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Expression of a Zinc-Binding Domain of Boar Spermatidal Transition Protein 2 in Escherichia coli
Protein Expression and Purification 16, 454 – 462 (1999) Article ID prep.1999.1095, available online at http://www.idealibrary.com on
Expression of a Zinc-Binding Domain of Boar Spermatidal Transition Protein 2 in Escherichia coli Hiroki Sato,* Kuniko Akama,* Shuichi Kojima,† Kin-ichiro Miura,† Atsushi Sekine,* and Minoru Nakano* *Graduate School of Science and Technology, Chiba University, Chiba, Chiba 263-8522, Japan; and †Institute for Biomolecular Science, Gakushuin University, Mejiro, Tokyo 171-0031, Japan
Received March 1, 1999, and in revised form April 29, 1999
Transition protein 2 (TP2; 137 amino acid residues) from boar late spermatid nuclei has three potential zinc finger motifs in the N-terminal 43 region. Gel shift assays revealed that boar TP2 recognized a CpG island sequence in a zinc-dependent manner. However, there was some nonspecific recognition of the oligonucleotide. Then, we constructed the expression system of zinc-binding domain of TP2 (TP2Z) (residues 1–103) in Escherichia coli. Double-stranded DNA fragments encoding TP2Z were synthesized as 18 fragments with 103 residues, annealed, and cloned into the expression plasmid pET11d. TP2Z was expressed upon induction with 1 mM isopropylthiogalactoside and extracted with acid including 0.71 M 2-mercaptoethanol. TP2Z was purified by ion-exchange chromatography on Fractogel EMD SO 32 and HPLC on Nucleosil 300 7C18 and on Diol-120. Atomic absorption and CD spectroscopy showed that TP2Z bound three atoms of zinc per molecule of the protein and underwent a zinc-dependent conformational change in a manner similar to that for intact TP2. Gel shift assays indicated that TP2Z recognized a CpG island sequence more specifically than intact TP2 and that the specificity is dependent on zinc. © 1999 Academic Press Key Words: CpG-rich sequence; gene engineering; transition protein; zinc-binding domain.
The chromatin structure undergoes extensive changes during mammalian spermiogenesis. Transition proteins (TP1– 4) appear during the elongating phase of mammalian spermiogenesis. Nucleosomal histones are transiently replaced by transition proteins and, finally, by protamines (1– 4). At that time, transformation of the nucleosomal-type chromatin into a smooth chromatin fiber, initiation of chromatin condensation, and cessation of transcription occur (5). Rat 454
TP1, having higher affinity for single-stranded DNA, induces local melting of DNA (6,7). Rat TP2, with two potential zinc finger motifs in the amino terminal region, is a DNA-condensing protein and prefers zincdependently GC-rich sequences, including a human nonmethylated CpG island sequence (8 –12). Recently, we have developed methods of isolating boar TP1– 4 (13–15) and have reported that boar TP1 and TP3, having higher affinity for single-stranded DNA, and TP4, having higher affinity for double-stranded DNA, are DNA-melting proteins mediated through the stacking of Tyr 32, Trp 18, and Trp 126 with nucleic acid bases, respectively (16,17). Boar TP2 has three potential zinc finger motifs in the N-terminal 43 region and binds three atoms of zinc per molecule of the protein (15). The sequence of boar TP2 has two insertions of 11 amino acids at position 36, and of 10 amino acids at position 50, into that of rat and mouse TP2 (18), although boar, rat, and mouse TP2 have a highly conserved carboxy-terminal basic region. In this paper, we describe the nonspecific interaction of boar TP2 with a CpG island sequence by gel shift assays, the construction of an expression system of zinc-binding domain (TP2Z) with three zinc finger motifs of boar TP2 (residues 1–103) (Fig. 1) in Escherichia coli, and some characteristics of TP2Z. TP2Z contained three atoms of zinc bound per molecule and underwent a zinc-dependent secondary structural change similar to intact TP2 (137 residues), TP2Z recognized a CpG island sequence more preferentially than intact TP2, and its specificity is dependent on zinc. MATERIALS AND METHODS
Fractogel EMD SO 32 650 (M), Bio-gel P-2, Nucleosil 300 7C18, and Diol-120 were products of Merck, BioRad, Nagel, and YMC, respectively. The synthetic polynucleotides, poly(dG-dC) z poly(dG-dC), poly(dA1046-5928/99 $30.00 Copyright © 1999 by Academic Press All rights of reproduction in any form reserved.
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FIG. 1. A scheme of boar TP2 with three potential zinc finger motifs (15) and its zinc-binding domain (TP2Z).
dT) z poly(dA-dT), poly(dI-dC) z poly(dI-dC), poly(dG) z poly(dC), and poly(dA) z poly(dT), were purchased from Pharmacia. The complementary oligonucleotides (40mer) containing the human CpG island sequence 39 downstream of the d-aminolevulinic acid dehydratase gene (Fig. 2) were synthesized on a Sci-media gene assembler. [g- 32P]ATP (sp act of .7000 Ci/mmol) was from ICN Pharmaceuticals, Inc. T4 polynucleotide kinase was from Takara. Preparation of Boar TP2 Boar transition protein 2 was isolated from the late spermatid nuclei and refolded by our previously published method (13,15). The freshly refolded TP2 was used in the experiments. Gel Shift Assays The sequence of the oligonucleotide containing the human CpG island sequence used in binding studies of boar TP2 and TP2Z is shown in Fig. 2. The complementary strands were annealed in 1 mM EDTA/10 mM Tris–HCl, pH 7.5, by heating at 95°C for 20 min and slow cooling at 25°C for 4 h. The duplex oligonucleotide was purified on a nondenaturing 15% polyacrylamide gel, and labeled with [g- 32P]ATP using T4 polynucleotide kinase. The binding of TP2 to the nucleotide was carried out in a buffer (5 ml) containing 150 mM NaCl/5 mM MgCl 2/10 mM Tris–HCl, pH 7.5, or 50 mM NaCl/5 mM MgCl 2/10 mM Tris–HCl, pH 7.5, at 37°C for 1 h. In each binding reaction, 25,000 cpm of the nucleotide
FIG. 2. The sequence of a 40-mer oligonucleotide defined as a CpG island that is present in the 39-downstream of the human d-aminolevulinic acid dehydratase gene (12) used for gel shift assays.
(approx 1 ng) and 10 –25 ng of the refolded TP2 were used. After the reaction, samples were electrophoresed on a 6% native polyacrylamide gel (acrylamide:bisacrylamide 5 19:1) in 6.6 mM Tris–HCl/3.3 mM sodium acetate, pH 7.5. The gel was preelectrophoresed at 20 V/cm for 30 min at 15°C. After the samples were loaded, electrophoresis was carried out at 20 V/cm for 1 h. The gel was subsequently dried and autoradiographed. For competition experiments, the competitor nucleic acid was added to the complex of TP2–CpG island oligonucleotide and further incubated at 37°C for 1 h. In the case of TP2Z, freshly refolded TP2Z or preincubated TP2Z in 150 mM NaCl/5 mM MgCl 2/10 mM Tris–HCl, pH 7.5, at 37°C overnight with shaking to oxidize TP2Z was used. The binding reactions of TP2Z (10 – 45 ng) to the nucleotide (25,000 cpm, approximately 1 ng) were carried out at 37°C for 1 h in 150 mM NaCl/5 mM MgCl 2/10 mM Tris–HCl, pH 7.5 (5 ml). After the reaction, samples were electrophoresed on a 10% native polyacrylamide gel for 2 h. Construction of an Expression Vector for Boar TP2– Zinc-Binding Domain Double-stranded DNA fragments encoding TP2Z with three potential zinc finger motifs were synthesized as 18 fragments coding for 103 amino acid residues. Their sequences are shown in Fig. 3A. After phosphorylation of the 59-termini using T4 polynucleotide kinase, the synthetic oligonucleotides were annealed and cloned into the EcoRI–BamHI sites of pUC18 using E. coli XL1-blue cells (Toyobo) (Fig. 3B), in order to verify the nucleotide sequences of the constructed genes by dideoxy sequencing of the plasmids. The plasmids were digested with NcoI and BamHI, and the DNA fragments encoding the TP2Z were purified
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FIG. 3. (A) Nucleotide sequences corresponding to boar TP2Z. The total nucleotide sequence encoding TP2Z was resolved into 18 fragments. The numbers of fragments (1–9) correspond to the order from the 59-terminal to the 39-terminal. The sense and anti-sense strands are shown as (1) and (2), respectively. DNA fragments were synthesized. (B) Construction of an expression vector for boar TP2Z.
