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Molecular cloning, functional expression and characterization of p15, a novel fungal protein with potent neurite-inducing activity in PC12 cells
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Molecular cloning, functional expression and characterization of p15, a novel fungal protein with potent neurite-inducing activity in PC12 cells Shuji Wakatsuki 1 , Tatsuya Yokoyama, Satoru Nakashima, Akiyoshi Nishimura, Manabu Arioka *, Katsuhiko Kitamoto Department of Biotechnology, University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan Received 5 July 2001; received in revised form 4 September 2001 ; accepted 5 September 2001
Abstract p15 is a novel fungal protein which induces neurite outgrowth and neuronal differentiation of PC12 cells. In the present study, we report molecular cloning, functional expression and characterization of the gene encoding p15. The deduced amino acid sequence suggested that p15 is synthesized as a precursor with 31 extra amino-terminal amino acids including a putative signal sequence, and 20 carboxy-terminal amino acids, in addition to the 118 amino acids-long mature region with neurite-inducing activity. From the poly(A) RNA prepared from the producing fungal strain, a cDNA fragment encoding the mature region of p15 was amplified and His6 -tagged recombinant p15 was produced in Escherichia coli. The recombinant protein purified by a single step on Ni2 agarose column chromatography exhibited comparable specific activity as native p15 in the PC12 neurite extension assay. The effect of His6 -p15 was blocked by nicardipine, suggesting that Ca2 influx through the L-type Ca2 channels is essential for its neurite-inducing activity. In addition, mutational analysis of His6 -p15 demonstrated that both intramolecular disulfide bonds are essential for its biological activity. ß 2001 Elsevier Science B.V. All rights reserved. Keywords : Neurite outgrowth ; Pheochromocytoma 12 cell; Neuronal di¡erentiation; L-type Ca2 channel
1. Introduction Rat pheochromocytoma PC12 cells di¡erentiate into sympathetic neuron-like phenotypes in response to stimulation such as nerve growth factor (NGF) [1], ¢broblast growth factors, bone morphogenetic protein-2 [2], neural cell adhesion molecule (NCAM) and interleukin 6 [3]. Among these, NGF-induced neuronal di¡erentiation of PC12 cells has been most extensively studied. Upon treatment with NGF, PC12 cells di¡erentiate into sympathetic neuron-like phenotypes characterized by cessation of cell growth, elongation of neurites and acquirement of electrical excitability. NGF binds to and activates its receptor, tyrosine kinase Trk, whose autophosphorylation then Abbreviations : His6 , hexahistidine ; NGF, nerve growth factor * Corresponding author. Fax: +81-3-58-41-80-33. E-mail address : [email protected] (M. Arioka). 1 Present address: Department of Growth Regulation, Institute for Frontier Medical Sciences, Kyoto University, Shogo-in Kawahara-cho 53, Sakyo-ku, Kyoto 606-8507, Japan.
transmits signals to various intracellular targets including Ras, phospholipase CQ and phosphatidylinositol 3-kinase [4^11]. The activation of the Ras pathway involves the formation of Shc/Grb2 complex [12] and nucleotide exchange of Ras, which then activates downstream kinases in the MAP kinase cascade [13^15]. Sustained activation and nuclear translocation of MAP kinase initiates several transcription-dependent cellular events leading to the expression of cytoskeletal proteins and neurite outgrowth, which is considered to be the morphological hallmark of di¡erentiation of PC12 cells. In addition to growth factor stimulation, it has been shown that di¡erentiation of PC12 cells is induced by Ca2 in£ux followed by membrane depolarization. In neurons, the increase in intracellular Ca2 level initiates a variety of cellular responses such as di¡erentiation, survival, apoptosis, and synaptic plasticity. For example, previous studies demonstrated that the depolarization-induced in£ux of Ca2 through the voltage-dependent Ca2 channels results in the expression of several immediate early and late genes including c-fos proto-oncogene
0167-4781 / 01 / $ ^ see front matter ß 2001 Elsevier Science B.V. All rights reserved. PII: S 0 1 6 7 - 4 7 8 1 ( 0 1 ) 0 0 3 0 8 - 6
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[16,17]. More recently, elevation of intracellular Ca2 by depolarization has been shown to activate the Src-Ras signaling pathway that eventually leads to the activation of MAP kinase cascade [18,19]. Several mechanisms could account for Ca2 -mediated Ras activation, such as involvement of Pyk2, Shc phosphorylation and/or EGF receptor activation [20]. Although the contribution of other pathways elicited by Ca2 cannot be underestimated [17], it seems likely that Src-Ras-MAPK cascade plays a central role in depolarization-induced cellular responses in PC12 cells. Thus, growth factors and Ca2 activate distinct but overlapping intracellular signaling pathways. In previous studies we reported the identi¢cation, puri¢cation and characterization of a fungal protein that has potent neurite-inducing activity. This protein, named p15, is a putative secreted protein with an apparent molecular mass of 15 kDa [21,22]. At a concentration as low as 1 nM, p15 induces neurites and expression of neuro¢lament-M protein in PC12 cells. We showed that these e¡ects of p15 were dependent on the activation of voltage-dependent L-type Ca2 channels and subsequent activation of Src-Ras-MAP kinase cascade. In the present study, we have cloned the gene encoding p15 precursor and achieved the functional expression of hexahistidine (His6 )-tagged recombinant p15 in Escherichia coli. The recombinant His6 -p15 exhibited potent neurite-inducing activity in PC12 cells that is comparable to native p15. The e¡ect of His6 -p15 was inhibited by nicardipine, indicating that the e¡ect of p15 is mediated by Ca2 in£ux through the Ltype Ca2 channels. In addition, mutational analysis of His6 -p15 demonstrated that both intramolecular disul¢de bonds are essential for its biological activity. 2. Materials and methods 2.1. Materials PCR reactions were done using Ampli Taq Gold (Applied Biosystems) or Pfx DNA polymerase (Gibco BRL). Puri¢cation of DNA fragments was performed by GeneClean II (BIO101). Ni2 -NTA agarose was purchased from Qiagen. 2.2. Cell culture Rat pheochromocytoma PC12 cells were maintained in Dulbecco's modi¢ed Eagle's medium (DMEM) supplemented with 5% horse serum and 5% fetal calf serum. Cells were passaged every 3^4 days and maintained at 37³C in 10% CO2 in humidi¢ed air. PC12 cells were seeded in growth medium at 4.5U103 cells/cm2 in collagen type I (Becton Dickinson)-coated 24 well culture plates and allowed to grow for 24 h. Then p15 was added and neurite outgrowth was measured after 48 h. For quanti¢cation of neurite outgrowth, ¢ve random photographs were taken
