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Identification of the GGPS1 genes encoding geranylgeranyl diphosphate synthases from mouse and human
Biochimica et Biophysica Acta 1437 (1999) 333^340
Identi¢cation of the GGPS1 genes encoding geranylgeranyl diphosphate synthases from mouse and human Tomohiro Kainou, Kei Kawamura, Katsunori Tanaka, Hideyuki Matsuda, Makoto Kawamukai * Department of Applied Bioscience and Biotechnology, Faculty of Life and Environmental Science, Shimane University, 1060 Nishikawatsu, Matsue 690-8504, Japan Received 13 November 1998; received in revised form 1 February 1999; accepted 8 February 1999
Abstract E,E,E-Geranylgeranyl diphosphate (GGPP) is an important precursor of carotenoids and geranylgeranylated proteins such as small G proteins. In this study, we have identified mouse and human GGPP synthase genes. Sequence analysis showed that mouse and human GGPP synthases share a high level of amino acid identity (94%) with each other, and share a high level of similarity (45^50%) with GGPP synthases of lower eukaryotes, but only weak similarity (22^31%) to plant and prokaryotic GGPP synthases. Both of the newly identified GGPP synthase genes from mouse and human were expressed in Escherichia coli, and their gene products displayed GGPP synthase activity when isopentenyl diphosphate and farnesyl diphosphate were used as substrates. The GGPP synthase activity of these genes was also confirmed by demonstrating carotenoid synthesis after co-transformation of E. coli with a plasmid expressing the crt genes derived from Erwinia uredovora, and a plasmid expressing either the mouse or human GGPS1 gene. Southern blot analysis suggests that the human GGPS1 gene is a single copy gene. ß 1999 Elsevier Science B.V. All rights reserved. Keywords: Geranylgeranyl diphosphate; Isoprenoid; Carotenoid
1. Introduction The isoprenoid biosynthetic pathway produces important compounds including farnesyl diphosphate (FPP) and geranylgeranyl diphosphate (GGPP) in various organisms from bacteria to higher eukaryotes. FPP is a major branch point isoprenoid and is used as a precursor for squalene, cholesterols and the side chain of ubiquinone [1]. GGPP is a common Abbreviations: IPP, isopentenyl diphosphate; FPP, farnesyl diphosphate; GGPP, geranylgeranyl diphosphate; PCR, polymerase chain reaction * Corresponding author. Fax: +81 (852) 32-6499; E-mail: [email protected]
precursor of carotenoids, isoprenoid quinone and prenylated proteins [1]. Geranylgeranyl diphosphate synthase (GGPS) catalyzes the condensation of FPP with isopentenyl diphosphate to give GGPP. Allylic diphosphates such as DMAPP and GPP are also used as precursors of GGPS from plants, but not from animals. The genes for GGPP synthase have been isolated from many organisms, and comparison of these amino acid sequences revealed a common structure which consists notably of two conserved aspartate-rich motifs, DDXX(XX)D [2]. However, the latter aspartate-rich motif di¡ers slightly among the GGPP synthases from Gibberella fujikuroi [3], Neurospora crassa [4] and Saccharomyces cerevisiae [5]. In these species, the last aspartate residue is re-
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placed by asparagine. In the GGPP synthases from Arabidopsis thaliana [6] and Sulfolobus acidocaldarius [7], the aspartate residue is replaced by a glutamate and a glycine residue, respectively. One of the important roles of GGPP is to ensure a supply of prenylated proteins. Prenylated proteins represent about 0.5% of all proteins, and most known prenylated proteins are geranylgeranylated in mammalian tissue [8]. Geranylgeranyltransferase type I, which transfers geranylgeranyl moieties to the cysteine residues of carboxy-terminal CAAX motifs (C, cysteine; A, aliphatic amino acid; X, any amino acid) has been cloned and characterized from rat and human [9]. Geranylgeranyltransferase type II, which can modify Rab proteins containing a variety of carboxy-terminal cysteine motifs, e.g. XCXC, XXCC or CCXX, was also identi¢ed [10]. Despite the identi¢cation of these proteins of the isoprenoid biosynthetic pathway, the mammalian GGPP synthase genes have not yet been described, although the presence of GGPP synthase activity was reported in rat tissue [11] and in bovine brain [12]. In this paper, we describe the identi¢cation of the GGPP synthase genes from mouse and human and their functional expression in Escherichia coli. 2. Materials and methods 2.1. Materials Restriction enzymes and other DNA-modifying enzymes were purchased from Takara Shuzo Co. Ltd. and New England Biolabs Inc. IPP, E-farnesyl diphosphate (all E-FPP), geranylgeraniol and solanesol (all E-nonaprenol) were purchased from Sigma Chemical Co. [1-14 C]IPP (1.96 TBq/mol) was purchased from Amersham Co. Reversed phase LKC18 thin layer chromatography plates were purchased
from Whatman Chemical Separation, Inc. The GGPP synthase clone (GenBank accession number AA254082) from mouse lymph node and that (GenBank accession number H67793) from human liver and spleen were purchased from Genome Systems Inc. 2.2. Strain and plasmids E. coli DH10B was used for the general construction of plasmids [13]. Plasmids pBluescript KS3 and SK were used as vectors (Stratagene). pACCAR25vcrtE, which contains the gene cluster crtX, crtY, crtI, crtB and crtZ encoding carotenoid biosynthetic enzymes, and pORF2, which contains the crtE gene encoding GGPP synthase, were used for color production to test GGPP synthase activity [14]. 2.3. Genomic DNA preparation and Southern blot analysis Genomic DNA was prepared from human blood cells by Qiagen Genomic-tip (Qiagen Inc.). Genomic DNA (15 Wg in each case) was digested with restriction enzymes BamHI and EcoRI. 0.1 Wg cDNA of human GGPS1 was used as a probe. Southern blots were performed by standard methods as described before [13]. For hybridization and signal detection, the AlkPhos direct system (Amersham Life Science Ltd.) was used according to the attached protocol. In this system, the hybridization temperature was set at 55³C. 2.4. Construction of plasmids for expression of GGPP synthase The inserts of plasmids pAA254082 and pH67793 were cloned into the EcoRI-NotI and PacI-EcoRI sites of the modi¢ed pT7T3D vector, respectively.