and inserted into the NcoI–BamHI sites of plasmid pET11d (Novagen) (Fig. 3B). Expression of TP2Z This expression plasmid pET–TP2Z was transformed into HMS174 (DE3) E. coli competent cells. The E. coli were selected on an LB–agar plate containing 100 mg/ml ampicillin. A single colony was picked up and grown overnight in LB medium containing 100 mg/ml ampicillin. The overnight culture was diluted 1:100 with LB medium containing 100 mg/ml ampicillin and allowed to grow further until the A 600 reached 0.8 –1.0. Isopropylthiogalactoside (IPTG) was added at
a final concentration of 1 mM, and the cells were harvested after 6 h by centrifugation at 5000g for 10 min. The aliquot of the culture was SDS-treated and the total E. coli proteins were analyzed by 15% SDS–polyacrylamide gel electrophoresis (SDS–PAGE). The cells were resuspended in 1 mM EDTA/10 mM Tris–HCl, pH 7.5, and sonicated on ice six or seven times with 1-min bursts every 3 min at a 200-W output, using a Tomy UP-200 ultrasonic vibrator. Trichloroacetic acid (TCA) (80% (w/w)) was added to the sonicate to give a final concentration of 20%. The precipitate including DNA and proteins was washed with acidified acetone. The basic proteins were extracted from the precipitate
ZINC-BINDING DOMAIN OF BOAR TRANSITION PROTEIN 2
with 0.71 M 2-mercaptoethanol/0.2 M H 2SO 4. The extract was centrifuged at 15,000g for 10 min. To the supernatant, 80% TCA was added to bring the TCA concentration to 20%. The precipitate was recovered by centrifugation and washed with acidified acetone and dried. The dried material was suspended in 65 mM dithiothreitol (DTT)/7 M urea/0.1 M sodium acetate– HCl, pH 2.0. The suspension was incubated at 37°C for 30 min and centrifuged at 15,000g for 10 min. The supernatant was loaded onto ion-exchange chromatography on a Fractogel EMD SO 32 650 (M) column equilibrated with 50 mM NaCl/0.1 M sodium acetate–HCl buffer, pH 2.0. The column was washed with 5 vol of 50 mM NaCl/0.1 M sodium acetate–HCl buffer, pH 2.0, to remove DNA fragments thoroughly. The bound proteins including TP2Z were eluted in 0.1 M sodium acetate–HCl buffer, pH 2.0, with a 0.05–2.0 M NaCl linear gradient at a flow rate of 55 ml/h. TP2Z was eluted at about 0.8 M NaCl. The fractions containing TP2Z were desalted by gel filtration through Bio-gel P-2 in 0.2 M acetic acid. The fraction passed through the column was lyophilized. The lyophilized proteins were dissolved in 0.71 M 2-mercaptoethanol/7 M GuHCl/50 mM sodium acetate–HCl buffer, pH 2.0. The solution was incubated at 37°C for 15 min and subjected to reverse-phase HPLC on Nucleosil 300 7C18. The fractions containing TP2Z were lyophilized, and the lyophilized proteins were dissolved in 0.71 M 2-mercaptoethanol/7 M GuHCl/50 mM sodium acetate–HCl buffer, pH 2.0, and further purified by gel filtration HPLC on Diol-120. The purified TP2Z was refolded in the same manner as intact TP2 (15) by a slight modification of the method of Beaudette et al. (20). TP2Z was dissolved in 32 mM DTT/7 M GuHCl/10 mM Tris–HCl, pH 8.8, and incubated for 1 h at 40°C. After the pH was adjusted to 2.0 with 10% trifluoroacetic acid (TFA), the solution was dialyzed against 50 mM ZnCl 2/150 mM KF/0.1 mM DTT/10 mM Tricine– NaOH, pH 7.4, or 10 mM EDTA/150 mM KF/0.1 mM DTT/10 mM Tricine–NaOH, pH 7.4, for 24 h and then dialyzed against the same buffer without ZnCl 2 or EDTA for 48 h. The freshly refolded TP2Z was used in the experiments. Circular Dichroism CD was measured with a Jasco J-500 spectropolarimeter at 25°C with nitrogen flushing. The results were expressed in terms of mean residue ellipticity [u] in deg cm 2 dmol 21. Protein concentration of the solution was determined by amino acid analysis. Amino Acid Analysis Samples were hydrolyzed in vacuo with 6 M HCl at 110°C for 24 h. Amino acids were determined on a Hitachi 655 amino acid analyzer.
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Atomic Absorption Spectroscopy Analysis of the zinc content in TP2Z samples was carried out by the one-drop method in a Shimadzu AA-630-12 atomic absorption/flame emission spectrophotometer as described earlier (15). All glassware was washed with 0.1 M nitric acid and extensively rinsed with metal-free water (obtained with a Milli Q system). The solution of TP2Z was dialyzed exhaustively against metal-free water. The concentrations of zinc in the samples were adjusted to give readings between 200 –1000 ppb and determined as the mean value of triplicate analyses. Matrix-Assisted Laser Desorption and Ionization Time-of-Flight Mass Spectrometry Matrix-assisted laser desorption and ionization time-of-flight mass spectrometry were carried out using a Jeol JMS-LT1000 laser TOFMS instrument in the positive ion mode. Ten microliters of 50 mM a-cyano-4-hydroxycinnamic acid dissolved in 0.1% TFA/35% acetonitrile was mixed with 1 ml of TP2Z solution (1 mg/ml), and a 1-ml aliquot of the mixture was applied to the sample stage and allowed to air-dry. Approximately 250 shots were summed. Calibration of the mass scale was accomplished with bovine insulin (M r 5,733) and hen egg lysozyme (M r 14,306). RESULTS AND DISCUSSION
Gel Shift Assays with Boar TP2 and CpG Island Oligonucleotide Rat TP2 recognizes preferentially poly(dG-dC) z poly(dG-dC) and a human CpG island oligonucleotide containing six CpG doublets in a zinc-dependent manner (11,12). We examined whether boar TP2 recognized the CpG island oligonucleotide (40-mer) as a model substance containing GC-rich sequence (Fig. 2). Figure 4 shows the results of the gel shift experiments carried out with TP2 and CpG island oligonucleotide in the buffer containing 150 mM NaCl. The gel mobility shifts due to the complex formation appeared at 10 ng of TP2. The size and breadth of bands of the complexes increased with an increase in the amount of TP2 (Fig. 4). At the amount of TP2 between 15 and 20 ng, some aggregates of the complex were observed in the well. With an increase in the amount of TP2, the amount of the soluble complexes detected on the gel decreased (Fig. 4) and that of the insoluble complexes precipitated in the reaction buffer increased (data not shown). When the binding reaction was carried out in the buffer containing 50 mM NaCl, similar results were obtained (data not shown). The sequence preference in DNA recognition by TP2 was investigated by adding various synthetic polynucleotides as competitors in the gel shift experi-
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binding domain of boar TP2 recognized the CpG island oligonucleotide. The content of boar TP2 is estimated to be 4.3 nmol per 100 g of the testis and lowest among that of boar TP1-4 (21). Therefore, we constructed an expression system of the expected zinc binding domain (TP2Z) of boar TP2 with three potential zinc finger motifs (residues 1–103) (Fig. 1) and it expressed in E. coli. Expression and Purification of TP2Z The synthesized cDNA fragment was inserted between the NcoI and BamHI sites of pET11d expression FIG. 4. Gel shift assay with boar TP2 and the human CpG island oligonucleotide (Fig. 2). The binding reaction was done with 32P-endlabeled oligonucleotide (25,000 cpm, 1 ng) as described in the text. After the reaction, the complexes were analyzed on a 6% nondenaturing polyacrylamide gel. An arrowhead shows location of the gel wells.