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per well, and cells bearing processes longer than the cell diameter were judged to be positive. Preparation of cell extracts followed by Western blotting with anti-neuro¢lament-M antibody was performed as described [22]. 2.3. Cloning of genomic and complementary DNAs encoding p15 Chromosomal DNA from the producing fungal strain was prepared as follows. The fungus was cultured in medium (50 ml; 0.3% yeast extract, 0.3% malt extract, 0.5% polypeptone, 1% glucose, pH 6.0) with constant shaking at 120 rpm for 6 days at 25³C. At the end of the culture, Tween 80 was added at 0.1^0.2%. The cells were collected by centrifugation at 1700Ug for 15 min at room temperature, frozen in liquid nitrogen and ground to a ¢ne powder using a mortar and pestle. Twenty milliliters of solution I (50 mM EDTA, 0.5% SDS, 0.1 mg/ml proteinase K, pH 8.0) were added and incubated for 4 h at 50³C. Then the extract was treated twice with an equal volume of phenol, once with phenol/chloroform, and once with chloroform. One-tenth volume of 3 M sodium acetate (pH 5.2) was added, and chromosomal DNA was precipitated with isopropanol overnight at 4³C. After centrifugation at 1700Ug for 10 min, chromosomal DNA was rinsed once with 70% ethanol and dissolved in 10 ml of TE (10 mM Tris^HCl (pH 8.0), 1 mM EDTA). After RNase A treatment (10 Wg/ml for 30 min at 37³C), DNA was extracted with phenol/chloroform and precipitated with ethanol. The resultant chromosomal DNA was dissolved in 1 ml of TE. The genomic sequence encoding p15 precursor was cloned as follows. Three degenerate primers corresponding to the amino- and carboxy-termini of mature p15 were synthesized. Their sequences are 5P-GCNACNAT(A/T/ C)GA(A/C)GA(A/C)ACNAC-3P for the sense primer (which encodes the amino-terminal ATIEETT sequence of mature p15), 5P-(A/C)CTNCC(A/C)AA(T/G)GT(T/C)TTNACCGCCTC-3P and 5P-NGANCC(A/C)AA(T/G)GT(T/C)TTNACCGCCTC-3P for the antisense primers (which encode the carboxy-terminal EAVKTFGS sequence of mature p15), where N represents A, T, G, and C. Using the genomic DNA (1 Wg) as a template, PCR ampli¢cation was performed by a 35 cycle PCR protocol with a 30 s denaturing step at 94³C, a 30 s annealing step at 50³C, and a 30 s extension step at 72³C and its product (approx. 440 bp) was cloned into pT7Blue (TaKaRa). DNA sequencing analysis con¢rmed that the cloned DNA encodes p15 with an 81 bp intron. This DNA insert was used as a probe for the Southern blot analysis. The genomic DNA (5 Wg) was digested overnight with various restriction enzymes and blotted onto a Hybond-N ¢lter (Amersham-Pharmacia). Labeling of the probe and detection of the hybridized band was done according to the DIG-High Prime DNA Labeling and Detection Kit (Boehringer), and 5 kb and 2.5 kb bands were detected
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in the DNA digested with EcoRI and PstI, respectively. EcoRI- or PstI-digested genomic DNA fragments were run on agarose gel and the regions of agarose gel which corresponded to the hybridized bands were excised, DNA fragments were extracted and ligated to the pBluescript II SK(+) digested with EcoRI and PstI, respectively. After transformation, each E. coli colony was examined by colony PCR using the same primer set as described above. Out of 56 colonies thus examined, one colony obtained from PstI-digested genomic DNA (pP19) gave a positive signal, and the nucleotide sequence of its insert DNA (2 kb) was determined using a Shimadzu DSQ-1000 automated sequencer. The 0.5 kb AccI (blunt-ended)-PstI fragment of pP19 carrying the p15 precursor was further subcloned into pBluescript II SK(+) digested with EcoRV and PstI, generating pB8. For the isolation of cDNA encoding the mature region of p15, cells were grown in the production medium (4% sucrose, 2% pharmamedia, 2% dry yeast, 1% polypeptone, 0.2% K2 HPO4 , 0.2% CaCO3 , 0.1% Tween 80) for 7 days at 25³C. Cells (1 g) were frozen and homogenized as described above. RNA was extracted in 4 ml of 10 mM EDTA, 0.5% sodium SDS and then 4 ml of 10 mM EDTA, 0.1% sodium acetate were added. Water-saturated phenol (6 ml) was added, and the sample was mixed and centrifuged. The supernatant was recovered and re-extracted with phenol, and the total RNA was precipitated with ethanol. Typically V250 Wg of total RNA were isolated by this method. From this total RNA, poly(A) RNA (10 Wg) was obtained using the mRNA Puri¢cation Kit (Amersham-Pharmacia), and reverse-transcribed using the 1st Strand cDNA Synthesis Kit (Boehringer), RAV-2 reverse transcriptase (TaKaRa) and random primer p(dN)6 . The resultant single-stranded cDNA (1 Wg) was served as a PCR template. Two sets of degenerate primers corresponding to the amino- and carboxy-termini of mature p15 were synthesized for PCR ampli¢cation of p15 cDNA. The sequences of the sense and antisense primers were 5P-GC(A/G)AC(T/G/C)AT(A/T/C)GAGGAGACCACAGATA-3P and 5P-(A/T/G)(G/C)A(A/T/G)CCGAA(A/T/G)GTTTTGACCACCTCG-3P, respectively. DNA ampli¢cation was done by a 30 cycle PCR protocol with a 30 s denaturing step at 94³C, a 30 s annealing step at 50³C, and a 30 s extension step at 72³C. The ampli¢ed DNA was ligated into pT7Blue and sequenced, which con¢rmed that the cloned fragment is authentic cDNA for p15. 2.4. Expression and puri¢cation of recombinant p15 For the expression of hexahistidine-tagged (His6 ) p15, a set of PCR primers was designed by adding PstI and HindIII sites at the 5P end of sense and antisense primers, respectively. The sequences were 5P-AACTGCAGCAACGATTGAGGAGACCAC-3P and 5P-ATAAGCTTATGAGCCGAAGGTTTTGAC-3P. Using these primers and p15
cDNA carried on pT7Blue as a template, PCR was done as described above. The ampli¢ed DNA fragment was digested with PstI and HindIII and subcloned into PstIand HindIII-digested pRSETB vector (Invitrogen). The resultant plasmid named p15-pRSETB was introduced into E. coli BL21(DE3)pLysS. The cells were grown to mid-log phase in LB broth, and the His6 -p15 was induced by 0.3 mM IPTG for 3 h at 37³C. The induced cells were lysed by sonication, and the cell lysate was centrifuged at 100 000Ug for 30 min at 4³C to remove insoluble materials. His6 -p15 was found in both the soluble and insoluble fractions. The soluble fraction was collected and applied to a Ni2 -NTA agarose column (Qiagen). The column was extensively washed with bu¡er (20 mM sodium phosphate (pH 7.8), 0.5 M NaCl) containing 50 mM imidazole, and then His6 -p15 was eluted by bu¡er containing 200 mM imidazole. Insoluble fraction was solubilized, applied to the Ni2 -NTA column, washed, and eluted according to the standard procedure. The majority of His6 -p15 was eluted by bu¡er containing 20 mM sodium phosphate (pH 4.0), 8 M urea, 0.5 M NaCl. The proteins were analyzed by sodium dodecyl sulfate^polyacrylamide gel electrophoresis (SDS^PAGE) containing 15% acrylamide. The fractions containing His6 -p15 were collected and dialyzed against 20 mM sodium phosphate (pH 7.4). 2.5. PCR-based mutagenesis Site-directed mutagenesis by PCR was performed essentially according to the method of Imai [23]. To construct a double mutant in which both cysteine residues that are linked by disul¢de bridges are replaced with serine residues, a set of PCR primers, each being designed to code for cysteine to serine mutation, was synthesized. Their sequences are: for replacement of Cys38 and Cys54 with serine (C38S/C54S mutation), 5P-AATGGGTCATCTGGAGACGAGCTGGACC-3P (antisense) and 5P-CGGCTTCGACTTCTTATCCTCTTCCCAC-3P (sense); for replacement of Cys90 and Cys102 with serine (C90S/C102S mutation), 5P-ATATTGCTCGGTATTGGACTGG-3P (antisense) and 5P-CTTCACAAGAGCTGCCTCCAAAGC-3P (sense). Underlined sequences indicate the sites of mutations. To construct mutants carrying only one Cys-to-Ser mutation, a set of primers with wild type and mutant sequence was used. Using these primer sets and p15-pRSETB as a template, PCR reactions were conducted using Expand High-Fidelity PCR Systems (Roche Diagnostics). PCR products were recovered from agarose gel, processed by T4 DNA polymerase, T4 polynucleotide kinase, then circularized by ligation, and transformed into E. coli. Introduction of mutations was con¢rmed by DNA sequencing. The resultant six plasmids, carrying His6 -p15 with substitutions of C38S/C54S, C90S/C102S, C38S, C54S, C90S, and C102S, were introduced into E. coli BL21(DE3)pLysS. Induction and puri¢cation of mutant proteins were done as described above.
