Báo cáo Y học: Identification of mammalian-type transglutaminase in Physarum polycephalum Evidence from the cDNA sequence and involvement of GTP in the regulation of transamidating activity potx

Identification of mammalian-type transglutaminase in Physarum polycephalum Evidence from the cDNA sequence and involvement of GTP in the regulation of transamidating activity Fumitaka Wada 1 , Akio Nakamura 2 , Tomohiro Masutani 1 , Koji Ikura 3 , Masatoshi Maki 1 and Kiyotaka Hitomi 1 1 Department of Applied Biological Sciences, Graduate School of Bioagricultural Sciences, Nagoya University, Chikusa, Nagoya, Japan; 2 Department of Pharmacology, Gunma University School of Medicine, Gunma, Japan; 3 Department of Applied Biology, Faculty of Textile, Kyoto Institute of Technology, Kyoto, Japan Transglutaminase (TGase) catalyses the post-translational modification of proteins by transamidation of available glutamine residues. While several TGase genes of fish and arthropods have been cloned and appear to have similar structures to those of mammals, no homologous gene has been found in lower eukaryotes. We have cloned the acel- lular slime mold Physarum polycephalum TGase cDNA using RT-PCR with degenerated primers, based on the partial amino acid sequence of the purified enzyme. The cDNA contained a 2565-bp ORF encoding a 855-residue polypeptide. By Northern blotting, an mRNA of  2600 bases was detected. In comparison with primary sequences of mammalian TGases, surprisingly, significant similarity was observed including catalytic triad residues (Cys, His, Asn) and a GTP-binding region. The alignment of sequences and a phylogenetic tree also demonstrated that the structure of P. polycephalum TGase is similar to that of TGases of vertebrates. Furthermore, we observed that the purified TGase had GTP-hydrolysing activity and that GTP inhib- ited its transamidating activity, as in the case of mammalian tissue-type TGase (TGase 2). Keywords:GTP;GTPase;Physarum polycephalum;trans- glutaminase. Transglutaminase (TGase; EC 2.3.2.13) catalyses cross- linking between the c-carboxyamide of glutamine residues and the e-amino group of lysine residues or other primary amine. The reaction leads to the formation of an isopeptide bond between two proteins and the covalent incorporation of polyamine into proteins [1,2]. In mammals, TGases have a wide distribution in various organs, tissues, and body fluids, suggesting that they participate in a vast array of physiological processes. Cross-linking reactions are involved in clot formation, apoptosis, embryogenesis, angiogenesis, and skin formation [3–8]. Similar cross-linking has also been found in invertebrates, plants, unicellular eukaryotes, and bacteria [9,10]. In vertebrates and some invertebrates, Ca 2+ is required for the enzymatic reaction by exposing a cysteine residue in the active site domains, while the bacterial enzyme is not Ca 2+ dependent [11]. This suggests that there are structural differences responsible for the catalytic reactions in different organisms. In humans, nine isozymes of TGase have been found, and they form a large protein family [12]. In other mammals, several isozymes have also been found, and the primary sequences appear to be significantly similar, suggesting that these TGases evolved from a common ancestor gene. Among these TGases, tissue-type TGase (TGase 2), which is distributed ubiquitously, has been studied exten- sively [13–15]. In addition to its protein cross-linking activity, TGase 2 appears to have other functions. While GTP inhibits transamidating activity, TGase 2 also shares GTP-hydrolysing activity [16–19]. TGase 2 has been shown to function as a signal-transducing GTP-binding protein that couples activated receptors, resulting in stimulation of the effector enzyme [20,21]. Furthermore, TGase 2 was found to be to localized at the cell surface and to mediate the interaction of integrin with fibronectin [22,23]. The physio- logical significance of these multifunctional roles of TGase 2 is currently under investigation. TGase cDNAs have been isolated from other