ments (Fig. 5). As expected, addition of cold homologous oligonucleotide inhibited the complex formation of TP2 with the labeled oligonucleotide (Fig. 5A). That is, the homologous oligonucleotide was as effective as a competitor. Poly(dI-dC) z (dI-dC) did not inhibit the complex formation (Fig. 5B). Interestingly, the alternating copolymer poly(dG-dC) z poly(dG-dC) was a more effective competitor than the cold homologous oligonucleotide (Fig. 5D). This can be explained by the fact that this alternating copolymer provides more GC binding sites for TP2 than the CpG island oligonucleotide. However, poly(dA-dT) z poly(dA-dT), poly(dA) z poly(dT), and poly(dG) z poly(dC) showed weak competition at 20-fold weight excess (Figs. 5C, 5E, and 5F). When TP2 was preincubated with 10 mM EDTA, this metal chelator did not reduce the formation of the protein– oligonucleotide complex (lanes 1 in Figs. 6A and 6B). Poly(dI-dC) z poly(dI-dC) was also effective competitor similar to the cold homologous oligonucleotides (Fig. 6) in contrast to the case of TP2 refolded in the presence of Zn 21 (Fig. 5B). The other four synthetic polynucleotide competitors, poly(dA-dT) z poly(dA-dT), poly(dG-dC) z poly(dG-dC), poly(dA) z poly(dT), and poly(dG) z poly(dC), gave results similar to those of poly(dI-dC) z poly(dI-dC) (data not shown). When EDTA concentration was increased up to 25 mM, these properties remained unchanged (data not shown). These results indicate that by the EDTA-treatment of TP2 the binding specificity disappears. As described above, the complexes of TP2-CpG island oligonucleotide had a tendency to form aggregates and precipitate (Fig. 4), and there was some nonspecific binding of TP2 to the oligonucleotide (Figs. 5C, 5E, and 5F), making the results of gel shift assays obscure. Then we attempted to investigate whether the zinc-
FIG. 5. Effect of synthetic polynucleotides of defined sequences on the binding of boar TP2 to the human CpG island oligonucleotide. Gel shift assays were done as described in the legend to Fig. 4 in the presence of (A) cold homologous oligonucleotide, (B) poly(dI-dC) z poly(dI-dC), (C) poly(dA-dT) z poly(dA-dT), (D) poly(dG-dC) z poly(dGdC), (E) poly(dA) z poly(dT), and (F) poly(dG) z poly(dC). The fold weight excess of unlabeled competitor with regard to nucleotide concentration in lanes 1–5 is 0, 2.5, 5, 10, and 20, respectively. The free oligonucleotide probe is shown in lane P.
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CD Studies and Atomic Absorption Spectroscopy Figure 8 shows CD spectra of TP2Z refolded in the presence of 50 mM ZnCl 2 and 150 mM KF or in the presence of 10 mM EDTA and 150 mM KF. Potassium fluoride instead of NaCl was used for desired ionic strength to eliminate the Cl 2 interference (24). The CD spectrum of TP2Z refolded in the presence of 50 mM Zn 21 has a 204-nm minimum with a shoulder around
FIG. 6. Effect of removal of zinc from boar TP2 on its binding to the CpG island oligonucleotide. TP2 was preincubated with 10 mM EDTA and then used in the binding reaction in the presence of (A) cold homologous oligonucleotide and (B) poly(dI-dC) z poly(dI-dC). The fold weight excess with regard to nucleotide concentration is the same as that in Fig. 5.
vector. The recombinant plasmid was transformed into competent E. coli HMS174 (DE3) cells. Following induction with 1 mM IPTG, the cells were harvested after 6 h and the total E. coli proteins were analyzed by 15% SDS–PAGE (Fig. 7C, lane 2). TP2Z (M r 11,368) gave a band corresponding to a polypeptide with M r of 21,000. This may be due to the higher basic amino acid content of TP2Z than that of molecular-mass markers. After SDS–PAGE, the total proteins were transferred onto polyvinylidene difluoride membrane (22) and the polypeptide around 21 kDa was sequenced directly with a protein sequencer. As a result, the N-terminal amino acid sequence of TP2Z (residues 1–15) was confirmed. The basic proteins were then extracted with acid including 0.71 M 2-mercaptoethanol from the harvested cells. The extracted was chromatographed on Fractogel EMD SO 32 650 (M). The TP2Z-containing fraction was purified by HPLC on Nucleosil 300 7C18 (Fig. 7A). The TP2Z-containing fraction (peak a in Fig. 7A) was further purified by HPLC on Diol-120 (Fig. 7B). The purified TP2Z (peak b-protein in Fig. 7B) showed a single band on SDS–PAGE (Fig. 7C, lane 5). The matrix-assisted laser desorption ionization timeof-flight mass spectrometry spectrum of the purified TP2Z showed a peak indicating the protonated mass value of 11,370 6 89 Da (data not shown). Figure 7C and Table 1 show the process of purification of TP2Z. The yield of the purified TP2Z was approximately 7 mg/liter of the culture. Its yield is greater than that of rat TP2 expressed in E. coli (0.4 – 0.5 mg/liter) (23). This might be due to the fact that we designed the synthetic nucleotide sequence for TP2Z in which the third letters for the Ser and Cys were selected in order to avoid formation of the secondary structure interfering with translation of TP2Z mRNA into its polypeptide chain, when TP2Z mRNA was synthesized in E. coli.
FIG. 7. (A) Separation of the crude basic proteins from E. coli lysate by reverse-phase HPLC. The basic proteins containing TP2Z (1 mg) obtained by ion-exchange chromatography on Fractogel EMD SO 32 650(M) was chromatographed on a column of (4 3 150 mm) Nucleosil 300 7C18 in 0.1% TFA with a 0 – 80% acetonitrile (MeCN) linear gradient at 35°C at a flow rate of 1 ml/min. (B) Gel filtration of the TP2Z fraction on Diol-120. The proteins (0.2 mg) in peak a in Fig. 7A were chromatographed on a column (8 3 500 mm) of Diol-120 in 0.1% TFA/10% MeCN. Fractions were collected at a flow rate of 1 ml/min. (C) SDS–PAGE of the fractions obtained in the process of purification of TP2Z. Lanes: 1, total proteins of E. coli HMS174 (DE3) cells containing expression plasmid pET–TP2Z before induction with IPTG; 2, total E. coli proteins after induction with IPTG for 6 h; 3, acid extract from E. coli cells; 4, peak a-proteins (Fig. 7A); 5, peak b-protein (Fig. 7B). The mobilities of molecular-mass markers (rabbit muscle phosphorylase b, bovine serum albumin, hen egg white ovalbumin, bovine carbonic anhydrase, soy bean trypsin inhibitor, and hen egg white lysozyme), in kDa, are indicated by arrowheads.
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FIG. 8. CD spectra of TP2Z refolded in the presence and absence of Zn 21. 1 (- - -), TP2Z treated with 10 mM EDTA and refolded in the absence of Zn 21; 2 (—), TP2Z refolded in the presence of 50 mM Zn 21.
220 nm. The shape of the curve is similar to that of intact TP2 refolded in the presence of 50 mM Zn 21 (15), and typical of a type I b-turn which has been observed for Cbz-Gly-Gly-Pro-Gly-O-stearate in acetonitrile (25). When TP2Z was treated with 10 mM EDTA and refolded in the absence of Zn 21, there was an increase in the negative ellipticity at 204 nm. Furthermore, the ellipticity minimum was shifted to 202 nm and the shoulder around 220 nm deleted, in a manner similar to that for intact TP2 (15). These results suggest that zinc induces formation of antiparallel b-sheet and/or b-turn in TP2Z as well as in intact TP2 (15). On the other hand, removal of zinc from rat TP2 and its zincbinding domain decreases the negative ellipticity at 200 nm and deletes a shoulder at 222 nm (10). In order to determine the actual number of zinc atoms that can be bound to TP2Z in the nearly physiological ionic strength and pH, the TP2Z refolded in the presence of 50 mM ZnCl 2 and 150 mM KF was exhaustively dialyzed against metal-free water until the dialysate showed negligible amounts of zinc. TP2Z contained 2.65 atoms of zinc bound per molecule of the protein, as intact TP2 does (15). The TP2Z pretreated with 10 mM EDTA did not give any signal for zinc. These results indicate that TP2Z has zinc-binding domain of boar TP2. Gel Shift Assays with TP2Z and CpG Island Oligonucleotide We examined whether TP2Z recognizes the CpG island oligonucleotide as does boar TP2. Figures 9A and 9B show the results of the gel shift experiments carried out with freshly refolded TP2Z and CpG island oligonucleotide in the buffer containing 150 mM NaCl and with the oxidized TP2Z and CpG island oligonucleotide
in the same buffer, respectively. The complex of freshly refolded TP2Z–CpG island oligonucleotide appeared at TP2Z amounts larger than 20 ng (Fig. 9A). The amount of the complex formed remained constant between 25 and 40 ng of input TP2Z amount, and small amounts of aggregates of the complex were observed in the well. On the other hand, the complex of the oxidized TP2Z– CpG island oligonucleotide appeared only in the form of aggregates which were retained in the well. These results suggest that freshly refolded TP2Z under reducing conditions binds to the oligonucleotide specifically and that the oxidized TP2Z does nonspecifically and forms aggregates of the complex. Aggregation seen in Fig. 9B may be due to oxidation forming TP2Z dimers that form a network complex with DNA, since TP2Z dimer was detected as a main product of TP2Z oxidation on SDS–PAGE (data not shown). Cold homologous oligonucleotide showed effective competition (Fig. 10A), and poly(dI-dC) z poly(dI-dC) showed no competition (Fig. 10B), indicating that the complexes are specific in nature. Poly(dG-dC) z poly(dG-dC) was a most effective competitor among the synthetic polynucleotides used, and the degree of competition is stronger than that in the case with intact TP2 (Fig. 10D and Fig. 5D). Figure 10C shows the results obtained with the alternating copolymer poly(dA-dT) z poly(dA-dT) as the competitor. There was a decrease in the amount of the complex formed (Fig. 10C, lane 5, 20-fold weight excess). When the two homoduplexes, poly(dA) z poly(dT) and poly(dG) z poly(dC), were used as competitors in the gel shift assays, neither of these homoduplexes competed with the complex formation (Figs. 10E and 10F). The results in Figs. 10D and 10F suggest that GC base pairs within
TABLE 1 Summary of Purification of the Zinc-Binding Domain of Boar Transition Protein 2 from the Culture of E. coli HMS174(DE3) Cells Containing Expression Plasmid pET–TP2Z
Step
Total proteins a (mg)
TP2Z b (mg)
Relative purification (fold)
Yield (%)
culture Acid extract Fractogel EMD SO 32 Nucleosil 300 7C18 Diol-120
60 16 10 7.7 7.2
20 12 8.7 7.4 7.2 c
1.0 2.3 2.6 2.9 3.0
100 60 44 37 36
a
The total proteins were estimated by amino acid analysis. The amount of TP2Z during purification was estimated from the ratio of TP2Z to the total proteins: The ratio was calculated from the peak areas of TP2Z and other proteins in the microdensitometric tracings of the gel (Fig. 7C). c The amount of the purified TP2Z was determined by amino acid analysis. b
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FIG. 9. (A) Gel shift assays with freshly refolded TP2Z and CpG island oligonucleotide. (B) Gel shift assays with oxidized TP2Z and CpG island oligonucleotide. The binding reaction was done with 32P-end-labeled oligonucleotide (25,000 cpm, 1 ng) as described in the text. After the reaction, the complexes were analyzed on a 10% nondenaturing polyacrylamide gel. An arrowhead shows the location of the gel wells.