C
Fig. 1. Alignment of the amino acid sequences of geranylgeranyl diphosphate synthases and farnesyl diphosphate synthases. Abbreviations of species names, and the GenBank accession numbers are follows: MMGG, Mus musculus GGPP synthase, AB016044; HSGG, Homo sapiens GGPP synthase, AB016043; DMGG, Drosophila melanogaster, AF049659; SCGG, Saccharomyces cerevisiae GGPP synthase, U31632; NCGG, Neurospora crassa GGPP synthase, U20940; GFGG, Gibberella fujikuroi GGPP synthase, X96943; SAGG, Sulfolobus acidocaldarius GGPP synthase, D28748; HSFP, Homo sapiens FPP synthase, J05262; RRFP, Rattus rattus FPP synthase, M34477. Filled boxes indicate the presence of conserved residues in more than ¢ve out of nine sequences. The ¢rst (I) and second (II) aspartate-rich motifs are shown by underlining. Numbers on the right side indicate the position of amino acid residues.
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and 25 Wg/ml chloramphenicol. Chloramphenicoland ampicillin-resistant colonies were replicated on LB plates containing antibiotics and 0.1 mM of IPTG, and then incubated for 3^4 days at 28³C. 2.6. GGPP synthase activity and reaction product analysis
The A1 oligonucleotide primer (5P-GTGGATCCTATGGAGAAAACTAAAGA-3P; sense) for pAA254082 or the H1 oligonucleotide primer (5P-CAGGATCCTATGGAGAAGACTCAAGA-3P; sense) for pH67793 and the universal primer (5PGTAAAACGACGGCCAGT-3P; antisense), which anneals with the downstream sequence of the cloning site within the pT7T3D vector, were used to amplify the GGPS genes by polymerase chain reaction (PCR). PCR fragments were digested with BamHI and NotI, and then cloned into the same sites of pBluescript KS3 to give pBAA for mouse and pBH for human.
GGPP synthase activity was measured by the method described previously [15], in which incorporation of [1-14 C]IPP into reaction products is detected. E. coli DH10B harboring pBAA, pBH, pGGPS6-1 or pBluescript KS3 was incubated to late log phase in LB medium containing 50 Wg/ml ampicillin at 37³C. Cells were harvested by centrifugation, suspended in bu¡er A (100 mM potassium phosphate (pH 7.4), 5 mM EDTA, 1 mM 2-mercaptoethanol), and ruptured by sonication 6 times for 30 s at 30-s intervals in an ice bath. After centrifugation of the homogenate, the supernatant was used as a crude enzyme extract. The assay reaction mixture contained 1.0 mM of MgCl2 , 0.1% (w/v) of Triton X-100, 50 mM of potassium phosphate bu¡er (pH 7.5), 10 WM of [1-14 C]IPP (speci¢c activity 0.92 TBq/mol), 5 WM of FPP and 200 Wg of crude extract containing the enzyme in a ¢nal volume of 0.4 ml. Sample mixtures were incubated for 120 min at 30³C. Reaction products such as prenyl diphosphates were extracted with 1-butanol saturated water, and hydrolyzed with acid phosphatase [16]. The products of hydrolysis were extracted with hexane and analyzed by reversed phase thin layer chromatography with acetone/water (19:1, v/v). Radioactivity on the plate was detected with an imaging analyzer BAS1500Mac (Fuji Film Co.). The plate was exposed to iodine vapor to detect the spots of the marker prenols.