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3. Results We previously reported the complete amino acid sequence of the mature region of p15 by sequencing the chemically and enzymatically digested fragments of p15 puri¢ed from the culture broth of the producing fungal strain. Degenerate primers encoding the determined amino- and carboxy-terminal sequences were synthesized, and the partial genomic DNA encoding p15 was ampli¢ed by PCR using the chromosomal DNA of the producing fungal strain as a template. The nucleotide sequence analysis of this DNA fragment con¢rmed that this PCR fragment encodes p15. Using this DNA fragment as a probe, we isolated the whole genomic sequence of p15 precursor as described in Section 2. The entire coding region of p15 was carried on the 2.0 kb PstI fragment of the genomic DNA. The p15 precursor gene consists of a 588 bp open reading frame with an 81 bp intron, the position of which was determined by comparison with the cDNA sequence of the mature region of p15 (Fig. 1; see below). Thus the gene predicts a protein of 169 amino acids. Compared to the 118 amino acids-long mature region with neurite-inducing activity [22], the p15 precursor has 31 and 20 additional amino acids at the amino- and carboxy-terminus, respectively. According to the prediction by SMART [24], the 18 amino-terminal amino acids are assumed to be the signal sequence for secretion. No obvious TATA sequence
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was found within the 300 nucleotides upstream from the start codon (data not shown). In the 3P-noncoding region, a putative polyadenylation signal was found about 20 bp downstream from the stop codon. We next isolated the cDNA encoding mature region of p15 and expressed it in E. coli. Poly(A) RNA prepared from the producing fungal strain was reverse transcribed, and the cDNA encoding the mature region of p15 was isolated by PCR. Nucleotide sequence analysis indicated that the ampli¢ed product was authentic p15 cDNA. It was ligated to pRSETB vector to produce the amino-terminally His6 -tagged version of recombinant p15. This expression plasmid was introduced into E. coli strain BL21(DE3)pLysS and the expression of His6 -p15 was induced by IPTG. SDS^PAGE analysis of the lysed cells revealed the presence of a protein band with an approximate molecular mass of 20 kDa in the extract induced by IPTG, but not in the extract without induction (Fig. 2A). Since His6 -p15 has an extra MRGSH6 GMASMTGGQQMGRDLYDDDDKDPSSRSA at the amino-terminus compared with mature p15, which would permit the expression of a protein with the calculated molecular mass of 17.5 kDa, we concluded that this band was His6 -p15. The induced cell lysate was centrifuged to separate soluble and insoluble fractions, which demonstrated that roughly equal amounts of His6 -p15 occurred in the soluble and insoluble fractions. His6 -p15 from both fractions was pu-
Fig. 1. Nucleotide and deduced amino acid sequences of p15 precursor gene. The predicted amino acid sequence is shown below the nucleotide sequence. The putative signal sequence is italicized, and the amino acid sequence of mature p15 is underlined. The four cysteine residues mutated to serine are boxed, together with the amino acid number from the amino-terminus of mature p15. The intron sequence is shown in lower case, and the putative polyadenylation signal is indicated by the double underline. The nucleotide sequence has been deposited in the DDBJ database under accession no. AB071215.
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from the insoluble material, although the majority of the protein was His6 -p15. His6 -p15 thus puri¢ed was examined in a PC12 neurite extension assay (Fig. 2B,C). Cells were cultured in the presence of native or His6 -p15 for 48 h, and the number of cells with neurites longer than the cell diameter was counted. Native p15 puri¢ed from the culture broth of producing fungal strain exhibits neurite-inducing activity on PC12 cells as low as 0.01 nM and maximal activity was observed at 1 nM. His6 -p15 puri¢ed from both the soluble and insoluble fractions displayed similar activities, which were comparable to that observed with native p15 (Fig. 2C). We con¢rmed that the amino-terminal His6 tag had no e¡ect in this neurite extension assay, since removal of this region by enterokinase digestion did not alter the speci¢c activity of the recombinant protein (data not shown). We next examined the expression of neuro¢lament-M in His6 -p15-treated PC12 cells. Neuro¢lament-M is one of the di¡erentiation markers of PC12 cells that is speci¢cally expressed in neurons, and its protein level has been reported to be increased in response to NGF treatment [25]. As shown in Fig. 3, His6 -p15 potentiated the expression of neuro¢lament-M. Although the expression level of neuro¢lament-M was less than half of that observed in
Fig. 2. Neurite-inducing activity of His6 -p15. (A) SDS^PAGE analysis. Cell lysates from uninduced and induced cells (lane 1 and 2, respectively), His6 -p15 puri¢ed from the soluble fraction by Ni2 -agarose column chromatography (lane 3) and the native p15 partially puri¢ed from the culture broth of producing fungal strain (lane 4) were analyzed. The positions of His6 -p15 and native p15 are indicated by the arrow and the dot, respectively. Molecular mass markers are shown on the right. (B) PC12 cells treated for 48 h without (a) or with NGF (b, 50 ng/ml), His6 -p15 (c, 1 nM), or native p15 (d, 1 nM) are shown. Bar, 100 Wm. (C) PC12 cells were incubated with the indicated concentrations of native p15 (a) or His6 -p15 (b) for 48 h. Cells with neurites longer than one cell diameter were judged to be positive and counted. The results show the percentage of the total cell number.
ri¢ed by one-step a¤nity chromatography through Ni2 agarose. His6 -p15 from the soluble fraction was puri¢ed to almost homogeneity, whereas additional high molecular mass bands could be detected in the puri¢ed fraction
Fig. 3. Induction of neuro¢lament-M expression in His6 -p15-treated PC12 cells. PC12 cells were grown in the absence (lane 1) or presence of His6 -p15 (1 nM; lane 2) or NGF (50 ng/ml; lane 3) for 7 days. Cell extracts were prepared as described in Section 2 and analyzed for the expression of neuro¢lament-M using anti-neuro¢lament-M antibody. In the lower panel, the relative intensities of the bands compared to the control lane were quanti¢ed and are shown in the bar graph. The experiments were repeated at least three times with no signi¢cant di¡erence.
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Fig. 4. The e¡ects of nicardipine on neurite outgrowth induced by NGF, native p15 or His6 -p15. PC12 cells were treated for 48 h with native p15 (1 nM; a), His6 -p15 (1 nM; b) or NGF (50 ng/ml; O) in the presence of the indicated concentrations of nicardipine.
cells treated with NGF, the induction was signi¢cant compared to control cells. These results demonstrate that the processes induced in His6 -p15-treated PC12 cells are authentic neurites and that PC12 cells underwent neuronal di¡erentiation upon His6 -p15 treatment. We previously reported that the action of p15 on PC12 cells is blocked by treatment with nicardipine, an inhibitor of L-type Ca2 channels, but not by K-252a. This indicated that p15 activates a pathway that is distinct from NGF. We next asked if His6 -p15 also requires the activation of Ca2 signaling pathway. As shown in Fig. 4, neu-
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rite formation by native p15 as well as His6 -p15 is inhibited by nicardipine in a dose-dependent manner, but that by NGF treatment is not. This result indicates that His6 p15, like native p15, induces neurites through the activation of L-type Ca2 channels. In our previous study, we analyzed the primary structure of p15 by protein sequencing and showed that p15 has two intramolecular disul¢de bonds linking Cys38 to Cys54 and Cys90 to Cys102 (amino acid numbers are based on the sequence of mature p15). Preliminary observation with reduced, carboxymethylated p15 suggested that either one or both of these disul¢de bonds are essential for its biological activity (S.W., unpublished observation). We therefore examined if these disul¢de bridges are indeed necessary for its activity by substituting cysteine residues of His6 -p15 with serine. For this purpose, PCR-based sitedirected mutagenesis was performed to produce mutants C38S (in which Cys38 is replaced by a serine residue), C54S, C90S and C102S as well as double mutants C38S/ C54S (in which both Cys38 and Cys54 are replaced by serine residues) and C90S/C102S as described in Section 2 [23]. As shown in Fig. 5A, the single and double mutants of the amino-terminal disul¢de bond, C38S, C54S and C38S/C54S, exhibited no detectable neurite-inducing activity at concentrations as high as 10 nM, indicating that the amino-terminal disul¢de bond is essential. Similarly, disruption of the carboxy-terminal disul¢de bond in the C90S and C102S mutants also resulted in loss of activity (Fig. 5B). Interestingly, the C-terminal double mutant C90S/ C102S did not completely lose its neurite-inducing activity, although the activity was less than one-tenth of that of wild type. We reasoned that this is because the one re-
Fig. 5. Neurite-inducing activities of mutant His6 -p15. His6 -tagged mutant p15 proteins in which cysteine residues are replaced by serine were produced in E. coli and puri¢ed as wild type His6 -p15. PC12 cells were incubated with increasing concentrations of recombinant p15 for 48 h, and cells with neurites longer than one cell diameter were counted. (A) a, His6 -p15; E, C38S mutant; F, C54S mutant; O, C38S/C54S mutant. (B) a, His6 -p15; E, C90S mutant; F, C102S mutant; O, C90S/C102S mutant.