the CpG doublet, and not a single GC base pair, are important for recognition of the CpG rich oligonucleotide by TP2Z. These results show that TP2Z recognizes the CpG-rich sequence more preferentially than intact TP2. EDTA-treatment of TP2Z did not affect the binding ability to the CpG oligonucleotide (lanes 1 in Figs. 11A and 11B), but the binding specificity disappeared (Figs. 11A and 11B). Poly(dI-dC) z poly(dI-dC) showed similar competition to the cold homologous oligonucleotide, providing direct evidence for the involvement of zinc-induced polypeptide folding of TP2Z (Fig. 8) in the specific recognition of the oligonucleotide. In the case of rat TP2, more than 0.7-fold molar excess of poly(dA-dT) z poly(dA-dT) decreases the binding to the CpG island oligonucleotide to about 40%, but poly(dG) z poly(dC) or poly(dA) z poly(dT) does not show any competition, and removal of zinc inhibits the binding to the CpG oligonucleotide. Accordingly, boar TP2 and TP2Z recognize the CpG island sequence in a manner similar to but somewhat different than that for rat TP2 (12).
FIG. 10. Effect of synthetic polynucleotides of defined sequences on the binding of boar TP2Z to the CpG island oligonucleotide. Gel shift assays were done as described in the legend to Fig. 9 in the presence of (A) cold homologous oligonucleotide, (B) poly(dI-dC) z poly(dI-dC), (C) poly(dA-dT) z poly(dA-dT), (D) poly(dG-dC) z poly(dG-dC), (E) poly(dA) z poly(dT), and (F) poly(dG) z poly(dC). The fold weight excess with respect to nucleotide concentration is the same as that in Fig. 5.
FIG. 11. Effect of removal of zinc from TP2Z on its binding to the CpG island oligonucleotide. TP2Z was preincubated with 10 mM EDTA and then used in the binding reaction in the presence of (A) cold homologous oligonucleotide or (B) poly(dI-dC) z poly(dI-dC). The fold weight excess with regard to nucleotide concentration is the same as that in Fig. 5.
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The C-terminal one-third region of rat TP2 does not bind to zinc, and its CD spectra are unchanged in the presence or absence of zinc (10). The C-terminal basic region of TP2 is highly conserved between rat, mouse, boar, and bovine (18), suggesting that this region of boar TP2 (104–137 residues) lacks the zinc-binding motif. It is likely that this C-terminal domain does not show specific recognition to DNA, since basic peptides that contain several to about 30 arginine residues show nonspecific binding to DNA mainly by electrostatic interaction (26,27). On the basis of the preferential recognition of the CpG-rich oligonucleotide by rat TP2, Kundu and Rao have proposed that TP2 interacts with DNA using the CpG-rich nucleotide as the target sequence, which in turn may initiate chromatin condensation and repress transcriptional activity (12). Our expression system for isolating zinc-binding domain of boar TP2 will be useful for further study to identify the exact cysteine and histidine residues that coordinate with zinc to understand the tertiary structure of boar TP2. Such information will be valuable to understand biological functions of boar TP2 and its zinc-binding domain and to elucidate the essential structures and functions of mammalian TP2s by comparison of their structures and binding characteristic to DNA. ACKNOWLEDGMENTS The authors are indebted to Professor Koichi Oguma and Mr. Toshihiro Suzuki of Faculty of Engineering, Chiba University, Japan, for atomic absorption spectroscopy in the analysis of the zinc content in TP2Z.
REFERENCES 1. Meistrich, M. L., Brock, W. A., Grimes, S. R., Platz, R. D., and Hnilica, L. S. (1978) Nuclear transition proteins during spermatogenesis. Fed. Proc. 37, 2522–2525. 2. Balhorn, R., Weston, S., Thomas, C., and Wyrobeck, A. J. (1984) DNA packing in mouse spermatids. Synthesis of protamine variants and four transition proteins. Exp. Cell Res. 150, 298 –308. 3. Dupressoir, T., Sautie`re, P., Lanneau, M., and Loir, M. (1985) Isolation and characterization of the ram spermatidal nuclear proteins P1, 4 and T. Exp. Cell Res. 161, 63–74. 4. Unni, E., and Meistrich, M. L. (1992) Purification and characterization of the rat spermatid nuclear protein, TP4. J. Biol. Chem. 267, 25359 –25363. 5. Loir, M., Bouvier, D., Fornells, M., Lanneau, M., and Subirana, J. A. (1985) Interactions of nuclear proteins with DNA, during sperm differentiation in the ram. Chromosoma 92, 304 –312. 6. Singh, J., and Rao, M. R. S. (1987) Interaction of rat testis protein, TP, with nucleic acids in vitro: Fluorescence quenching, UV absorption, and thermal denaturation studies. J. Biol. Chem. 262, 734 –740. 7. Singh, J., and Rao, M. R. S. (1988) Interaction of rat testis protein TP1 with nucleosome core particles. Biochem. Int. 17, 701– 410. 8. Baskaran, R., and Rao, M. R. S. (1990) Interaction of spermatidspecific protein TP2 with nucleic acids, in vitro: A comparative study with TP1. J. Biol. Chem. 265, 21039 –21047.