2.5. Carotenoid expression test using the crt gene cluster
3. Results
Fig. 2. Southern blot analysis using human genomic DNA. Genomic DNA was digested with BamHI or EcoRI, as described in Section 2.3. The sizes of markers (kb) are shown on the left. Lane 1, BamHI; lane 2, EcoRI.
E. coli DH10B was transformed with pACCAR25vcrtE which contains the gene cluster crtX, crtY, crtI, crtB and crtZ encoding carotenoid biosynthetic enzymes, and plated on LB medium plates containing 25 Wg/ml of chloramphenicol. A transformant was retransformed with pBAA, pBH, pGGPS6-1 (a positive control) or pBluescript KS3 , and plated on plates containing 50 Wg/ml ampicillin
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3.1. Isolation and sequence analysis of GGPP synthase genes from mouse and human A number of GGPP synthase genes have been isolated from various organisms, and conserved amino acid sequence motifs in these GGPP synthases were observed [2,17]. To isolate and identify GGPP synthase genes from animals, we searched the EST data-
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Fig. 3. Carotenoid color production test in E. coli harboring both pACCAR25vcrtE and the plasmid expressing the GGPP synthase gene from mouse or human. The E. coli strain DH10B containing the carotenoid biosynthetic gene cluster (pACCAR25vcrtE) with pBluescript KS3 (A); pORF2 (B), which carries the crtE gene from E. uredovora; pBAA (C), which carries the GGPS1 from mouse; and pBH (D), which carries the GGPS1 gene from human, were grown for 3^4 days at 28³C.
base using Arabidopsis and fungus GGPP synthases as the template at the National Center for Biotechnology Information (NCBI) using the BLAST program. Interestingly, many clones with conserved sequence motifs to GGPP synthases were found in both the mouse and human EST databases. We selected a mouse cDNA clone (accession number AA254082) and a human cDNA clone (accession number H67793), and named them pAA254082 and pH67793, respectively, after obtaining them from Genome Systems Inc. Since only short sequences of these EST clones were known, we determined their complete sequence. Both the mouse cDNA and human cDNA were 903 bp in size, and were deposited in the DDBJ with the accession numbers AB016044 and AB016043, respectively. We designated both clones GGPS1. Mouse GGPS1 and human GGPS1 were 88.6% identical at the DNA level and their translated products were 94% identical at the amino acid level. Mouse and human GGPS1 displayed high similarity (45^50%) with GGPP synthases from G. fujikuroi [3], N. crassa [4] and S. cerevisiae [5], but were less similar (22^31%) to other GGPP synthases from bacteria and plants. Mouse and human GGPS1
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showed 22% similarity with FPP synthase from human [18] and rat [19], which share a high level of identity with each other at the amino acid sequence level (85%). In the mouse and human GGPS, the third aspartate residue in the second aspartate-rich motif (Fig. 1) is replaced by an asparagine, and the arginine residue found in the third position from the carboxy-terminus of most polyprenyl diphosphate synthases was replaced by a glutamate. The GGPP synthase from S. acidocaldarius and one of the six GGPP synthases from Arabidopsis [6] had the same amino acid, glutamate. These amino acid compositions were not found in other farnesyl or long-chain polyprenyl diphosphate synthases. GGPP synthase puri¢ed from bovine brain was reported to be a homo-oligomer (150^195 kDa) with a molecular mass of 37.5 kDa for the monomer [12]. This is consistent with the calculated molecular mass (35 kDa) of mouse and human GGPP synthases. 3.2. Southern blot analysis of GGPP synthase genes Southern blot analysis under low stringency conditions was performed using the 1.0 kb fragment of
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coli DH10B harboring pACCAR25vcrtE which contains the gene cluster crtX, crtY, crtI, crtB and crtZ, but not crtE which encodes GGPP synthase. Both transformants produced the expected yellow color pigment (Fig. 3), indicating that pBAA and pBH can substitute for the function of the crtE gene. These results suggest that the genes from mouse and human encode functional GGPP synthases.
Fig. 4. A reversed phase thin layer chromatogram of the products of GGPP synthase activity in E. coli. The enzyme reaction was carried out using [1-14 C]IPP and FPP as substrates and cell extract as crude enzyme. The reaction products were hydrolyzed by acid phosphatase and the resulting alcohols was analyzed by reversed phase thin layer chromatography. Arrowheads indicate the positions of the di¡erent alcohol products: GG, all-E-geranylgeraniol ; O, octaprenol; S.F., solvent front; Ori, origin. Lane 1, DH10B/pBluescript KS3 ; lane 2, DH10B/pORF2 ; lane 3, DH10B/pBAA; and lane 4, DH10B/pBH.
human GGPS1 to probe human genomic DNA digested with BamHI or EcoRI. This resulted in the appearance of bands of 5.0 kb and 2.2 kb for the BamHI and EcoRI digests, respectively (Fig. 2). We did not see any other bands under these conditions. This result suggests that the human GGPS1 gene is a single copy gene. 3.3. Detection of GGPP synthase activity using the carotenoid color production test in E. coli To determine if the mouse and human genes are functional, we tested their GGPP synthase activity by the carotenoid formation test. E. coli cells transformed with a plasmid containing the crtE, crtX, crtY, crtI, crtB and crtZ genes from Erwinia uredovora are able to accumulate zeaxanthin, which has a yellow color, only if GGPP is produced in E. coli. The genes from mouse and human were cloned into pBluescript KS3 to create a lacZ fusion, which yielded pBAA and pBH, respectively. The expression plasmids pBAA and pBH were transformed into E.