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maining cysteine residue in C90S or C102S mutants interferes with the formation of the amino-terminal disul¢de bond, resulting in the failure in the formation of both correct disul¢de bonds and complete loss of activity. In summary, both intramolecular disul¢de bridges are necessary for the full activity of His6 -p15 in neurite induction, and it is probable that the amino-terminal disul¢de bridge is more important than the carboxy-terminal one. 4. Discussion In this study we reported molecular cloning, functional expression, and mutational analysis of p15, which we found in a search for microbial metabolites with neurotrophin-like activity [21,22]. The structure of p15 precursor gene predicted that mature p15 is £anked by amino- and carboxy-terminal extensions of 31 and 20 amino acids, respectively. Consistent with its presence in the culture supernatant of the producing fungal strain, p15 precursor contained an amino-terminal sequence of 18 amino acids which is presumably a signal sequence for secretion. The presence of a dibasic Lys-Arg sequence close to the carboxy-terminus of mature p15 suggests that processing of the carboxy-terminus of p15 is performed in two steps during the course of secretion that might involve a Kex2-like endoprotease and a carboxypeptidase. We previously observed that reduced, carboxymethylated p15 lost its neurite-inducing activity. Consistent with this, substitution of both or one of the cysteine residues linked by disul¢de bonds with serine in His6 -p15 resulted in complete or marked loss of neurite-inducing activity, indicating that both intramolecular disul¢de bonds are critical for its activity. Since the speci¢c activity of His6 -p15 is comparable to that of native p15 puri¢ed from the culture broth of the producing fungal strain, it is reasonable to assume that the formation of correct pairs of disul¢de bonds is achieved during the course of puri¢cation of His6 -p15 that is produced intracellularly in E. coli. Although p15 displays no sequence similarity to neurotrophin family proteins, it exhibits potent neurite-inducing activity and induces the expression of neuro¢lament-M, one of the hallmarks of di¡erentiation of PC12 cells. We have shown that the e¡ect of His6 -p15 requires activation of the Ca2 signaling pathway by demonstrating that nicardipine treatment inhibits neuritogenesis. Walsh and coworkers have demonstrated that increased in£ux of extracellular Ca2 via Ca2 channels induces neurite outgrowth in PC12 cells stimulated with cell adhesion molecules such as neural cell adhesion molecule (N-CAM), L1 and Ncadherin [26^32]. These studies pointed to the activation of ¢broblast growth factor receptor-phospholipase CQ (PLCQ) cascade as being important for the neurite outgrowth responses stimulated by all three CAMs. Following recruitment and activation of PLCQ via interaction of
its SH-2 domain with the activated receptor, generation of diacylglycerol (DAG) and its conversion to arachidonic acid (AA) is thought to occur. This scenario is in good agreement with the fact that Ca2 channel blockers inhibit neurite outgrowth by CAMs, since AA has been shown to increase voltage-dependent Ca2 currents in cardiac myocytes [33]. Indeed, AA potentiates neurite outgrowth, which is inhibited by N- or L-type Ca2 channel antagonists [31]. In this context the key event downstream from the activation of PLCQ required for neurite growth appears to be the conversion of DAG to AA via DAG lipase. We pursued the possibility that p15-generated signal converges at some point in this pathway by examining the e¡ects of pertussis toxin and an inhibitor of DAG lipase, RHC-80267, on p15-induced neurite outgrowth. However, neither of these agents blocked His6 -p15-induced neurite outgrowth, indicating that the pathways that are activated by p15 and CAMs are distinct. Database search analysis revealed that an open reading frame from Streptomyces coelicolor, CAB38593.1, shares signi¢cant homology with p15. Our preliminary analysis demonstrated that this putative secreted protein indeed has neurite-inducing activity toward PC12, although its speci¢c activity was considerably low compared to that of p15. Interestingly, CAB38593.1 also has four cysteine residues and the positions of these residues are almost completely conserved between CAB38593.1 and p15, suggesting that these residues in CAB38593.1 are linked by disul¢de bridges and essential for neurite-inducing activity. Further structural comparison between these proteins will help specify the region responsible for their biological activity. Acknowledgements This work was supported by Grants-in-Aid for Scienti¢c Research (No. 12460041 and No. 12760048) from the Ministry of Education, Science, Sports and Culture of Japan, and by a grant from the Fujisawa Foundation. S.W. was supported by Research Fellowships of the Japan Society for the Promotion of Science for Young Scientists. References [1] L.A. Greene, A.S. Tischler, Proc. Natl. Acad. Sci. USA 73 (1976) 2424^2428. [2] S. Iwasaki, A. Hattori, M. Sato, M. Tsujimoto, M. Kohno, J. Biol. Chem. 271 (1996) 17360^17365. [3] S. Ihara, A. Iwamatsu, T. Fujiyoshi, A. Komi, T. Yamori, Y. Fukui, J. Biochem. 120 (1996) 865^868. [4] D.R. Kaplan, B.L. Hempstead, D. Martin-Zanca, M.V. Chao, L.F. Parada, Science 252 (1991) 554^558. [5] D.R. Kaplan, D. Martin-Zanca, L.F. Parada, Nature 350 (1991) 158^ 160. [6] D.J. Robbins, M. Cheng, E. Zhen, C.A. Vanderbilt, L.A. Feig, M.H. Cobb, Proc. Natl. Acad. Sci. USA 89 (1992) 6924^6928.
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S. Wakatsuki et al. / Biochimica et Biophysica Acta 1522 (2001) 74^81 [7] S.M. Thomas, M. DeMarco, G. D'Arcangelo, S. Halegoua, J.S. Brugge, Cell 68 (1992) 1031^1040. [8] K. Wood, C. Sarnecki, T.M. Roberts, J. Blenis, Cell 68 (1992) 1041^ 1050. [9] A. Obermeier, H. Halfter, K.H. Wiesmuller, G. Jung, J. Schlessinger, A. Ullrich, EMBO J. 12 (1993) 933^941. [10] A. Obermeier, R. Lammers, K.H. Wiesmuller, G. Jung, J. Schlessinger, A. Ullrich, J. Biol. Chem. 268 (1993) 22963^22966. [11] A. Obermeier, R.A. Bradshaw, K. Seedorf, A. Choidas, J. Schlessinger, A. Ullrich, EMBO J. 13 (1994) 1585^1590. [12] M. Rozakis-Adcock, J. McGlade, G. Mbamalu, G. Pelicci, R. Daly, W. Li, A. Batzer, S. Thomas, J. Brugge, P.G. Pelicci, J. Schlessinger, T. Pawson, Nature 360 (1992) 689^692. [13] N.W. Gale, S. Kaplan, E.J. Lowenstein, J. Schlessinger, D. Bar-Sagi, Nature 363 (1993) 88^92. [14] N. Li, A. Batzer, R. Daly, V. Yajnik, E. Skolnik, P. Chardin, D. BarSagi, B. Margolis, J. Schlessinger, Nature 363 (1993) 85^88. [15] M. Rozakis-Adcock, R. Fernley, J. Wade, T. Pawson, D. Bowtell, Nature 363 (1993) 83^85. [16] J.I. Morgan, T. Curran, Nature 322 (1986) 552^555. [17] A. Ghosh, M.E. Greenberg, Science 268 (1995) 239^247. [18] L.B. Rosen, D.D. Ginty, M.J. Weber, M.E. Greenberg, Neuron 12 (1994) 1207^1221. [19] G. Rusanescu, H. Qi, S.M. Thomas, J.S. Brugge, S. Halegoua, Neuron 15 (1995) 1415^1425. [20] S. Finkbeiner, M.E. Greenberg, Neuron 16 (1996) 233^236.
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[21] T. Hanada, T. Sato, M. Arioka, M. Uramoto, M. Yamasaki, Biochem. Biophys. Res. Commun. 228 (1996) 209^215. [22] S. Wakatsuki, M. Arioka, N. Dohmae, K. Takio, M. Yamasaki, K. Kitamoto, J. Biochem. 126 (1999) 1151^1160. [23] Y. Imai, Y. Matsushima, T. Sugimura, M. Terada, Nucleic Acids Res. 19 (1991) 2785. [24] J. Schultz, F. Milpetz, P. Bork, C.P. Ponting, Proc. Natl. Acad. Sci. USA 95 (1998) 5857^5864. [25] M.H. Lindenbaum, S. Carbonetto, F. Grosveld, D. Flavell, W.E. Mushynski, J. Biol. Chem. 263 (1988) 5662^5667. [26] P. Doherty, S.V. Ashton, S.E. Moore, F.S. Walsh, Cell 67 (1991) 21^ 33. [27] J.L. Sa¡ell, F.S. Walsh, P. Doherty, J. Cell Biol. 118 (1992) 663^ 670. [28] E.J. Williams, P. Doherty, G. Turner, R.A. Reid, J.J. Hemperly, F.S. Walsh, J. Cell Biol. 119 (1992) 883^892. [29] E.J. Williams, J. Furness, F.S. Walsh, P. Doherty, Neuron 13 (1994) 583^594. [30] E.J. Williams, J. Furness, F.S. Walsh, P. Doherty, Development 120 (1994) 1685^1693. [31] E.J. Williams, F.S. Walsh, P. Doherty, J. Neurochem. 62 (1994) 1231^1234. [32] E.J. Williams, F.S. Walsh, P. Doherty, J. Cell Biol. 124 (1994) 1029^ 1037. [33] J.M. Huang, H. Xian, M. Bacaner, Proc. Natl. Acad. Sci. USA 89 (1992) 6452^6456.