9. Baskaran, R., and Rao, M. R. S. (1991) Mammalian spermatid specific protein, TP2, is a zinc metalloprotein with two finger motifs. Biochem. Biophys. Res. Commun. 179, 1491–1499. 10. Kundu, T. K., and Rao, M. R. S. (1994) Characterization of the zinc-metalloprotein nature of rat spermatidal protein TP2. FEBS Lett. 351, 6 –10. 11. Kundu, T. K., and Rao, M. R. S. (1995) DNA condensation by the rat spermatidal protein TP2 shows GC-rich sequence preference and is zinc dependent. Biochemistry 34, 5143–5150. 12. Kundu, T. K., and Rao, M. R. S. (1996) Zinc dependent recognition of a human CpG island sequence by the mammalian spermatidal protein TP2. Biochemistry 35, 15626 –15632. 13. Akama, K., Kosuge, M., Sato, H., Yamaoka, Y., Nakano, M., and Tobita, T. (1994) Isolation of intact transition protein 1 and 3 from boar late spermatid nuclei. Biochem. Mol. Biol. Int. 32, 615– 622. 14. Akama, K., Ichimura, H., Kosuge, M., Sato, H., Yamaoka, Y., Nakano, M., and Tobita, T. (1994) Isolation of intact transition protein 4 from boar late spermatid nuclei. Biochem. Mol. Biol. Int. 34, 315–321. 15. Akama, K., Sato, H., Oguma, K., and Nakano, M. (1997) Isolation of intact transition protein 2 with three zinc finger motifs from boar late spermatid nuclei. Biochem. Mol. Biol. Int. 42, 865– 872. 16. Akama, K., Sato, H., Frihata-Yamauchi, M., Komatsu, Y., Tobita, T., and Nakano, M. (1996) Interaction of nucleosome core DNA with transition proteins 1 and 3 from boar late spermatid nuclei. J. Biochem. 119, 448 – 455. 17. Akama, K., Ichimura, H., Kosuge, M., Sato, H., Kojima, S., Miura, K.-I., Hayashi, H., Komatsu, Y., and Nakano, M. (1995) The amino acid sequence and interaction with the nucleosome core DNA of transition protein 4 from boar late spermatid nuclei. Eur. J. Biochem. 233, 179 –185. 18. Keime, S., Kumm, S., Luerssen, H., and Engel, W. (1992) The nucleotide sequence of boar transition protein 2 (TNP2) cDNA and haploid expression of the gene during spermatogenesis. Animal Genet. 23, 373–378. 19. O’Farrel, P. H. (1975) High resolution two-dimensional electrophoresis of proteins. J. Biol. Chem. 250, 4007– 4021. 20. Beaudette, N. V., Fulmer, A. W., Okabayashi, H., and Fasman, G. D. (1981) Study of conformational states and reversibility of histone complexes. Biochemistry 20, 6526 – 6535. 21. Akama, K., Maruyama, R., Mochizuki, H., and Tobita, T. (1990) Boar transition protein 2 and 4 isolated from late spermatid nuclei by high-performance liquid chromatography. Biochem. Biophys. Acta 1041, 264 –268. 22. Hirano, H. (1989) Microsequence analysis of winged been seed proteins electroblotted from two-dimensional gel. J. Protein Chem. 8, 115–130. 23. Meetei, A. R., and Rao, M. R. S. (1996) Cloning of cDNA encoding rat spermatidal protein TP2 and expression in Escherichia coli. Protein Expr. Purif. 8, 409 – 415. 24. Yang, J. T., Wu, C.-S. C., and Martinez, H. M. (1986) Calculation of protein conformation from circular dichroism. Methods in Enzymology (Hirs, C. H. W., and Timasheff, S. N., Eds.), Vol. 130, pp. 208 –269, Academic Press, New York. 25. Garnier, J., Osguthorpe, D. J., and Robson, B. (1978) Analysis of the accuracy and implications of simple methods for predicting the secondary structure of globular proteins. J. Mol. Biol. 120, 97–120. 26. Kawashima, S., and Ando, T. (1978) Interaction of basic oligo-Lamino acids with deoxyribonucleic acids: Oligo-L-arginines of various chain lengths and herring sperm DNA. J. Biochem. 84, 343–350. 27. Churchill, M. E., and Travers, A. A. (1991) Protein motifs that recognize structural features of DNA. Trends Biochem. Sci. 16, 92–97.
Expression of a Zinc-Binding Domain of Boar Spermatidal Transition Protein 2 in Escherichia coli Hiroki Sato,* Kuniko Akama,* Shuichi Kojima,† Kin-ichiro Miura,† Atsushi Sekine,* and Minoru Nakano* *Graduate School of Science and Technology, Chiba University, Chiba, Chiba 263-8522, Japan; and †Institute for Biomolecular Science, Gakushuin University, Mejiro, Tokyo 171-0031, Japan
Received March 1, 1999, and in revised form April 29, 1999
Transition protein 2 (TP2; 137 amino acid residues) from boar late spermatid nuclei has three potential zinc finger motifs in the N-terminal 43 region. Gel shift assays revealed that boar TP2 recognized a CpG island sequence in a zinc-dependent manner. However, there was some nonspecific recognition of the oligonucleotide. Then, we constructed the expression system of zinc-binding domain of TP2 (TP2Z) (residues 1–103) in Escherichia coli. Double-stranded DNA fragments encoding TP2Z were synthesized as 18 fragments with 103 residues, annealed, and cloned into the expression plasmid pET11d. TP2Z was expressed upon induction with 1 mM isopropylthiogalactoside and extracted with acid including 0.71 M 2-mercaptoethanol. TP2Z was purified by ion-exchange chromatography on Fractogel EMD SO 32 and HPLC on Nucleosil 300 7C18 and on Diol-120. Atomic absorption and CD spectroscopy showed that TP2Z bound three atoms of zinc per molecule of the protein and underwent a zinc-dependent conformational change in a manner similar to that for intact TP2. Gel shift assays indicated that TP2Z recognized a CpG island sequence more specifically than intact TP2 and that the specificity is dependent on zinc. © 1999 Academic Press Key Words: CpG-rich sequence; gene engineering; transition protein; zinc-binding domain.
The chromatin structure undergoes extensive changes during mammalian spermiogenesis. Transition proteins (TP1– 4) appear during the elongating phase of mammalian spermiogenesis. Nucleosomal histones are transiently replaced by transition proteins and, finally, by protamines (1– 4). At that time, transformation of the nucleosomal-type chromatin into a smooth chromatin fiber, initiation of chromatin condensation, and cessation of transcription occur (5). Rat 454
TP1, having higher affinity for single-stranded DNA, induces local melting of DNA (6,7). Rat TP2, with two potential zinc finger motifs in the amino terminal region, is a DNA-condensing protein and prefers zincdependently GC-rich sequences, including a human nonmethylated CpG island sequence (8 –12). Recently, we have developed methods of isolating boar TP1– 4 (13–15) and have reported that boar TP1 and TP3, having higher affinity for single-stranded DNA, and TP4, having higher affinity for double-stranded DNA, are DNA-melting proteins mediated through the stacking of Tyr 32, Trp 18, and Trp 126 with nucleic acid bases, respectively (16,17). Boar TP2 has three potential zinc finger motifs in the N-terminal 43 region and binds three atoms of zinc per molecule of the protein (15). The sequence of boar TP2 has two insertions of 11 amino acids at position 36, and of 10 amino acids at position 50, into that of rat and mouse TP2 (18), although boar, rat, and mouse TP2 have a highly conserved carboxy-terminal basic region. In this paper, we describe the nonspecific interaction of boar TP2 with a CpG island sequence by gel shift assays, the construction of an expression system of zinc-binding domain (TP2Z) with three zinc finger motifs of boar TP2 (residues 1–103) (Fig. 1) in Escherichia coli, and some characteristics of TP2Z. TP2Z contained three atoms of zinc bound per molecule and underwent a zinc-dependent secondary structural change similar to intact TP2 (137 residues), TP2Z recognized a CpG island sequence more preferentially than intact TP2, and its specificity is dependent on zinc. MATERIALS AND METHODS
Fractogel EMD SO 32 650 (M), Bio-gel P-2, Nucleosil 300 7C18, and Diol-120 were products of Merck, BioRad, Nagel, and YMC, respectively. The synthetic polynucleotides, poly(dG-dC) z poly(dG-dC), poly(dA1046-5928/99 $30.00 Copyright © 1999 by Academic Press All rights of reproduction in any form reserved.
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FIG. 1. A scheme of boar TP2 with three potential zinc finger motifs (15) and its zinc-binding domain (TP2Z).
dT) z poly(dA-dT), poly(dI-dC) z poly(dI-dC), poly(dG) z poly(dC), and poly(dA) z poly(dT), were purchased from Pharmacia. The complementary oligonucleotides (40mer) containing the human CpG island sequence 39 downstream of the d-aminolevulinic acid dehydratase gene (Fig. 2) were synthesized on a Sci-media gene assembler. [g- 32P]ATP (sp act of .7000 Ci/mmol) was from ICN Pharmaceuticals, Inc. T4 polynucleotide kinase was from Takara. Preparation of Boar TP2 Boar transition protein 2 was isolated from the late spermatid nuclei and refolded by our previously published method (13,15). The freshly refolded TP2 was used in the experiments. Gel Shift Assays The sequence of the oligonucleotide containing the human CpG island sequence used in binding studies of boar TP2 and TP2Z is shown in Fig. 2. The complementary strands were annealed in 1 mM EDTA/10 mM Tris–HCl, pH 7.5, by heating at 95°C for 20 min and slow cooling at 25°C for 4 h. The duplex oligonucleotide was purified on a nondenaturing 15% polyacrylamide gel, and labeled with [g- 32P]ATP using T4 polynucleotide kinase. The binding of TP2 to the nucleotide was carried out in a buffer (5 ml) containing 150 mM NaCl/5 mM MgCl 2/10 mM Tris–HCl, pH 7.5, or 50 mM NaCl/5 mM MgCl 2/10 mM Tris–HCl, pH 7.5, at 37°C for 1 h. In each binding reaction, 25,000 cpm of the nucleotide
FIG. 2. The sequence of a 40-mer oligonucleotide defined as a CpG island that is present in the 39-downstream of the human d-aminolevulinic acid dehydratase gene (12) used for gel shift assays.