lower vertebrates, such as fish, and the genes have been found to have structural similarity with those of mammalian genes [24,25]. TGase cDNAs of a few invertebrates, such as ascidians [26], grasshopper (annulin) [27], and limulus [28], have also been cloned. While the structures of these genes have been shown to be homologous to those of the mammalian gene, a TGase with a structure similar to a mammalian-type has not been found in lower invertebrates such as Caenorhabditis elegans. On the whole, protein Correspondence to K. Hitomi, Department of Applied Biological Sciences, Graduate School of Bioagricultural Sciences, Nagoya University, Chikusa, Nagoya, 464-8601, Japan. Fax: + 81 52 789 5542, Tel.: + 81 52 789 5541, E-mail: hitomi@agr.nagoya-u.ac.jp Abbreviations: TGase, transglutaminase; PpTGase, Physarum polycephalum transglutaminase; PLC, phospholipase; AMV, avian myeloblastosis virus. Enzyme: transglutaminase (EC 2.3.2.13). Note: The nucleotide sequence of Physarum polycephalum TGase in this paper has been submitted to the DDBJ/EMBL/GenBank under accession number AB076663. (Received 4 February 2002, revised 24 May 2002, accepted 29 May 2002) Eur. J. Biochem. 269, 3451–3460 (2002) Ó FEBS 2002 doi:10.1046/j.1432-1033.2002.03026.x disulfide isomerase has been reported to play a role in transamidating activity in C. elegans and phylarial parasites [29–31]. TGase genes in bacteria have also been cloned, but the sequences were found to be completely different from those of mammalian genes [32–34]. In Escherichia coli, cytotoxic necrotizing factor 1 possesses TGase activity to deamidate Rho A [35]. The results of those studies suggest that the lower eukaryotes and bacterial enzymes evolved as a separate lineage from the mammalian TGases. The physiological roles of these invertebrate and bacterial TGases also remain unclear. Physarum polycephalum is a true slime mold and has been used mainly in studies of cell motility [36,37]. This is one of the lowest eukaryotes with a unique life cycle that is characterized by spores, amoebae, and plasmodia. The plasmodia are giant, multinuclear cells in which vigorous cytoplasmic streaming is observed. Starvation of macro- plasmodia causes differentiation into sporangia, which undergo meiosis to form haploid spores. Germinating spores form amoebae, which can fuse to produce diploid plasmodia. Although there have been reports on identifica- tion and purification of P. polycephalum TGase, no struc- tural information has been presented [38,39]. To find out more about lower eukaryote TGases, their physiological roles, and evolutionary relationship to other TGases, we attempted the molecular cloning of P. poly- cephalum TGase (PpTGase). In this study, based on the partial amino acid sequences of the purified enzyme, a cDNA clone encoding PpTGase was isolated. Unexpected- ly, the primary structure deduced from its cDNA sequence appeared to be significantly similar to those of mammalian TGases. Furthermore, GTP inhibited the enzymatic activity of PpTGase, which also displayed GTP-hydrolysing activ- ity. We conclude that P. polycephalum is the lowest organism that has characteristics of mammalian TGase 2. MATERIALS AND METHODS Culture of plasmodia Plasmodia of P. polycephalum (strain Ng-1) were grown on Quaker Oatmeal (Quaker Oats Company, Chicago, IL, USA) in the dark [37]. The migrating sheets of plasmodia were collected and used for experiments. Purification of PpTGase Purification was performed essentially as described by Mottahedeh and Marsh with some modification [39]. All procedures were performed at 4 °C. The plasmodia growing as migrating sheets were collected and washed twice with a solution of 0.4% glycerol, 20 m M sodium citrate, 10 m M NaPO 4 (pH 5.0). After suspension in 2.5-pellet vols of TEN buffer (20 m M Tris/HCl, 2 m M EDTA, 80 m M NaCl, 5 m M 2-mercaptoethanol, 0.2 m M phenylmethanesulfonyl fluor- ide, pH 8.0), the cells were homogenized. The homogenate was centrifuged at 10 000 g for 20 min, and the supernatant was further centrifuged at 100 000 g for 40 min. Strepto- mycin