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Fig. 5. The phylogenetic relationship between known GGPP synthases. XXGG indicates GGPP synthase and XXFP indicates FPP synthase. The abbreviations of species names and the GenBank accession numbers that were not shown in Fig. 1 are as follows: MT, Methanobacterium thermoautotrophicum, S75695; AT, Arabidopsis thaliana, AB000835; CA, Capsicum annuum, X80267; CR, Catharanthus roseus, X92893; LA, Lupinus albus, U15778 ; EH, Erwinia herbicola, M87280; EU, Erwinia uredovora, D90087. The amino acid sequences were aligned by the DDBJ CLUSTAL W program [24]. The numbers were obtained by bootstrap analysis (out of 100).
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3.4. In vitro analysis of GGPP synthase activity from mouse and human In order to demonstrate GGPP synthase activity directly, crude extracts from E. coli expressing the GGPS1 genes from mouse and human were examined for GGPP synthase activity using [1-14 C]IPP and FPP as substrates. The reaction products were hydrolyzed by acid phosphatase and analyzed by reversed phase thin layer chromatography. While octaprenyl diphosphate synthesized by the endogenous IspB gene product [20] was detected in E. coli DH10B harboring the vector alone (Fig. 4, lane 1), geranylgeraniol resulting from GGPP activity was detected in DH10B harboring pBAA (Fig. 4, lane 3) and pBH (Fig. 4, lane 4). These results indicated that the genes from mouse and human produced GGPP as a result of condensation of IPP and FPP as the allylic substrates. 4. Discussion In this study, we have identi¢ed the GGPS1 genes encoding geranylgeranyl diphosphate synthase from mouse and human. Two lines of evidence demonstrate that these genes encode GGPP synthases. One is the direct measurement of GGPP synthase activity and the other is the indirect detection of GGPP synthase activity in E. coli strains in the form of carotenoid pigmentation. The latter method was shown to be an e¤cient method for the detection of GGPP synthase activity in our previous work [14,21]. The sequences of GGPS1 genes from mouse and human show them to be closely related to their eukaryotic counterparts, but not to plant and bacterial GGPP synthases. The phylogenetic analysis shown in Fig. 5 suggests that there are two types of GGPP synthase. One is of eukaryote origin and includes mouse and human genes, and the other is of prokaryote origin. The eukaryotic type of GGPP synthases which form one cluster (Fig. 5) has the DDXXN motif but not DDXXD in the second aspartate-rich motif. The archaebacterial S. acidocaldarius GGPS which possesses the DDXXG motif has been classi¢ed in the other cluster because it is likely to be of
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bacterial origin and to have a lower level of similarity with eukaryotic GGPP synthases. Plants have many di¡erent types of GGPP synthase such as Arabidopsis which has six GGPS genes [21]. But so far only one GGPP synthase gene has been identi¢ed in mouse and human as shown in this study. When we again searched the current EST database using the entire GGPS1s from mouse and human as probes, 64 and 15 clones from mouse and human, respectively, gave a high score. Taking into consideration the level of sequence error in the EST sequences, it seems that all of these clones are either of mouse or human GGPS1 gene origin. We did not ¢nd any other types of clone with the GGPS motif in the EST database. Southern blot analysis suggests that the human GGPS1 is a single copy gene. Thus, only one type of animal GGPP synthase gene appears to exist, which is in contrast to the many GGPP synthase genes found in plants. It is a somewhat surprising gene organization for higher eukaryotes if human only contains a single gene of GGPS. Rat GGPS activity was detected in the cytosol [11] and consistent with this, PSORT program analysis of the sequences of mouse and human GGPS1 indicates that they are localized in the cytoplasm. Recently, GGPP was shown to regulate cell cycle progression in human lymphocytes [22] and geranylgeraniol, which is the alcohol form of GGPP, was found to induce apoptosis of tumor cells [23]. The GGPP synthase genes of mouse and human identi¢ed here should prove useful for the study of many aspects of cellular regulation such as protein geranylgeranylation, cell cycle progression and apoptosis. 5. Note Recently, the GGPP synthase gene from human was reported by Ericsson et al. [25], while we were submitting this manuscript. We saw some sequence discrepancy between ours and theirs. Acknowledgements This work was supported by a Grant-in-Aid from
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the Ministry of Education, Science and Culture of Japan. [15]
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Identi¢cation of the GGPS1 genes encoding geranylgeranyl diphosphate synthases from mouse and human Tomohiro Kainou, Kei Kawamura, Katsunori Tanaka, Hideyuki Matsuda, Makoto Kawamukai * Department of Applied Bioscience and Biotechnology, Faculty of Life and Environmental Science, Shimane University, 1060 Nishikawatsu, Matsue 690-8504, Japan Received 13 November 1998; received in revised form 1 February 1999; accepted 8 February 1999