(approx 1 ng) and 10 –25 ng of the refolded TP2 were used. After the reaction, samples were electrophoresed on a 6% native polyacrylamide gel (acrylamide:bisacrylamide 5 19:1) in 6.6 mM Tris–HCl/3.3 mM sodium acetate, pH 7.5. The gel was preelectrophoresed at 20 V/cm for 30 min at 15°C. After the samples were loaded, electrophoresis was carried out at 20 V/cm for 1 h. The gel was subsequently dried and autoradiographed. For competition experiments, the competitor nucleic acid was added to the complex of TP2–CpG island oligonucleotide and further incubated at 37°C for 1 h. In the case of TP2Z, freshly refolded TP2Z or preincubated TP2Z in 150 mM NaCl/5 mM MgCl 2/10 mM Tris–HCl, pH 7.5, at 37°C overnight with shaking to oxidize TP2Z was used. The binding reactions of TP2Z (10 – 45 ng) to the nucleotide (25,000 cpm, approximately 1 ng) were carried out at 37°C for 1 h in 150 mM NaCl/5 mM MgCl 2/10 mM Tris–HCl, pH 7.5 (5 ml). After the reaction, samples were electrophoresed on a 10% native polyacrylamide gel for 2 h. Construction of an Expression Vector for Boar TP2– Zinc-Binding Domain Double-stranded DNA fragments encoding TP2Z with three potential zinc finger motifs were synthesized as 18 fragments coding for 103 amino acid residues. Their sequences are shown in Fig. 3A. After phosphorylation of the 59-termini using T4 polynucleotide kinase, the synthetic oligonucleotides were annealed and cloned into the EcoRI–BamHI sites of pUC18 using E. coli XL1-blue cells (Toyobo) (Fig. 3B), in order to verify the nucleotide sequences of the constructed genes by dideoxy sequencing of the plasmids. The plasmids were digested with NcoI and BamHI, and the DNA fragments encoding the TP2Z were purified
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FIG. 3. (A) Nucleotide sequences corresponding to boar TP2Z. The total nucleotide sequence encoding TP2Z was resolved into 18 fragments. The numbers of fragments (1–9) correspond to the order from the 59-terminal to the 39-terminal. The sense and anti-sense strands are shown as (1) and (2), respectively. DNA fragments were synthesized. (B) Construction of an expression vector for boar TP2Z.
and inserted into the NcoI–BamHI sites of plasmid pET11d (Novagen) (Fig. 3B). Expression of TP2Z This expression plasmid pET–TP2Z was transformed into HMS174 (DE3) E. coli competent cells. The E. coli were selected on an LB–agar plate containing 100 mg/ml ampicillin. A single colony was picked up and grown overnight in LB medium containing 100 mg/ml ampicillin. The overnight culture was diluted 1:100 with LB medium containing 100 mg/ml ampicillin and allowed to grow further until the A 600 reached 0.8 –1.0. Isopropylthiogalactoside (IPTG) was added at
a final concentration of 1 mM, and the cells were harvested after 6 h by centrifugation at 5000g for 10 min. The aliquot of the culture was SDS-treated and the total E. coli proteins were analyzed by 15% SDS–polyacrylamide gel electrophoresis (SDS–PAGE). The cells were resuspended in 1 mM EDTA/10 mM Tris–HCl, pH 7.5, and sonicated on ice six or seven times with 1-min bursts every 3 min at a 200-W output, using a Tomy UP-200 ultrasonic vibrator. Trichloroacetic acid (TCA) (80% (w/w)) was added to the sonicate to give a final concentration of 20%. The precipitate including DNA and proteins was washed with acidified acetone. The basic proteins were extracted from the precipitate
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with 0.71 M 2-mercaptoethanol/0.2 M H 2SO 4. The extract was centrifuged at 15,000g for 10 min. To the supernatant, 80% TCA was added to bring the TCA concentration to 20%. The precipitate was recovered by centrifugation and washed with acidified acetone and dried. The dried material was suspended in 65 mM dithiothreitol (DTT)/7 M urea/0.1 M sodium acetate– HCl, pH 2.0. The suspension was incubated at 37°C for 30 min and centrifuged at 15,000g for 10 min. The supernatant was loaded onto ion-exchange chromatography on a Fractogel EMD SO 32 650 (M) column equilibrated with 50 mM NaCl/0.1 M sodium acetate–HCl buffer, pH 2.0. The column was washed with 5 vol of 50 mM NaCl/0.1 M sodium acetate–HCl buffer, pH 2.0, to remove DNA fragments thoroughly. The bound proteins including TP2Z were eluted in 0.1 M sodium acetate–HCl buffer, pH 2.0, with a 0.05–2.0 M NaCl linear gradient at a flow rate of 55 ml/h. TP2Z was eluted at about 0.8 M NaCl. The fractions containing TP2Z were desalted by gel filtration through Bio-gel P-2 in 0.2 M acetic acid. The fraction passed through the column was lyophilized. The lyophilized proteins were dissolved in 0.71 M 2-mercaptoethanol/7 M GuHCl/50 mM sodium acetate–HCl buffer, pH 2.0. The solution was incubated at 37°C for 15 min and subjected to reverse-phase HPLC on Nucleosil 300 7C18. The fractions containing TP2Z were lyophilized, and the lyophilized proteins were dissolved in 0.71 M 2-mercaptoethanol/7 M GuHCl/50 mM sodium acetate–HCl buffer, pH 2.0, and further purified by gel filtration HPLC on Diol-120. The purified TP2Z was refolded in the same manner as intact TP2 (15) by a slight modification of the method of Beaudette et al. (20). TP2Z was dissolved in 32 mM DTT/7 M GuHCl/10 mM Tris–HCl, pH 8.8, and incubated for 1 h at 40°C. After the pH was adjusted to 2.0 with 10% trifluoroacetic acid (TFA), the solution was dialyzed against 50 mM ZnCl 2/150 mM KF/0.1 mM DTT/10 mM Tricine– NaOH, pH 7.4, or 10 mM EDTA/150 mM KF/0.1 mM DTT/10 mM Tricine–NaOH, pH 7.4, for 24 h and then dialyzed against the same buffer without ZnCl 2 or EDTA for 48 h. The freshly refolded TP2Z was used in the experiments. Circular Dichroism CD was measured with a Jasco J-500 spectropolarimeter at 25°C with nitrogen flushing. The results were expressed in terms of mean residue ellipticity [u] in deg cm 2 dmol 21. Protein concentration of the solution was determined by amino acid analysis. Amino Acid Analysis Samples were hydrolyzed in vacuo with 6 M HCl at 110°C for 24 h. Amino acids were determined on a Hitachi 655 amino acid analyzer.