sulfate was slowly added to the resultant supernatant to a final concentration of 2 mgÆmL )1 , and the mixture was placed on ice for 30 min. Insoluble material was removed by centrifugation at 20 000 g for 15 min. The supernatant was mixed with an equal volume of 10% glycerol and then applied to a DEAE-cellulose column (Amersham Pharma- cia Biotech) equilibrated with buffer A (40 m M NaCl, 10 m M Tris/HCl, 2.5 m M 2-mercaptoethanol, pH 8.0). The column was washed with  1 column vol. buffer A contain- ing 5% glycerol. CaCl 2 was added to the flow-through fraction to a final concentration of 1.2 m M , and this solution was passed through a phenyl–Sepharose column (Amersham Pharmacia Biotech) equilibrated with buffer A containing 0.5 m M CaCl 2 . The column was washed with 4 col. vol. equilibration buffer containing 10% glycerol followed by 4 col. vol. equilibration buffer containing 80 m M NaCl and 10% glycerol. Bound proteins were eluted with 80 m M NaCl, 20 m M Tris/HCl, 2 m M dithiothreitol, 2 m M MgCl 2 ,1m M EDTA, 10% glycerol, pH 8.0. The eluted solution was concentrated using a Centricon-50 concentrator (Millipore) and then fractionated by gel filtration using a Superdex-200 column (Amersham Pharmacia Biotech) for further purifi- cation. The fractions with TGase activity were collected, concentrated, and used for the experiments. Samples were analysed by SDS/PAGE in a 7.5% acrylamide gel and stained with Coomassie brilliant blue. Cleavage of the PpTGase with CNBr and amino acid sequencing The purified TGase was dissolved in 70% formic acid and treated with CNBr at room temperature for 24 h in the dark. The reaction product was separated by SDS/PAGE in a subjected to 12.5% acrylamide gel and transferred to poly(vinylidene difluoride) membrane (Millipore). Protein bands of interest were excised and sequenced by automated Edman degradation. 3¢ RACE 3¢ RACE was performed using an RNA LA PCR TM Kit (AMV) Verson 1.1 (TAKARA Biomedicals, Tokyo, Japan). Total RNA from plasmodia was obtained by the acid guanidium/phenol/chloroform (AGPC) method. The first-strand cDNA was synthesized using 1 lgtotalRNAin a reaction mixture of 0.5 m M dNTPs, 40 U RNasin, 4 U avian myeloblastosis virus (AMV) reverse transcriptase, and an oligo dT-adaptor primer in the buffer supplied. The resulting cDNAs were subjected to PCR with M13 primer M4 and a degenerated primer, 5¢-GTTCCTATCACC GCCGT(A/T/G/C)AA(A/G)GT(A/T/G/C)GG(A/T/G/C) GA(A/G)AA-3¢, which was designed on the basis of amino acid sequence, VPISAVKV GEK. Amplification conditions were as follows: 30 cycles at 94 °Cfor0.5min,55°C for 1 min, and 72 °C for 1 min. Using the reaction products as a template, nested PCR was performed with M4 and another degenerated primer, 5¢-AA(A/G)GT(A/T/ G/C)GG(A/C/T)GA(A/G)AA(A/G)GG(A/T/G/C)AT-3¢, designed from the amino acid sequence, KVGEKGI. The amplification conditions were as follows: 30 cycle at 94 °C for 0.5 min, 52 °Cfor1min,and72°C for 1 min. The PCR products obtained from 3¢ RACE was cloned into a TA-cloning vector pCR-TOPO (Invitrogen, USA) accord- ing to the manufacturer’s instructions. The nucleotide sequences of the isolated clones were determined with an automated fluorescent sequencer, ABI PRISM 310 (PE Applied Biosystems), using a Bigdye TM terminator cycle sequencing ready reaction kit (PE Applied Biosystems). 3452 F. Wada et al. (Eur. J. Biochem. 269) Ó FEBS 2002 5¢ RACE 5¢ RACE was performed using reverse transcriptase and RNA ligase, according to the manufacturer’s protocols (5¢-Full RACE Core Set, TAKARA Biomedicals, Tokyo, Japan) [40]. First-strand cDNA was synthesized from 0.35 lg poly(A) + RNA and purified with an oligo(dT) cellulose column using an AMV reverse transcriptase XL with a specific primer, 5¢-GCGAGCATTGGTGCCTACA G-3¢ (antisense, nucleotide sequence positions 1857–1876), which was phosphorylated by T4 polynucleotide kinase. After degradation of the template poly(A) + RNA with RNase H at 30 °C for 1 h, the resulting single-stranded cDNA was precipitated with ethanol and dissolved in 40 lL of a reaction mixture containing 