Abstract E,E,E-Geranylgeranyl diphosphate (GGPP) is an important precursor of carotenoids and geranylgeranylated proteins such as small G proteins. In this study, we have identified mouse and human GGPP synthase genes. Sequence analysis showed that mouse and human GGPP synthases share a high level of amino acid identity (94%) with each other, and share a high level of similarity (45^50%) with GGPP synthases of lower eukaryotes, but only weak similarity (22^31%) to plant and prokaryotic GGPP synthases. Both of the newly identified GGPP synthase genes from mouse and human were expressed in Escherichia coli, and their gene products displayed GGPP synthase activity when isopentenyl diphosphate and farnesyl diphosphate were used as substrates. The GGPP synthase activity of these genes was also confirmed by demonstrating carotenoid synthesis after co-transformation of E. coli with a plasmid expressing the crt genes derived from Erwinia uredovora, and a plasmid expressing either the mouse or human GGPS1 gene. Southern blot analysis suggests that the human GGPS1 gene is a single copy gene. ß 1999 Elsevier Science B.V. All rights reserved. Keywords: Geranylgeranyl diphosphate; Isoprenoid; Carotenoid
1. Introduction The isoprenoid biosynthetic pathway produces important compounds including farnesyl diphosphate (FPP) and geranylgeranyl diphosphate (GGPP) in various organisms from bacteria to higher eukaryotes. FPP is a major branch point isoprenoid and is used as a precursor for squalene, cholesterols and the side chain of ubiquinone [1]. GGPP is a common Abbreviations: IPP, isopentenyl diphosphate; FPP, farnesyl diphosphate; GGPP, geranylgeranyl diphosphate; PCR, polymerase chain reaction * Corresponding author. Fax: +81 (852) 32-6499; E-mail: [email protected]
precursor of carotenoids, isoprenoid quinone and prenylated proteins [1]. Geranylgeranyl diphosphate synthase (GGPS) catalyzes the condensation of FPP with isopentenyl diphosphate to give GGPP. Allylic diphosphates such as DMAPP and GPP are also used as precursors of GGPS from plants, but not from animals. The genes for GGPP synthase have been isolated from many organisms, and comparison of these amino acid sequences revealed a common structure which consists notably of two conserved aspartate-rich motifs, DDXX(XX)D [2]. However, the latter aspartate-rich motif di¡ers slightly among the GGPP synthases from Gibberella fujikuroi [3], Neurospora crassa [4] and Saccharomyces cerevisiae [5]. In these species, the last aspartate residue is re-
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placed by asparagine. In the GGPP synthases from Arabidopsis thaliana [6] and Sulfolobus acidocaldarius [7], the aspartate residue is replaced by a glutamate and a glycine residue, respectively. One of the important roles of GGPP is to ensure a supply of prenylated proteins. Prenylated proteins represent about 0.5% of all proteins, and most known prenylated proteins are geranylgeranylated in mammalian tissue [8]. Geranylgeranyltransferase type I, which transfers geranylgeranyl moieties to the cysteine residues of carboxy-terminal CAAX motifs (C, cysteine; A, aliphatic amino acid; X, any amino acid) has been cloned and characterized from rat and human [9]. Geranylgeranyltransferase type II, which can modify Rab proteins containing a variety of carboxy-terminal cysteine motifs, e.g. XCXC, XXCC or CCXX, was also identi¢ed [10]. Despite the identi¢cation of these proteins of the isoprenoid biosynthetic pathway, the mammalian GGPP synthase genes have not yet been described, although the presence of GGPP synthase activity was reported in rat tissue [11] and in bovine brain [12]. In this paper, we describe the identi¢cation of the GGPP synthase genes from mouse and human and their functional expression in Escherichia coli. 2. Materials and methods 2.1. Materials Restriction enzymes and other DNA-modifying enzymes were purchased from Takara Shuzo Co. Ltd. and New England Biolabs Inc. IPP, E-farnesyl diphosphate (all E-FPP), geranylgeraniol and solanesol (all E-nonaprenol) were purchased from Sigma Chemical Co. [1-14 C]IPP (1.96 TBq/mol) was purchased from Amersham Co. Reversed phase LKC18 thin layer chromatography plates were purchased
from Whatman Chemical Separation, Inc. The GGPP synthase clone (GenBank accession number AA254082) from mouse lymph node and that (GenBank accession number H67793) from human liver and spleen were purchased from Genome Systems Inc. 2.2. Strain and plasmids E. coli DH10B was used for the general construction of plasmids [13]. Plasmids pBluescript KS3 and SK were used as vectors (Stratagene). pACCAR25vcrtE, which contains the gene cluster crtX, crtY, crtI, crtB and crtZ encoding carotenoid biosynthetic enzymes, and pORF2, which contains the crtE gene encoding GGPP synthase, were used for color production to test GGPP synthase activity [14]. 2.3. Genomic DNA preparation and Southern blot analysis Genomic DNA was prepared from human blood cells by Qiagen Genomic-tip (Qiagen Inc.). Genomic DNA (15 Wg in each case) was digested with restriction enzymes BamHI and EcoRI. 0.1 Wg cDNA of human GGPS1 was used as a probe. Southern blots were performed by standard methods as described before [13]. For hybridization and signal detection, the AlkPhos direct system (Amersham Life Science Ltd.) was used according to the attached protocol. In this system, the hybridization temperature was set at 55³C. 2.4. Construction of plasmids for expression of GGPP synthase The inserts of plasmids pAA254082 and pH67793 were cloned into the EcoRI-NotI and PacI-EcoRI sites of the modi¢ed pT7T3D vector, respectively.
