb U-244 (lanes 3, 4, and 7), MAb T-7 (lanes 5 and 6), MAb P-96 (lane 8), MAb 0-55 (lane 9), MAb P-78 (lane 10), and MAb 0-137 (lanes 11 and 12). Immunocompetition with purified p35 is shown in lanes 2, 4, 6, and 12. Lanes 1-6 and lanes 7-12 were The relative mobilities of the protein standards are run on separate gels. shown. (B) Peptide mapping by partial digestion with staphylococcal V8 protease. The ets-2 protein p56 detected by the six MAbs was excised from the gel and analyzed as described (22). Lanes 1-3 and lanes 4-8 were analyzed on separate gels. A 30 ng sample of the enzyme was added to each lane.
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Immunoblot Detection of the Human ets-2 Protein by MAbs. Jurkat FIGURE 4. cells were treated with TPA (10 nM) for 6 h (lanes 2 and 4) and analyzed as described in Materials and Methods. Same number of the cells were processed similarly but without TPA treatment (lanes 1 and 3). The filter was probed with the 0-137 antibody (lanes 3 and 4) or purified murine IgGl, used as a negative control (lanes 1 and 2). The ets-2 protein is indicated by an arrow. The p56 and p54 proteins were not well separated in this experiment. The reactivity of these MAbs on immunoblot analysis was tested using the human T-lymphocyte line Jurkat, as described in Materials and Methods. The T7 antibody detected the ets-2 protein from Jurkat cells treated with the tumorpromoting phorbol ester 12-0-tetradecanoylphorbol-13-acetate (TPA), while the protein was below the detectable level in the control cells (Figure 4). This is consistent with our previous finding that protein kinase C activation increases the half-life of ets-2 protein and thereby elevates its steady state level. The results of this study also indicate that the steady-state level of the ets-2 protein can be measured by immunoblot analysis using the MAb. Our quantitative study showed that control Jurkat cells have approximately 1500 molecules of the ets-2 protein, which is close to the threshold level observed in immunoblot analysis using the avidin-biotin system (S. Koizumi, manuscript in preparation). The U-244 antibody gave a similar result (data not shown).
Immunopeptide Mapping To address the question of whether these six MAbs recognize the same epitope different epitopes on the ets-2 protein, we tested the reactivity of each antibody with a mixture of peptide fragments generated by partial digestion of p35 with staphylococcal V8 protease. The MAbs could be divided into three groups, according to their patterns of reactivity with the peptides (Figure 5). The U-244 and 0-55 antibodies recognize a common set of peptides, while the 0137 and T-7 antibodies reacted with a different set of fragments. The P-96 antibody detected still another set of peptides. Results not shown in Figure 5 indicate that the P-78 antibody recognized the same combination of peptides or
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FIGURE 5. Epitope Mapping by the ets-2 MAbs. (A) A 1 /tg sample of purified p35 protein was partially digested with 100 ng of staphylococcal V8 protease. The peptides were fractionated by SDS-PAGE and electroblotted on nitrocellulose membrane. The filter was then cut into strips and probed with the MAbs, as indicated at the top of the panel. Epitopes were visualized with 125I-labeled second antibodies. (B) A 1 /ig sample of p35 protein was digested with the amount of the protease indicated and the epitope was detected by the MAb U-244 as in
(A).
detected
by U-244 and 0-55. These results are summarized in Table 1. As included two anti-peptide antibodies, F-13-W and L-13-D, directed to known amino acid sequences in the ets-2 primary protein structure (Figure 1). The F-13-W antibody recognizes the amino acid sequence FKLSDPDEVARRW (amino acids 391-403) and L-13-D is directed to the carboxy-terminal thirteen amino acids LHAILGVQPDTED (7). These two reference antibodies recognized almost identical combinations of peptides, that are different from any of the combinations observed with the MAbs (Figure 5). Thus these results indicate that the six MAbs recognize at least three different epitopes on the ets-2 protein. These studies also suggest that the epitopes detected by the MAbs are located somewhere between the Trp residue (amino acid 190, see ref. 7) which starts the p35 protein and the F-13-W sequence, because the F-13-W and L-13-D (carboxy terminal) antibodies recognized almost identical combinations of peptide fragments and these as
standards,
we
combinations are different from any of those observed with the six MAbs. It remains to be seen whether the MAbs within each group recognize an identical epitope or closely spaced epitopes that remained joined on the smallest peptide of several thousand daltons generated by the protease digestion.
Specificity of the MAbs for Human ets-2 Protein To test whether the MAbs recognize ets-2 proteins from other species, we prepared [35S]methionine-labeled lysates of chicken embryo fibroblasts, rat embryo fibroblasts, mouse thymocytes, and African green monkey Vero cells. In the immunoprecipitation using these lysates (Figure 6), the L-13-D antibody
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