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Molecular cloning, functional expression and characterization of p15, a novel fungal protein with potent neurite-inducing activity in PC12 cells Shuji Wakatsuki 1 , Tatsuya Yokoyama, Satoru Nakashima, Akiyoshi Nishimura, Manabu Arioka *, Katsuhiko Kitamoto Department of Biotechnology, University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan Received 5 July 2001; received in revised form 4 September 2001 ; accepted 5 September 2001
Abstract p15 is a novel fungal protein which induces neurite outgrowth and neuronal differentiation of PC12 cells. In the present study, we report molecular cloning, functional expression and characterization of the gene encoding p15. The deduced amino acid sequence suggested that p15 is synthesized as a precursor with 31 extra amino-terminal amino acids including a putative signal sequence, and 20 carboxy-terminal amino acids, in addition to the 118 amino acids-long mature region with neurite-inducing activity. From the poly(A) RNA prepared from the producing fungal strain, a cDNA fragment encoding the mature region of p15 was amplified and His6 -tagged recombinant p15 was produced in Escherichia coli. The recombinant protein purified by a single step on Ni2 agarose column chromatography exhibited comparable specific activity as native p15 in the PC12 neurite extension assay. The effect of His6 -p15 was blocked by nicardipine, suggesting that Ca2 influx through the L-type Ca2 channels is essential for its neurite-inducing activity. In addition, mutational analysis of His6 -p15 demonstrated that both intramolecular disulfide bonds are essential for its biological activity. ß 2001 Elsevier Science B.V. All rights reserved. Keywords : Neurite outgrowth ; Pheochromocytoma 12 cell; Neuronal di¡erentiation; L-type Ca2 channel
1. Introduction Rat pheochromocytoma PC12 cells di¡erentiate into sympathetic neuron-like phenotypes in response to stimulation such as nerve growth factor (NGF) [1], ¢broblast growth factors, bone morphogenetic protein-2 [2], neural cell adhesion molecule (NCAM) and interleukin 6 [3]. Among these, NGF-induced neuronal di¡erentiation of PC12 cells has been most extensively studied. Upon treatment with NGF, PC12 cells di¡erentiate into sympathetic neuron-like phenotypes characterized by cessation of cell growth, elongation of neurites and acquirement of electrical excitability. NGF binds to and activates its receptor, tyrosine kinase Trk, whose autophosphorylation then Abbreviations : His6 , hexahistidine ; NGF, nerve growth factor * Corresponding author. Fax: +81-3-58-41-80-33. E-mail address : [email protected] (M. Arioka). 1 Present address: Department of Growth Regulation, Institute for Frontier Medical Sciences, Kyoto University, Shogo-in Kawahara-cho 53, Sakyo-ku, Kyoto 606-8507, Japan.
transmits signals to various intracellular targets including Ras, phospholipase CQ and phosphatidylinositol 3-kinase [4^11]. The activation of the Ras pathway involves the formation of Shc/Grb2 complex [12] and nucleotide exchange of Ras, which then activates downstream kinases in the MAP kinase cascade [13^15]. Sustained activation and nuclear translocation of MAP kinase initiates several transcription-dependent cellular events leading to the expression of cytoskeletal proteins and neurite outgrowth, which is considered to be the morphological hallmark of di¡erentiation of PC12 cells. In addition to growth factor stimulation, it has been shown that di¡erentiation of PC12 cells is induced by Ca2 in£ux followed by membrane depolarization. In neurons, the increase in intracellular Ca2 level initiates a variety of cellular responses such as di¡erentiation, survival, apoptosis, and synaptic plasticity. For example, previous studies demonstrated that the depolarization-induced in£ux of Ca2 through the voltage-dependent Ca2 channels results in the expression of several immediate early and late genes including c-fos proto-oncogene
0167-4781 / 01 / $ ^ see front matter ß 2001 Elsevier Science B.V. All rights reserved. PII: S 0 1 6 7 - 4 7 8 1 ( 0 1 ) 0 0 3 0 8 - 6
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[16,17]. More recently, elevation of intracellular Ca2 by depolarization has been shown to activate the Src-Ras signaling pathway that eventually leads to the activation of MAP kinase cascade [18,19]. Several mechanisms could account for Ca2 -mediated Ras activation, such as involvement of Pyk2, Shc phosphorylation and/or EGF receptor activation [20]. Although the contribution of other pathways elicited by Ca2 cannot be underestimated [17], it seems likely that Src-Ras-MAPK cascade plays a central role in depolarization-induced cellular responses in PC12 cells. Thus, growth factors and Ca2 activate distinct but overlapping intracellular signaling pathways. In previous studies we reported the identi¢cation, puri¢cation and characterization of a fungal protein that has potent neurite-inducing activity. This protein, named p15, is a putative secreted protein with an apparent molecular mass of 15 kDa [21,22]. At a concentration as low as 1 nM, p15 induces neurites and expression of neuro¢lament-M protein in PC12 cells. We showed that these e¡ects of p15 were dependent on the activation of voltage-dependent L-type Ca2 channels and subsequent activation of Src-Ras-MAP kinase cascade. In the present study, we have cloned the gene encoding p15 precursor and achieved the functional expression of hexahistidine (His6 )-tagged recombinant p15 in Escherichia coli. The recombinant His6 -p15 exhibited potent neurite-inducing activity in PC12 cells that is comparable to native p15. The e¡ect of His6 -p15 was inhibited by nicardipine, indicating that the e¡ect of p15 is mediated by Ca2 in£ux through the Ltype Ca2 channels. In addition, mutational analysis of His6 -p15 demonstrated that both intramolecular disul¢de bonds are essential for its biological activity. 2. Materials and methods 2.1. Materials PCR reactions were done using Ampli Taq Gold (Applied Biosystems) or Pfx DNA polymerase (Gibco BRL). Puri¢cation of DNA fragments was performed by GeneClean II (BIO101). Ni2 -NTA agarose was purchased from Qiagen. 2.2. Cell culture Rat pheochromocytoma PC12 cells were maintained in Dulbecco's modi¢ed Eagle's medium (DMEM) supplemented with 5% horse serum and 5% fetal calf serum. Cells were passaged every 3^4 days and maintained at 37³C in 10% CO2 in humidi¢ed air. PC12 cells were seeded in growth medium at 4.5U103 cells/cm2 in collagen type I (Becton Dickinson)-coated 24 well culture plates and allowed to grow for 24 h. Then p15 was added and neurite outgrowth was measured after 48 h. For quanti¢cation of neurite outgrowth, ¢ve random photographs were taken