C
Fig. 1. Alignment of the amino acid sequences of geranylgeranyl diphosphate synthases and farnesyl diphosphate synthases. Abbreviations of species names, and the GenBank accession numbers are follows: MMGG, Mus musculus GGPP synthase, AB016044; HSGG, Homo sapiens GGPP synthase, AB016043; DMGG, Drosophila melanogaster, AF049659; SCGG, Saccharomyces cerevisiae GGPP synthase, U31632; NCGG, Neurospora crassa GGPP synthase, U20940; GFGG, Gibberella fujikuroi GGPP synthase, X96943; SAGG, Sulfolobus acidocaldarius GGPP synthase, D28748; HSFP, Homo sapiens FPP synthase, J05262; RRFP, Rattus rattus FPP synthase, M34477. Filled boxes indicate the presence of conserved residues in more than ¢ve out of nine sequences. The ¢rst (I) and second (II) aspartate-rich motifs are shown by underlining. Numbers on the right side indicate the position of amino acid residues.
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and 25 Wg/ml chloramphenicol. Chloramphenicoland ampicillin-resistant colonies were replicated on LB plates containing antibiotics and 0.1 mM of IPTG, and then incubated for 3^4 days at 28³C. 2.6. GGPP synthase activity and reaction product analysis
The A1 oligonucleotide primer (5P-GTGGATCCTATGGAGAAAACTAAAGA-3P; sense) for pAA254082 or the H1 oligonucleotide primer (5P-CAGGATCCTATGGAGAAGACTCAAGA-3P; sense) for pH67793 and the universal primer (5PGTAAAACGACGGCCAGT-3P; antisense), which anneals with the downstream sequence of the cloning site within the pT7T3D vector, were used to amplify the GGPS genes by polymerase chain reaction (PCR). PCR fragments were digested with BamHI and NotI, and then cloned into the same sites of pBluescript KS3 to give pBAA for mouse and pBH for human.
GGPP synthase activity was measured by the method described previously [15], in which incorporation of [1-14 C]IPP into reaction products is detected. E. coli DH10B harboring pBAA, pBH, pGGPS6-1 or pBluescript KS3 was incubated to late log phase in LB medium containing 50 Wg/ml ampicillin at 37³C. Cells were harvested by centrifugation, suspended in bu¡er A (100 mM potassium phosphate (pH 7.4), 5 mM EDTA, 1 mM 2-mercaptoethanol), and ruptured by sonication 6 times for 30 s at 30-s intervals in an ice bath. After centrifugation of the homogenate, the supernatant was used as a crude enzyme extract. The assay reaction mixture contained 1.0 mM of MgCl2 , 0.1% (w/v) of Triton X-100, 50 mM of potassium phosphate bu¡er (pH 7.5), 10 WM of [1-14 C]IPP (speci¢c activity 0.92 TBq/mol), 5 WM of FPP and 200 Wg of crude extract containing the enzyme in a ¢nal volume of 0.4 ml. Sample mixtures were incubated for 120 min at 30³C. Reaction products such as prenyl diphosphates were extracted with 1-butanol saturated water, and hydrolyzed with acid phosphatase [16]. The products of hydrolysis were extracted with hexane and analyzed by reversed phase thin layer chromatography with acetone/water (19:1, v/v). Radioactivity on the plate was detected with an imaging analyzer BAS1500Mac (Fuji Film Co.). The plate was exposed to iodine vapor to detect the spots of the marker prenols.