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Atomic Absorption Spectroscopy Analysis of the zinc content in TP2Z samples was carried out by the one-drop method in a Shimadzu AA-630-12 atomic absorption/flame emission spectrophotometer as described earlier (15). All glassware was washed with 0.1 M nitric acid and extensively rinsed with metal-free water (obtained with a Milli Q system). The solution of TP2Z was dialyzed exhaustively against metal-free water. The concentrations of zinc in the samples were adjusted to give readings between 200 –1000 ppb and determined as the mean value of triplicate analyses. Matrix-Assisted Laser Desorption and Ionization Time-of-Flight Mass Spectrometry Matrix-assisted laser desorption and ionization time-of-flight mass spectrometry were carried out using a Jeol JMS-LT1000 laser TOFMS instrument in the positive ion mode. Ten microliters of 50 mM a-cyano-4-hydroxycinnamic acid dissolved in 0.1% TFA/35% acetonitrile was mixed with 1 ml of TP2Z solution (1 mg/ml), and a 1-ml aliquot of the mixture was applied to the sample stage and allowed to air-dry. Approximately 250 shots were summed. Calibration of the mass scale was accomplished with bovine insulin (M r 5,733) and hen egg lysozyme (M r 14,306). RESULTS AND DISCUSSION
Gel Shift Assays with Boar TP2 and CpG Island Oligonucleotide Rat TP2 recognizes preferentially poly(dG-dC) z poly(dG-dC) and a human CpG island oligonucleotide containing six CpG doublets in a zinc-dependent manner (11,12). We examined whether boar TP2 recognized the CpG island oligonucleotide (40-mer) as a model substance containing GC-rich sequence (Fig. 2). Figure 4 shows the results of the gel shift experiments carried out with TP2 and CpG island oligonucleotide in the buffer containing 150 mM NaCl. The gel mobility shifts due to the complex formation appeared at 10 ng of TP2. The size and breadth of bands of the complexes increased with an increase in the amount of TP2 (Fig. 4). At the amount of TP2 between 15 and 20 ng, some aggregates of the complex were observed in the well. With an increase in the amount of TP2, the amount of the soluble complexes detected on the gel decreased (Fig. 4) and that of the insoluble complexes precipitated in the reaction buffer increased (data not shown). When the binding reaction was carried out in the buffer containing 50 mM NaCl, similar results were obtained (data not shown). The sequence preference in DNA recognition by TP2 was investigated by adding various synthetic polynucleotides as competitors in the gel shift experi-
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binding domain of boar TP2 recognized the CpG island oligonucleotide. The content of boar TP2 is estimated to be 4.3 nmol per 100 g of the testis and lowest among that of boar TP1-4 (21). Therefore, we constructed an expression system of the expected zinc binding domain (TP2Z) of boar TP2 with three potential zinc finger motifs (residues 1–103) (Fig. 1) and it expressed in E. coli. Expression and Purification of TP2Z The synthesized cDNA fragment was inserted between the NcoI and BamHI sites of pET11d expression FIG. 4. Gel shift assay with boar TP2 and the human CpG island oligonucleotide (Fig. 2). The binding reaction was done with 32P-endlabeled oligonucleotide (25,000 cpm, 1 ng) as described in the text. After the reaction, the complexes were analyzed on a 6% nondenaturing polyacrylamide gel. An arrowhead shows location of the gel wells.
ments (Fig. 5). As expected, addition of cold homologous oligonucleotide inhibited the complex formation of TP2 with the labeled oligonucleotide (Fig. 5A). That is, the homologous oligonucleotide was as effective as a competitor. Poly(dI-dC) z (dI-dC) did not inhibit the complex formation (Fig. 5B). Interestingly, the alternating copolymer poly(dG-dC) z poly(dG-dC) was a more effective competitor than the cold homologous oligonucleotide (Fig. 5D). This can be explained by the fact that this alternating copolymer provides more GC binding sites for TP2 than the CpG island oligonucleotide. However, poly(dA-dT) z poly(dA-dT), poly(dA) z poly(dT), and poly(dG) z poly(dC) showed weak competition at 20-fold weight excess (Figs. 5C, 5E, and 5F). When TP2 was preincubated with 10 mM EDTA, this metal chelator did not reduce the formation of the protein– oligonucleotide complex (lanes 1 in Figs. 6A and 6B). Poly(dI-dC) z poly(dI-dC) was also effective competitor similar to the cold homologous oligonucleotides (Fig. 6) in contrast to the case of TP2 refolded in the presence of Zn 21 (Fig. 5B). The other four synthetic polynucleotide competitors, poly(dA-dT) z poly(dA-dT), poly(dG-dC) z poly(dG-dC), poly(dA) z poly(dT), and poly(dG) z poly(dC), gave results similar to those of poly(dI-dC) z poly(dI-dC) (data not shown). When EDTA concentration was increased up to 25 mM, these properties remained unchanged (data not shown). These results indicate that by the EDTA-treatment of TP2 the binding specificity disappears. As described above, the complexes of TP2-CpG island oligonucleotide had a tendency to form aggregates and precipitate (Fig. 4), and there was some nonspecific binding of TP2 to the oligonucleotide (Figs. 5C, 5E, and 5F), making the results of gel shift assays obscure. Then we attempted to investigate whether the zinc-
FIG. 5. Effect of synthetic polynucleotides of defined sequences on the binding of boar TP2 to the human CpG island oligonucleotide. Gel shift assays were done as described in the legend to Fig. 4 in the presence of (A) cold homologous oligonucleotide, (B) poly(dI-dC) z poly(dI-dC), (C) poly(dA-dT) z poly(dA-dT), (D) poly(dG-dC) z poly(dGdC), (E) poly(dA) z poly(dT), and (F) poly(dG) z poly(dC). The fold weight excess of unlabeled competitor with regard to nucleotide concentration in lanes 1–5 is 0, 2.5, 5, 10, and 20, respectively. The free oligonucleotide probe is shown in lane P.
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CD Studies and Atomic Absorption Spectroscopy Figure 8 shows CD spectra of TP2Z refolded in the presence of 50 mM ZnCl 2 and 150 mM KF or in the presence of 10 mM EDTA and 150 mM KF. Potassium fluoride instead of NaCl was used for desired ionic strength to eliminate the Cl 2 interference (24). The CD spectrum of TP2Z refolded in the presence of 50 mM Zn 21 has a 204-nm minimum with a shoulder around
FIG. 6. Effect of removal of zinc from boar TP2 on its binding to the CpG island oligonucleotide. TP2 was preincubated with 10 mM EDTA and then used in the binding reaction in the presence of (A) cold homologous oligonucleotide and (B) poly(dI-dC) z poly(dI-dC). The fold weight excess with regard to nucleotide concentration is the same as that in Fig. 5.
vector. The recombinant plasmid was transformed into competent E. coli HMS174 (DE3) cells. Following induction with 1 mM IPTG, the cells were harvested after 6 h and the total E. coli proteins were analyzed by 15% SDS–PAGE (Fig. 7C, lane 2). TP2Z (M r 11,368) gave a band corresponding to a polypeptide with M r of 21,000. This may be due to the higher basic amino acid content of TP2Z than that of molecular-mass markers. After SDS–PAGE, the total proteins were transferred onto polyvinylidene difluoride membrane (22) and the polypeptide around 21 kDa was sequenced directly with a protein sequencer. As a result, the N-terminal amino acid sequence of TP2Z (residues 1–15) was confirmed. The basic proteins were then extracted with acid including 0.71 M 2-mercaptoethanol from the harvested cells. The extracted was chromatographed on Fractogel EMD SO 32 650 (M). The TP2Z-containing fraction was purified by HPLC on Nucleosil 300 7C18 (Fig. 7A). The TP2Z-containing fraction (peak a in Fig. 7A) was further purified by HPLC on Diol-120 (Fig. 7B). The purified TP2Z (peak b-protein in Fig. 7B) showed a single band on SDS–PAGE (Fig. 7C, lane 5). The matrix-assisted laser desorption ionization timeof-flight mass spectrometry spectrum of the purified TP2Z showed a peak indicating the protonated mass value of 11,370 6 89 Da (data not shown). Figure 7C and Table 1 show the process of purification of TP2Z. The yield of the purified TP2Z was approximately 7 mg/liter of the culture. Its yield is greater than that of rat TP2 expressed in E. coli (0.4 – 0.5 mg/liter) (23). This might be due to the fact that we designed the synthetic nucleotide sequence for TP2Z in which the third letters for the Ser and Cys were selected in order to avoid formation of the secondary structure interfering with translation of TP2Z mRNA into its polypeptide chain, when TP2Z mRNA was synthesized in E. coli.
FIG. 7. (A) Separation of the crude basic proteins from E. coli lysate by reverse-phase HPLC. The basic proteins containing TP2Z (1 mg) obtained by ion-exchange chromatography on Fractogel EMD SO 32 650(M) was chromatographed on a column of (4 3 150 mm) Nucleosil 300 7C18 in 0.1% TFA with a 0 – 80% acetonitrile (MeCN) linear gradient at 35°C at a flow rate of 1 ml/min. (B) Gel filtration of the TP2Z fraction on Diol-120. The proteins (0.2 mg) in peak a in Fig. 7A were chromatographed on a column (8 3 500 mm) of Diol-120 in 0.1% TFA/10% MeCN. Fractions were collected at a flow rate of 1 ml/min. (C) SDS–PAGE of the fractions obtained in the process of purification of TP2Z. Lanes: 1, total proteins of E. coli HMS174 (DE3) cells containing expression plasmid pET–TP2Z before induction with IPTG; 2, total E. coli proteins after induction with IPTG for 6 h; 3, acid extract from E. coli cells; 4, peak a-proteins (Fig. 7A); 5, peak b-protein (Fig. 7B). The mobilities of molecular-mass markers (rabbit muscle phosphorylase b, bovine serum albumin, hen egg white ovalbumin, bovine carbonic anhydrase, soy bean trypsin inhibitor, and hen egg white lysozyme), in kDa, are indicated by arrowheads.