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per well, and cells bearing processes longer than the cell diameter were judged to be positive. Preparation of cell extracts followed by Western blotting with anti-neuro¢lament-M antibody was performed as described [22]. 2.3. Cloning of genomic and complementary DNAs encoding p15 Chromosomal DNA from the producing fungal strain was prepared as follows. The fungus was cultured in medium (50 ml; 0.3% yeast extract, 0.3% malt extract, 0.5% polypeptone, 1% glucose, pH 6.0) with constant shaking at 120 rpm for 6 days at 25³C. At the end of the culture, Tween 80 was added at 0.1^0.2%. The cells were collected by centrifugation at 1700Ug for 15 min at room temperature, frozen in liquid nitrogen and ground to a ¢ne powder using a mortar and pestle. Twenty milliliters of solution I (50 mM EDTA, 0.5% SDS, 0.1 mg/ml proteinase K, pH 8.0) were added and incubated for 4 h at 50³C. Then the extract was treated twice with an equal volume of phenol, once with phenol/chloroform, and once with chloroform. One-tenth volume of 3 M sodium acetate (pH 5.2) was added, and chromosomal DNA was precipitated with isopropanol overnight at 4³C. After centrifugation at 1700Ug for 10 min, chromosomal DNA was rinsed once with 70% ethanol and dissolved in 10 ml of TE (10 mM Tris^HCl (pH 8.0), 1 mM EDTA). After RNase A treatment (10 Wg/ml for 30 min at 37³C), DNA was extracted with phenol/chloroform and precipitated with ethanol. The resultant chromosomal DNA was dissolved in 1 ml of TE. The genomic sequence encoding p15 precursor was cloned as follows. Three degenerate primers corresponding to the amino- and carboxy-termini of mature p15 were synthesized. Their sequences are 5P-GCNACNAT(A/T/ C)GA(A/C)GA(A/C)ACNAC-3P for the sense primer (which encodes the amino-terminal ATIEETT sequence of mature p15), 5P-(A/C)CTNCC(A/C)AA(T/G)GT(T/C)TTNACCGCCTC-3P and 5P-NGANCC(A/C)AA(T/G)GT(T/C)TTNACCGCCTC-3P for the antisense primers (which encode the carboxy-terminal EAVKTFGS sequence of mature p15), where N represents A, T, G, and C. Using the genomic DNA (1 Wg) as a template, PCR ampli¢cation was performed by a 35 cycle PCR protocol with a 30 s denaturing step at 94³C, a 30 s annealing step at 50³C, and a 30 s extension step at 72³C and its product (approx. 440 bp) was cloned into pT7Blue (TaKaRa). DNA sequencing analysis con¢rmed that the cloned DNA encodes p15 with an 81 bp intron. This DNA insert was used as a probe for the Southern blot analysis. The genomic DNA (5 Wg) was digested overnight with various restriction enzymes and blotted onto a Hybond-N ¢lter (Amersham-Pharmacia). Labeling of the probe and detection of the hybridized band was done according to the DIG-High Prime DNA Labeling and Detection Kit (Boehringer), and 5 kb and 2.5 kb bands were detected
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in the DNA digested with EcoRI and PstI, respectively. EcoRI- or PstI-digested genomic DNA fragments were run on agarose gel and the regions of agarose gel which corresponded to the hybridized bands were excised, DNA fragments were extracted and ligated to the pBluescript II SK(+) digested with EcoRI and PstI, respectively. After transformation, each E. coli colony was examined by colony PCR using the same primer set as described above. Out of 56 colonies thus examined, one colony obtained from PstI-digested genomic DNA (pP19) gave a positive signal, and the nucleotide sequence of its insert DNA (2 kb) was determined using a Shimadzu DSQ-1000 automated sequencer. The 0.5 kb AccI (blunt-ended)-PstI fragment of pP19 carrying the p15 precursor was further subcloned into pBluescript II SK(+) digested with EcoRV and PstI, generating pB8. For the isolation of cDNA encoding the mature region of p15, cells were grown in the production medium (4% sucrose, 2% pharmamedia, 2% dry yeast, 1% polypeptone, 0.2% K2 HPO4 , 0.2% CaCO3 , 0.1% Tween 80) for 7 days at 25³C. Cells (1 g) were frozen and homogenized as described above. RNA was extracted in 4 ml of 10 mM EDTA, 0.5% sodium SDS and then 4 ml of 10 mM EDTA, 0.1% sodium acetate were added. Water-saturated phenol (6 ml) was added, and the sample was mixed and centrifuged. The supernatant was recovered and re-extracted with phenol, and the total RNA was precipitated with ethanol. Typically V250 Wg of total RNA were isolated by this method. From this total RNA, poly(A) RNA (10 Wg) was obtained using the mRNA Puri¢cation Kit (Amersham-Pharmacia), and reverse-transcribed using the 1st Strand cDNA Synthesis Kit (Boehringer), RAV-2 reverse transcriptase (TaKaRa) and random primer p(dN)6 . The resultant single-stranded cDNA (1 Wg) was served as a PCR template. Two sets of degenerate primers corresponding to the amino- and carboxy-termini of mature p15 were synthesized for PCR ampli¢cation of p15 cDNA. The sequences of the sense and antisense primers were 5P-GC(A/G)AC(T/G/C)AT(A/T/C)GAGGAGACCACAGATA-3P and 5P-(A/T/G)(G/C)A(A/T/G)CCGAA(A/T/G)GTTTTGACCACCTCG-3P, respectively. DNA ampli¢cation was done by a 30 cycle PCR protocol with a 30 s denaturing step at 94³C, a 30 s annealing step at 50³C, and a 30 s extension step at 72³C. The ampli¢ed DNA was ligated into pT7Blue and sequenced, which con¢rmed that the cloned fragment is authentic cDNA for p15. 2.4. Expression and puri¢cation of recombinant p15 For the expression of hexahistidine-tagged (His6 ) p15, a set of PCR primers was designed by adding PstI and HindIII sites at the 5P end of sense and antisense primers, respectively. The sequences were 5P-AACTGCAGCAACGATTGAGGAGACCAC-3P and 5P-ATAAGCTTATGAGCCGAAGGTTTTGAC-3P. Using these primers and p15
cDNA carried on pT7Blue as a template, PCR was done as described above. The ampli¢ed DNA fragment was digested with PstI and HindIII and subcloned into PstIand HindIII-digested pRSETB vector (Invitrogen). The resultant plasmid named p15-pRSETB was introduced into E. coli BL21(DE3)pLysS. The cells were grown to mid-log phase in LB broth, and the His6 -p15 was induced by 0.3 mM IPTG for 3 h at 37³C. The induced cells were lysed by sonication, and the cell lysate was centrifuged at 100 000Ug for 30 min at 4³C to remove insoluble materials. His6 -p15 was found in both the soluble and insoluble fractions. The soluble fraction was collected and applied to a Ni2 -NTA agarose column (Qiagen). The column was extensively washed with bu¡er (20 mM sodium phosphate (pH 7.8), 0.5 M NaCl) containing 50 mM imidazole, and then His6 -p15 was eluted by bu¡er containing 200 mM imidazole. Insoluble fraction was solubilized, applied to the Ni2 -NTA column, washed, and eluted according to the standard procedure. The majority of His6 -p15 was eluted by bu¡er containing 20 mM sodium phosphate (pH 4.0), 8 M urea, 0.5 M NaCl. The proteins were analyzed by sodium dodecyl sulfate^polyacrylamide gel electrophoresis (SDS^PAGE) containing 15% acrylamide. The fractions containing His6 -p15 were collected and dialyzed against 20 mM sodium phosphate (pH 7.4). 2.5. PCR-based mutagenesis Site-directed mutagenesis by PCR was performed essentially according to the method of Imai [23]. To construct a double mutant in which both cysteine residues that are linked by disul¢de bridges are replaced with serine residues, a set of PCR primers, each being designed to code for cysteine to serine mutation, was synthesized. Their sequences are: for replacement of Cys38 and Cys54 with serine (C38S/C54S mutation), 5P-AATGGGTCATCTGGAGACGAGCTGGACC-3P (antisense) and 5P-CGGCTTCGACTTCTTATCCTCTTCCCAC-3P (sense); for replacement of Cys90 and Cys102 with serine (C90S/C102S mutation), 5P-ATATTGCTCGGTATTGGACTGG-3P (antisense) and 5P-CTTCACAAGAGCTGCCTCCAAAGC-3P (sense). Underlined sequences indicate the sites of mutations. To construct mutants carrying only one Cys-to-Ser mutation, a set of primers with wild type and mutant sequence was used. Using these primer sets and p15-pRSETB as a template, PCR reactions were conducted using Expand High-Fidelity PCR Systems (Roche Diagnostics). PCR products were recovered from agarose gel, processed by T4 DNA polymerase, T4 polynucleotide kinase, then circularized by ligation, and transformed into E. coli. Introduction of mutations was con¢rmed by DNA sequencing. The resultant six plasmids, carrying His6 -p15 with substitutions of C38S/C54S, C90S/C102S, C38S, C54S, C90S, and C102S, were introduced into E. coli BL21(DE3)pLysS. Induction and puri¢cation of mutant proteins were done as described above.