2.5. Carotenoid expression test using the crt gene cluster
3. Results
Fig. 2. Southern blot analysis using human genomic DNA. Genomic DNA was digested with BamHI or EcoRI, as described in Section 2.3. The sizes of markers (kb) are shown on the left. Lane 1, BamHI; lane 2, EcoRI.
E. coli DH10B was transformed with pACCAR25vcrtE which contains the gene cluster crtX, crtY, crtI, crtB and crtZ encoding carotenoid biosynthetic enzymes, and plated on LB medium plates containing 25 Wg/ml of chloramphenicol. A transformant was retransformed with pBAA, pBH, pGGPS6-1 (a positive control) or pBluescript KS3 , and plated on plates containing 50 Wg/ml ampicillin
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3.1. Isolation and sequence analysis of GGPP synthase genes from mouse and human A number of GGPP synthase genes have been isolated from various organisms, and conserved amino acid sequence motifs in these GGPP synthases were observed [2,17]. To isolate and identify GGPP synthase genes from animals, we searched the EST data-
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Fig. 3. Carotenoid color production test in E. coli harboring both pACCAR25vcrtE and the plasmid expressing the GGPP synthase gene from mouse or human. The E. coli strain DH10B containing the carotenoid biosynthetic gene cluster (pACCAR25vcrtE) with pBluescript KS3 (A); pORF2 (B), which carries the crtE gene from E. uredovora; pBAA (C), which carries the GGPS1 from mouse; and pBH (D), which carries the GGPS1 gene from human, were grown for 3^4 days at 28³C.
base using Arabidopsis and fungus GGPP synthases as the template at the National Center for Biotechnology Information (NCBI) using the BLAST program. Interestingly, many clones with conserved sequence motifs to GGPP synthases were found in both the mouse and human EST databases. We selected a mouse cDNA clone (accession number AA254082) and a human cDNA clone (accession number H67793), and named them pAA254082 and pH67793, respectively, after obtaining them from Genome Systems Inc. Since only short sequences of these EST clones were known, we determined their complete sequence. Both the mouse cDNA and human cDNA were 903 bp in size, and were deposited in the DDBJ with the accession numbers AB016044 and AB016043, respectively. We designated both clones GGPS1. Mouse GGPS1 and human GGPS1 were 88.6% identical at the DNA level and their translated products were 94% identical at the amino acid level. Mouse and human GGPS1 displayed high similarity (45^50%) with GGPP synthases from G. fujikuroi [3], N. crassa [4] and S. cerevisiae [5], but were less similar (22^31%) to other GGPP synthases from bacteria and plants. Mouse and human GGPS1
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showed 22% similarity with FPP synthase from human [18] and rat [19], which share a high level of identity with each other at the amino acid sequence level (85%). In the mouse and human GGPS, the third aspartate residue in the second aspartate-rich motif (Fig. 1) is replaced by an asparagine, and the arginine residue found in the third position from the carboxy-terminus of most polyprenyl diphosphate synthases was replaced by a glutamate. The GGPP synthase from S. acidocaldarius and one of the six GGPP synthases from Arabidopsis [6] had the same amino acid, glutamate. These amino acid compositions were not found in other farnesyl or long-chain polyprenyl diphosphate synthases. GGPP synthase puri¢ed from bovine brain was reported to be a homo-oligomer (150^195 kDa) with a molecular mass of 37.5 kDa for the monomer [12]. This is consistent with the calculated molecular mass (35 kDa) of mouse and human GGPP synthases. 3.2. Southern blot analysis of GGPP synthase genes Southern blot analysis under low stringency conditions was performed using the 1.0 kb fragment of
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coli DH10B harboring pACCAR25vcrtE which contains the gene cluster crtX, crtY, crtI, crtB and crtZ, but not crtE which encodes GGPP synthase. Both transformants produced the expected yellow color pigment (Fig. 3), indicating that pBAA and pBH can substitute for the function of the crtE gene. These results suggest that the genes from mouse and human encode functional GGPP synthases.
Fig. 4. A reversed phase thin layer chromatogram of the products of GGPP synthase activity in E. coli. The enzyme reaction was carried out using [1-14 C]IPP and FPP as substrates and cell extract as crude enzyme. The reaction products were hydrolyzed by acid phosphatase and the resulting alcohols was analyzed by reversed phase thin layer chromatography. Arrowheads indicate the positions of the di¡erent alcohol products: GG, all-E-geranylgeraniol ; O, octaprenol; S.F., solvent front; Ori, origin. Lane 1, DH10B/pBluescript KS3 ; lane 2, DH10B/pORF2 ; lane 3, DH10B/pBAA; and lane 4, DH10B/pBH.
human GGPS1 to probe human genomic DNA digested with BamHI or EcoRI. This resulted in the appearance of bands of 5.0 kb and 2.2 kb for the BamHI and EcoRI digests, respectively (Fig. 2). We did not see any other bands under these conditions. This result suggests that the human GGPS1 gene is a single copy gene. 3.3. Detection of GGPP synthase activity using the carotenoid color production test in E. coli To determine if the mouse and human genes are functional, we tested their GGPP synthase activity by the carotenoid formation test. E. coli cells transformed with a plasmid containing the crtE, crtX, crtY, crtI, crtB and crtZ genes from Erwinia uredovora are able to accumulate zeaxanthin, which has a yellow color, only if GGPP is produced in E. coli. The genes from mouse and human were cloned into pBluescript KS3 to create a lacZ fusion, which yielded pBAA and pBH, respectively. The expression plasmids pBAA and pBH were transformed into E.