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FIG. 8. CD spectra of TP2Z refolded in the presence and absence of Zn 21. 1 (- - -), TP2Z treated with 10 mM EDTA and refolded in the absence of Zn 21; 2 (—), TP2Z refolded in the presence of 50 mM Zn 21.
220 nm. The shape of the curve is similar to that of intact TP2 refolded in the presence of 50 mM Zn 21 (15), and typical of a type I b-turn which has been observed for Cbz-Gly-Gly-Pro-Gly-O-stearate in acetonitrile (25). When TP2Z was treated with 10 mM EDTA and refolded in the absence of Zn 21, there was an increase in the negative ellipticity at 204 nm. Furthermore, the ellipticity minimum was shifted to 202 nm and the shoulder around 220 nm deleted, in a manner similar to that for intact TP2 (15). These results suggest that zinc induces formation of antiparallel b-sheet and/or b-turn in TP2Z as well as in intact TP2 (15). On the other hand, removal of zinc from rat TP2 and its zincbinding domain decreases the negative ellipticity at 200 nm and deletes a shoulder at 222 nm (10). In order to determine the actual number of zinc atoms that can be bound to TP2Z in the nearly physiological ionic strength and pH, the TP2Z refolded in the presence of 50 mM ZnCl 2 and 150 mM KF was exhaustively dialyzed against metal-free water until the dialysate showed negligible amounts of zinc. TP2Z contained 2.65 atoms of zinc bound per molecule of the protein, as intact TP2 does (15). The TP2Z pretreated with 10 mM EDTA did not give any signal for zinc. These results indicate that TP2Z has zinc-binding domain of boar TP2. Gel Shift Assays with TP2Z and CpG Island Oligonucleotide We examined whether TP2Z recognizes the CpG island oligonucleotide as does boar TP2. Figures 9A and 9B show the results of the gel shift experiments carried out with freshly refolded TP2Z and CpG island oligonucleotide in the buffer containing 150 mM NaCl and with the oxidized TP2Z and CpG island oligonucleotide
in the same buffer, respectively. The complex of freshly refolded TP2Z–CpG island oligonucleotide appeared at TP2Z amounts larger than 20 ng (Fig. 9A). The amount of the complex formed remained constant between 25 and 40 ng of input TP2Z amount, and small amounts of aggregates of the complex were observed in the well. On the other hand, the complex of the oxidized TP2Z– CpG island oligonucleotide appeared only in the form of aggregates which were retained in the well. These results suggest that freshly refolded TP2Z under reducing conditions binds to the oligonucleotide specifically and that the oxidized TP2Z does nonspecifically and forms aggregates of the complex. Aggregation seen in Fig. 9B may be due to oxidation forming TP2Z dimers that form a network complex with DNA, since TP2Z dimer was detected as a main product of TP2Z oxidation on SDS–PAGE (data not shown). Cold homologous oligonucleotide showed effective competition (Fig. 10A), and poly(dI-dC) z poly(dI-dC) showed no competition (Fig. 10B), indicating that the complexes are specific in nature. Poly(dG-dC) z poly(dG-dC) was a most effective competitor among the synthetic polynucleotides used, and the degree of competition is stronger than that in the case with intact TP2 (Fig. 10D and Fig. 5D). Figure 10C shows the results obtained with the alternating copolymer poly(dA-dT) z poly(dA-dT) as the competitor. There was a decrease in the amount of the complex formed (Fig. 10C, lane 5, 20-fold weight excess). When the two homoduplexes, poly(dA) z poly(dT) and poly(dG) z poly(dC), were used as competitors in the gel shift assays, neither of these homoduplexes competed with the complex formation (Figs. 10E and 10F). The results in Figs. 10D and 10F suggest that GC base pairs within
TABLE 1 Summary of Purification of the Zinc-Binding Domain of Boar Transition Protein 2 from the Culture of E. coli HMS174(DE3) Cells Containing Expression Plasmid pET–TP2Z
Step
Total proteins a (mg)
TP2Z b (mg)
Relative purification (fold)
Yield (%)
culture Acid extract Fractogel EMD SO 32 Nucleosil 300 7C18 Diol-120
60 16 10 7.7 7.2
20 12 8.7 7.4 7.2 c
1.0 2.3 2.6 2.9 3.0
100 60 44 37 36
a
The total proteins were estimated by amino acid analysis. The amount of TP2Z during purification was estimated from the ratio of TP2Z to the total proteins: The ratio was calculated from the peak areas of TP2Z and other proteins in the microdensitometric tracings of the gel (Fig. 7C). c The amount of the purified TP2Z was determined by amino acid analysis. b
ZINC-BINDING DOMAIN OF BOAR TRANSITION PROTEIN 2
461
FIG. 9. (A) Gel shift assays with freshly refolded TP2Z and CpG island oligonucleotide. (B) Gel shift assays with oxidized TP2Z and CpG island oligonucleotide. The binding reaction was done with 32P-end-labeled oligonucleotide (25,000 cpm, 1 ng) as described in the text. After the reaction, the complexes were analyzed on a 10% nondenaturing polyacrylamide gel. An arrowhead shows the location of the gel wells.
the CpG doublet, and not a single GC base pair, are important for recognition of the CpG rich oligonucleotide by TP2Z. These results show that TP2Z recognizes the CpG-rich sequence more preferentially than intact TP2. EDTA-treatment of TP2Z did not affect the binding ability to the CpG oligonucleotide (lanes 1 in Figs. 11A and 11B), but the binding specificity disappeared (Figs. 11A and 11B). Poly(dI-dC) z poly(dI-dC) showed similar competition to the cold homologous oligonucleotide, providing direct evidence for the involvement of zinc-induced polypeptide folding of TP2Z (Fig. 8) in the specific recognition of the oligonucleotide. In the case of rat TP2, more than 0.7-fold molar excess of poly(dA-dT) z poly(dA-dT) decreases the binding to the CpG island oligonucleotide to about 40%, but poly(dG) z poly(dC) or poly(dA) z poly(dT) does not show any competition, and removal of zinc inhibits the binding to the CpG oligonucleotide. Accordingly, boar TP2 and TP2Z recognize the CpG island sequence in a manner similar to but somewhat different than that for rat TP2 (12).
FIG. 10. Effect of synthetic polynucleotides of defined sequences on the binding of boar TP2Z to the CpG island oligonucleotide. Gel shift assays were done as described in the legend to Fig. 9 in the presence of (A) cold homologous oligonucleotide, (B) poly(dI-dC) z poly(dI-dC), (C) poly(dA-dT) z poly(dA-dT), (D) poly(dG-dC) z poly(dG-dC), (E) poly(dA) z poly(dT), and (F) poly(dG) z poly(dC). The fold weight excess with respect to nucleotide concentration is the same as that in Fig. 5.
FIG. 11. Effect of removal of zinc from TP2Z on its binding to the CpG island oligonucleotide. TP2Z was preincubated with 10 mM EDTA and then used in the binding reaction in the presence of (A) cold homologous oligonucleotide or (B) poly(dI-dC) z poly(dI-dC). The fold weight excess with regard to nucleotide concentration is the same as that in Fig. 5.
462
SATO ET AL.
The C-terminal one-third region of rat TP2 does not bind to zinc, and its CD spectra are unchanged in the presence or absence of zinc (10). The C-terminal basic region of TP2 is highly conserved between rat, mouse, boar, and bovine (18), suggesting that this region of boar TP2 (104–137 residues) lacks the zinc-binding motif. It is likely that this C-terminal domain does not show specific recognition to DNA, since basic peptides that contain several to about 30 arginine residues show nonspecific binding to DNA mainly by electrostatic interaction (26,27). On the basis of the preferential recognition of the CpG-rich oligonucleotide by rat TP2, Kundu and Rao have proposed that TP2 interacts with DNA using the CpG-rich nucleotide as the target sequence, which in turn may initiate chromatin condensation and repress transcriptional activity (12). Our expression system for isolating zinc-binding domain of boar TP2 will be useful for further study to identify the exact cysteine and histidine residues that coordinate with zinc to understand the tertiary structure of boar TP2. Such information will be valuable to understand biological functions of boar TP2 and its zinc-binding domain and to elucidate the essential structures and functions of mammalian TP2s by comparison of their structures and binding characteristic to DNA. ACKNOWLEDGMENTS The authors are indebted to Professor Koichi Oguma and Mr. Toshihiro Suzuki of Faculty of Engineering, Chiba University, Japan, for atomic absorption spectroscopy in the analysis of the zinc content in TP2Z.
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