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3. Results We previously reported the complete amino acid sequence of the mature region of p15 by sequencing the chemically and enzymatically digested fragments of p15 puri¢ed from the culture broth of the producing fungal strain. Degenerate primers encoding the determined amino- and carboxy-terminal sequences were synthesized, and the partial genomic DNA encoding p15 was ampli¢ed by PCR using the chromosomal DNA of the producing fungal strain as a template. The nucleotide sequence analysis of this DNA fragment con¢rmed that this PCR fragment encodes p15. Using this DNA fragment as a probe, we isolated the whole genomic sequence of p15 precursor as described in Section 2. The entire coding region of p15 was carried on the 2.0 kb PstI fragment of the genomic DNA. The p15 precursor gene consists of a 588 bp open reading frame with an 81 bp intron, the position of which was determined by comparison with the cDNA sequence of the mature region of p15 (Fig. 1; see below). Thus the gene predicts a protein of 169 amino acids. Compared to the 118 amino acids-long mature region with neurite-inducing activity [22], the p15 precursor has 31 and 20 additional amino acids at the amino- and carboxy-terminus, respectively. According to the prediction by SMART [24], the 18 amino-terminal amino acids are assumed to be the signal sequence for secretion. No obvious TATA sequence
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was found within the 300 nucleotides upstream from the start codon (data not shown). In the 3P-noncoding region, a putative polyadenylation signal was found about 20 bp downstream from the stop codon. We next isolated the cDNA encoding mature region of p15 and expressed it in E. coli. Poly(A) RNA prepared from the producing fungal strain was reverse transcribed, and the cDNA encoding the mature region of p15 was isolated by PCR. Nucleotide sequence analysis indicated that the ampli¢ed product was authentic p15 cDNA. It was ligated to pRSETB vector to produce the amino-terminally His6 -tagged version of recombinant p15. This expression plasmid was introduced into E. coli strain BL21(DE3)pLysS and the expression of His6 -p15 was induced by IPTG. SDS^PAGE analysis of the lysed cells revealed the presence of a protein band with an approximate molecular mass of 20 kDa in the extract induced by IPTG, but not in the extract without induction (Fig. 2A). Since His6 -p15 has an extra MRGSH6 GMASMTGGQQMGRDLYDDDDKDPSSRSA at the amino-terminus compared with mature p15, which would permit the expression of a protein with the calculated molecular mass of 17.5 kDa, we concluded that this band was His6 -p15. The induced cell lysate was centrifuged to separate soluble and insoluble fractions, which demonstrated that roughly equal amounts of His6 -p15 occurred in the soluble and insoluble fractions. His6 -p15 from both fractions was pu-
Fig. 1. Nucleotide and deduced amino acid sequences of p15 precursor gene. The predicted amino acid sequence is shown below the nucleotide sequence. The putative signal sequence is italicized, and the amino acid sequence of mature p15 is underlined. The four cysteine residues mutated to serine are boxed, together with the amino acid number from the amino-terminus of mature p15. The intron sequence is shown in lower case, and the putative polyadenylation signal is indicated by the double underline. The nucleotide sequence has been deposited in the DDBJ database under accession no. AB071215.
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from the insoluble material, although the majority of the protein was His6 -p15. His6 -p15 thus puri¢ed was examined in a PC12 neurite extension assay (Fig. 2B,C). Cells were cultured in the presence of native or His6 -p15 for 48 h, and the number of cells with neurites longer than the cell diameter was counted. Native p15 puri¢ed from the culture broth of producing fungal strain exhibits neurite-inducing activity on PC12 cells as low as 0.01 nM and maximal activity was observed at 1 nM. His6 -p15 puri¢ed from both the soluble and insoluble fractions displayed similar activities, which were comparable to that observed with native p15 (Fig. 2C). We con¢rmed that the amino-terminal His6 tag had no e¡ect in this neurite extension assay, since removal of this region by enterokinase digestion did not alter the speci¢c activity of the recombinant protein (data not shown). We next examined the expression of neuro¢lament-M in His6 -p15-treated PC12 cells. Neuro¢lament-M is one of the di¡erentiation markers of PC12 cells that is speci¢cally expressed in neurons, and its protein level has been reported to be increased in response to NGF treatment [25]. As shown in Fig. 3, His6 -p15 potentiated the expression of neuro¢lament-M. Although the expression level of neuro¢lament-M was less than half of that observed in
Fig. 2. Neurite-inducing activity of His6 -p15. (A) SDS^PAGE analysis. Cell lysates from uninduced and induced cells (lane 1 and 2, respectively), His6 -p15 puri¢ed from the soluble fraction by Ni2 -agarose column chromatography (lane 3) and the native p15 partially puri¢ed from the culture broth of producing fungal strain (lane 4) were analyzed. The positions of His6 -p15 and native p15 are indicated by the arrow and the dot, respectively. Molecular mass markers are shown on the right. (B) PC12 cells treated for 48 h without (a) or with NGF (b, 50 ng/ml), His6 -p15 (c, 1 nM), or native p15 (d, 1 nM) are shown. Bar, 100 Wm. (C) PC12 cells were incubated with the indicated concentrations of native p15 (a) or His6 -p15 (b) for 48 h. Cells with neurites longer than one cell diameter were judged to be positive and counted. The results show the percentage of the total cell number.
ri¢ed by one-step a¤nity chromatography through Ni2 agarose. His6 -p15 from the soluble fraction was puri¢ed to almost homogeneity, whereas additional high molecular mass bands could be detected in the puri¢ed fraction
Fig. 3. Induction of neuro¢lament-M expression in His6 -p15-treated PC12 cells. PC12 cells were grown in the absence (lane 1) or presence of His6 -p15 (1 nM; lane 2) or NGF (50 ng/ml; lane 3) for 7 days. Cell extracts were prepared as described in Section 2 and analyzed for the expression of neuro¢lament-M using anti-neuro¢lament-M antibody. In the lower panel, the relative intensities of the bands compared to the control lane were quanti¢ed and are shown in the bar graph. The experiments were repeated at least three times with no signi¢cant di¡erence.
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Fig. 4. The e¡ects of nicardipine on neurite outgrowth induced by NGF, native p15 or His6 -p15. PC12 cells were treated for 48 h with native p15 (1 nM; a), His6 -p15 (1 nM; b) or NGF (50 ng/ml; O) in the presence of the indicated concentrations of nicardipine.
cells treated with NGF, the induction was signi¢cant compared to control cells. These results demonstrate that the processes induced in His6 -p15-treated PC12 cells are authentic neurites and that PC12 cells underwent neuronal di¡erentiation upon His6 -p15 treatment. We previously reported that the action of p15 on PC12 cells is blocked by treatment with nicardipine, an inhibitor of L-type Ca2 channels, but not by K-252a. This indicated that p15 activates a pathway that is distinct from NGF. We next asked if His6 -p15 also requires the activation of Ca2 signaling pathway. As shown in Fig. 4, neu-
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rite formation by native p15 as well as His6 -p15 is inhibited by nicardipine in a dose-dependent manner, but that by NGF treatment is not. This result indicates that His6 p15, like native p15, induces neurites through the activation of L-type Ca2 channels. In our previous study, we analyzed the primary structure of p15 by protein sequencing and showed that p15 has two intramolecular disul¢de bonds linking Cys38 to Cys54 and Cys90 to Cys102 (amino acid numbers are based on the sequence of mature p15). Preliminary observation with reduced, carboxymethylated p15 suggested that either one or both of these disul¢de bonds are essential for its biological activity (S.W., unpublished observation). We therefore examined if these disul¢de bridges are indeed necessary for its activity by substituting cysteine residues of His6 -p15 with serine. For this purpose, PCR-based sitedirected mutagenesis was performed to produce mutants C38S (in which Cys38 is replaced by a serine residue), C54S, C90S and C102S as well as double mutants C38S/ C54S (in which both Cys38 and Cys54 are replaced by serine residues) and C90S/C102S as described in Section 2 [23]. As shown in Fig. 5A, the single and double mutants of the amino-terminal disul¢de bond, C38S, C54S and C38S/C54S, exhibited no detectable neurite-inducing activity at concentrations as high as 10 nM, indicating that the amino-terminal disul¢de bond is essential. Similarly, disruption of the carboxy-terminal disul¢de bond in the C90S and C102S mutants also resulted in loss of activity (Fig. 5B). Interestingly, the C-terminal double mutant C90S/ C102S did not completely lose its neurite-inducing activity, although the activity was less than one-tenth of that of wild type. We reasoned that this is because the one re-
Fig. 5. Neurite-inducing activities of mutant His6 -p15. His6 -tagged mutant p15 proteins in which cysteine residues are replaced by serine were produced in E. coli and puri¢ed as wild type His6 -p15. PC12 cells were incubated with increasing concentrations of recombinant p15 for 48 h, and cells with neurites longer than one cell diameter were counted. (A) a, His6 -p15; E, C38S mutant; F, C54S mutant; O, C38S/C54S mutant. (B) a, His6 -p15; E, C90S mutant; F, C102S mutant; O, C90S/C102S mutant.