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Fig. 5. The phylogenetic relationship between known GGPP synthases. XXGG indicates GGPP synthase and XXFP indicates FPP synthase. The abbreviations of species names and the GenBank accession numbers that were not shown in Fig. 1 are as follows: MT, Methanobacterium thermoautotrophicum, S75695; AT, Arabidopsis thaliana, AB000835; CA, Capsicum annuum, X80267; CR, Catharanthus roseus, X92893; LA, Lupinus albus, U15778 ; EH, Erwinia herbicola, M87280; EU, Erwinia uredovora, D90087. The amino acid sequences were aligned by the DDBJ CLUSTAL W program [24]. The numbers were obtained by bootstrap analysis (out of 100).
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3.4. In vitro analysis of GGPP synthase activity from mouse and human In order to demonstrate GGPP synthase activity directly, crude extracts from E. coli expressing the GGPS1 genes from mouse and human were examined for GGPP synthase activity using [1-14 C]IPP and FPP as substrates. The reaction products were hydrolyzed by acid phosphatase and analyzed by reversed phase thin layer chromatography. While octaprenyl diphosphate synthesized by the endogenous IspB gene product [20] was detected in E. coli DH10B harboring the vector alone (Fig. 4, lane 1), geranylgeraniol resulting from GGPP activity was detected in DH10B harboring pBAA (Fig. 4, lane 3) and pBH (Fig. 4, lane 4). These results indicated that the genes from mouse and human produced GGPP as a result of condensation of IPP and FPP as the allylic substrates. 4. Discussion In this study, we have identi¢ed the GGPS1 genes encoding geranylgeranyl diphosphate synthase from mouse and human. Two lines of evidence demonstrate that these genes encode GGPP synthases. One is the direct measurement of GGPP synthase activity and the other is the indirect detection of GGPP synthase activity in E. coli strains in the form of carotenoid pigmentation. The latter method was shown to be an e¤cient method for the detection of GGPP synthase activity in our previous work [14,21]. The sequences of GGPS1 genes from mouse and human show them to be closely related to their eukaryotic counterparts, but not to plant and bacterial GGPP synthases. The phylogenetic analysis shown in Fig. 5 suggests that there are two types of GGPP synthase. One is of eukaryote origin and includes mouse and human genes, and the other is of prokaryote origin. The eukaryotic type of GGPP synthases which form one cluster (Fig. 5) has the DDXXN motif but not DDXXD in the second aspartate-rich motif. The archaebacterial S. acidocaldarius GGPS which possesses the DDXXG motif has been classi¢ed in the other cluster because it is likely to be of
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bacterial origin and to have a lower level of similarity with eukaryotic GGPP synthases. Plants have many di¡erent types of GGPP synthase such as Arabidopsis which has six GGPS genes [21]. But so far only one GGPP synthase gene has been identi¢ed in mouse and human as shown in this study. When we again searched the current EST database using the entire GGPS1s from mouse and human as probes, 64 and 15 clones from mouse and human, respectively, gave a high score. Taking into consideration the level of sequence error in the EST sequences, it seems that all of these clones are either of mouse or human GGPS1 gene origin. We did not ¢nd any other types of clone with the GGPS motif in the EST database. Southern blot analysis suggests that the human GGPS1 is a single copy gene. Thus, only one type of animal GGPP synthase gene appears to exist, which is in contrast to the many GGPP synthase genes found in plants. It is a somewhat surprising gene organization for higher eukaryotes if human only contains a single gene of GGPS. Rat GGPS activity was detected in the cytosol [11] and consistent with this, PSORT program analysis of the sequences of mouse and human GGPS1 indicates that they are localized in the cytoplasm. Recently, GGPP was shown to regulate cell cycle progression in human lymphocytes [22] and geranylgeraniol, which is the alcohol form of GGPP, was found to induce apoptosis of tumor cells [23]. The GGPP synthase genes of mouse and human identi¢ed here should prove useful for the study of many aspects of cellular regulation such as protein geranylgeranylation, cell cycle progression and apoptosis. 5. Note Recently, the GGPP synthase gene from human was reported by Ericsson et al. [25], while we were submitting this manuscript. We saw some sequence discrepancy between ours and theirs. Acknowledgements This work was supported by a Grant-in-Aid from
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the Ministry of Education, Science and Culture of Japan. [15]
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