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maining cysteine residue in C90S or C102S mutants interferes with the formation of the amino-terminal disul¢de bond, resulting in the failure in the formation of both correct disul¢de bonds and complete loss of activity. In summary, both intramolecular disul¢de bridges are necessary for the full activity of His6 -p15 in neurite induction, and it is probable that the amino-terminal disul¢de bridge is more important than the carboxy-terminal one. 4. Discussion In this study we reported molecular cloning, functional expression, and mutational analysis of p15, which we found in a search for microbial metabolites with neurotrophin-like activity [21,22]. The structure of p15 precursor gene predicted that mature p15 is £anked by amino- and carboxy-terminal extensions of 31 and 20 amino acids, respectively. Consistent with its presence in the culture supernatant of the producing fungal strain, p15 precursor contained an amino-terminal sequence of 18 amino acids which is presumably a signal sequence for secretion. The presence of a dibasic Lys-Arg sequence close to the carboxy-terminus of mature p15 suggests that processing of the carboxy-terminus of p15 is performed in two steps during the course of secretion that might involve a Kex2-like endoprotease and a carboxypeptidase. We previously observed that reduced, carboxymethylated p15 lost its neurite-inducing activity. Consistent with this, substitution of both or one of the cysteine residues linked by disul¢de bonds with serine in His6 -p15 resulted in complete or marked loss of neurite-inducing activity, indicating that both intramolecular disul¢de bonds are critical for its activity. Since the speci¢c activity of His6 -p15 is comparable to that of native p15 puri¢ed from the culture broth of the producing fungal strain, it is reasonable to assume that the formation of correct pairs of disul¢de bonds is achieved during the course of puri¢cation of His6 -p15 that is produced intracellularly in E. coli. Although p15 displays no sequence similarity to neurotrophin family proteins, it exhibits potent neurite-inducing activity and induces the expression of neuro¢lament-M, one of the hallmarks of di¡erentiation of PC12 cells. We have shown that the e¡ect of His6 -p15 requires activation of the Ca2 signaling pathway by demonstrating that nicardipine treatment inhibits neuritogenesis. Walsh and coworkers have demonstrated that increased in£ux of extracellular Ca2 via Ca2 channels induces neurite outgrowth in PC12 cells stimulated with cell adhesion molecules such as neural cell adhesion molecule (N-CAM), L1 and Ncadherin [26^32]. These studies pointed to the activation of ¢broblast growth factor receptor-phospholipase CQ (PLCQ) cascade as being important for the neurite outgrowth responses stimulated by all three CAMs. Following recruitment and activation of PLCQ via interaction of
its SH-2 domain with the activated receptor, generation of diacylglycerol (DAG) and its conversion to arachidonic acid (AA) is thought to occur. This scenario is in good agreement with the fact that Ca2 channel blockers inhibit neurite outgrowth by CAMs, since AA has been shown to increase voltage-dependent Ca2 currents in cardiac myocytes [33]. Indeed, AA potentiates neurite outgrowth, which is inhibited by N- or L-type Ca2 channel antagonists [31]. In this context the key event downstream from the activation of PLCQ required for neurite growth appears to be the conversion of DAG to AA via DAG lipase. We pursued the possibility that p15-generated signal converges at some point in this pathway by examining the e¡ects of pertussis toxin and an inhibitor of DAG lipase, RHC-80267, on p15-induced neurite outgrowth. However, neither of these agents blocked His6 -p15-induced neurite outgrowth, indicating that the pathways that are activated by p15 and CAMs are distinct. Database search analysis revealed that an open reading frame from Streptomyces coelicolor, CAB38593.1, shares signi¢cant homology with p15. Our preliminary analysis demonstrated that this putative secreted protein indeed has neurite-inducing activity toward PC12, although its speci¢c activity was considerably low compared to that of p15. Interestingly, CAB38593.1 also has four cysteine residues and the positions of these residues are almost completely conserved between CAB38593.1 and p15, suggesting that these residues in CAB38593.1 are linked by disul¢de bridges and essential for neurite-inducing activity. Further structural comparison between these proteins will help specify the region responsible for their biological activity. Acknowledgements This work was supported by Grants-in-Aid for Scienti¢c Research (No. 12460041 and No. 12760048) from the Ministry of Education, Science, Sports and Culture of Japan, and by a grant from the Fujisawa Foundation. S.W. was supported by Research Fellowships of the Japan Society for the Promotion of Science for Young Scientists. References [1] L.A. Greene, A.S. Tischler, Proc. Natl. Acad. Sci. USA 73 (1976) 2424^2428. [2] S. Iwasaki, A. Hattori, M. Sato, M. Tsujimoto, M. Kohno, J. Biol. Chem. 271 (1996) 17360^17365. [3] S. Ihara, A. Iwamatsu, T. Fujiyoshi, A. Komi, T. Yamori, Y. Fukui, J. Biochem. 120 (1996) 865^868. [4] D.R. Kaplan, B.L. Hempstead, D. Martin-Zanca, M.V. Chao, L.F. Parada, Science 252 (1991) 554^558. [5] D.R. Kaplan, D. Martin-Zanca, L.F. Parada, Nature 350 (1991) 158^ 160. [6] D.J. Robbins, M. Cheng, E. Zhen, C.A. Vanderbilt, L.A. Feig, M.H. Cobb, Proc. Natl. Acad. Sci. USA 89 (1992) 6924^6928.
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S. Wakatsuki et al. / Biochimica et Biophysica Acta 1522 (2001) 74^81 [7] S.M. Thomas, M. DeMarco, G. D'Arcangelo, S. Halegoua, J.S. Brugge, Cell 68 (1992) 1031^1040. [8] K. Wood, C. Sarnecki, T.M. Roberts, J. Blenis, Cell 68 (1992) 1041^ 1050. [9] A. Obermeier, H. Halfter, K.H. Wiesmuller, G. Jung, J. Schlessinger, A. Ullrich, EMBO J. 12 (1993) 933^941. [10] A. Obermeier, R. Lammers, K.H. Wiesmuller, G. Jung, J. Schlessinger, A. Ullrich, J. Biol. Chem. 268 (1993) 22963^22966. [11] A. Obermeier, R.A. Bradshaw, K. Seedorf, A. Choidas, J. Schlessinger, A. Ullrich, EMBO J. 13 (1994) 1585^1590. [12] M. Rozakis-Adcock, J. McGlade, G. Mbamalu, G. Pelicci, R. Daly, W. Li, A. Batzer, S. Thomas, J. Brugge, P.G. Pelicci, J. Schlessinger, T. Pawson, Nature 360 (1992) 689^692. [13] N.W. Gale, S. Kaplan, E.J. Lowenstein, J. Schlessinger, D. Bar-Sagi, Nature 363 (1993) 88^92. [14] N. Li, A. Batzer, R. Daly, V. Yajnik, E. Skolnik, P. Chardin, D. BarSagi, B. Margolis, J. Schlessinger, Nature 363 (1993) 85^88. [15] M. Rozakis-Adcock, R. Fernley, J. Wade, T. Pawson, D. Bowtell, Nature 363 (1993) 83^85. [16] J.I. Morgan, T. Curran, Nature 322 (1986) 552^555. [17] A. Ghosh, M.E. Greenberg, Science 268 (1995) 239^247. [18] L.B. Rosen, D.D. Ginty, M.J. Weber, M.E. Greenberg, Neuron 12 (1994) 1207^1221. [19] G. Rusanescu, H. Qi, S.M. Thomas, J.S. Brugge, S. Halegoua, Neuron 15 (1995) 1415^1425. [20] S. Finkbeiner, M.E. Greenberg, Neuron 16 (1996) 233^236.
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[21] T. Hanada, T. Sato, M. Arioka, M. Uramoto, M. Yamasaki, Biochem. Biophys. Res. Commun. 228 (1996) 209^215. [22] S. Wakatsuki, M. Arioka, N. Dohmae, K. Takio, M. Yamasaki, K. Kitamoto, J. Biochem. 126 (1999) 1151^1160. [23] Y. Imai, Y. Matsushima, T. Sugimura, M. Terada, Nucleic Acids Res. 19 (1991) 2785. [24] J. Schultz, F. Milpetz, P. Bork, C.P. Ponting, Proc. Natl. Acad. Sci. USA 95 (1998) 5857^5864. [25] M.H. Lindenbaum, S. Carbonetto, F. Grosveld, D. Flavell, W.E. Mushynski, J. Biol. Chem. 263 (1988) 5662^5667. [26] P. Doherty, S.V. Ashton, S.E. Moore, F.S. Walsh, Cell 67 (1991) 21^ 33. [27] J.L. Sa¡ell, F.S. Walsh, P. Doherty, J. Cell Biol. 118 (1992) 663^ 670. [28] E.J. Williams, P. Doherty, G. Turner, R.A. Reid, J.J. Hemperly, F.S. Walsh, J. Cell Biol. 119 (1992) 883^892. [29] E.J. Williams, J. Furness, F.S. Walsh, P. Doherty, Neuron 13 (1994) 583^594. [30] E.J. Williams, J. Furness, F.S. Walsh, P. Doherty, Development 120 (1994) 1685^1693. [31] E.J. Williams, F.S. Walsh, P. Doherty, J. Neurochem. 62 (1994) 1231^1234. [32] E.J. Williams, F.S. Walsh, P. Doherty, J. Cell Biol. 124 (1994) 1029^ 1037. [33] J.M. Huang, H. Xian, M. Bacaner, Proc. Natl. Acad. Sci. USA 89 (1992) 6452^6456.
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