20% poly(ethylene glycol) 6000, RNA ligation buffer, and 1 U T4 RNA ligase. To change the cDNAs to circular and/or concatamer cDNAs, the reaction solution was incubated at 22 °C for 16 h. The cDNAs were used directly as a template for the first PCR amplification with primers 5¢-GGCGGATATAGACTTG TCAGG-3¢ (sense, 1796–1816) and 5¢-CTCGTCAGCATT CACTTCCG-3¢ (antisense, 1752–1771), which correspond to the cDNA sequence obtained by 3¢ RACE. The reaction was carried out for 25 cycles under the following conditions: 94 °C for 30 s, 55 °C for 30 s and 72 °C for 1 min. The resulting PCR product was diluted 100-fold with sterile H 2 O, and a 1-lL aliquot was used as a template for the second nested PCR amplification with primers 5¢-GGA CAATTACAGATTCAGTGGGAAAG-3¢ (sense, 1815– 1840) and 5¢-CGAGTATACGAAATCGATGTCGTAG- 3¢ (anti sense, 1729–1753) under the same conditions. In the second 5¢ RACE, first-strand cDNA was synthesized with another oligonucleotide primer, 5¢-CCCCTCCTAATAGC GAAGAA-3¢ (antisense, 692–711). The first PCR amplifi- cation was performed with gene-specific primers: 5¢-GGTC ATTCAGTCGATCGATTTAC-3¢ (sense, 616–638) and 5¢-TGGAACAACTGGAACGGGTGCTG-3¢ (antisense, 522–544). For the nested PCR, the primers 5¢-CAAGTCG AGAAGAATAGAGC-3¢ (sense, 638–657) and 5¢-CGAG TAAAGGTTTGGTGCCTGTT-3¢ (antisense, 485–507) were used. Cloning and nucleotide sequencing were carried out as described for the 3¢ RACE. Computer analyses Multiple sequence alignment was performed using the CLUSTAL X program released from the European Bioinfor- matics Institute [41], and phylogenetic trees were displayed using the tree-viewing program NJPLOT [42]. Northern blot analysis Total RNA extracted from plasmodia was electrophoresed in a 1% agarose-formaldehyde gel and transferred to a Hybond N + nylon membrane (Amersham Pharmacia Biotech). The membrane was hybridized with a 32 P-labelled probe and washed at 65 °C for normal stringency. Expression of recombinant PpTGase in E. coli Recombinant proteins were produced using partial C-terminal region (PpTGase-C: corresponding to 554Gly)855Val) and full-length cDNA for preparation of antiserum and analysis of PpTGase, respectively. To prepare antiserum against PpTGase-C, we generated a hexahistidine (His 6 )–PpTGase-C fusion protein as an antigen. PCR was performed using the PpTGase cDNA as a template with primers 5¢-CGGGATCCATATGGGACC CGTGCCTATTTCTGCT-3¢ and 5¢-CCGGATCCTTAA ACGACAATAACTTGGGCTTG-3¢. The PCR product was digested with NdeIandBamHIandtheninserted between the same sites of the pET19b vector (Novagen, Madison, USA). E. coli host strain BL21(DE3) trans- formed with the expression plasmid was grown in Luria– Bertani medium to an optical density of 0.5 at 600 nm, and the expression was induced with 1 m M isopropyl thio- b- D -galactoside by cultivation at 37 °C for 3 h. The His 6 –PpTGase-C fusion protein was purified from E. coli according to the manufacturer’s instructions. Antisera were produced in two rabbits by immunization with an emulsion containing approximately 1 mg His 6 –PpTGase-C protein in Freund’s complete adjuvant. Rabbits were inoculated by subcutaneous injection into the shaven back. One mg purified protein in Freund’s incomplete adjuvant was used for subsequent boosts. Three booster injections were given at 2-week intervals after the primary injection. Two weeks after the last immunization, blood was collected from the heart. For expression of the full-length cDNA in E. coli, the same system was used except for the vector. PCR was performed to insert the restriction enzyme sites (BamHI and SalI) at the termini of the amplified DNA with primers 5¢-GTGGATCCTATGACTACCGTATTCTTT-3¢ and 5¢-ATAGTCGACTTAAACGACAATAACTTG-3¢.Next, the resulting DNA fragment was inserted into BamHI and SalI of pET-24d vector (Novagen), which was modified by attaching a His 6 tag at the N-terminus of the expressed protein. For analysis of the expressed protein, harvested cells were washed and lysed in SDS sample buffer. The sample was treated with sonication and heated for 3 min. The sample was analysed by SDS/PAGE on 7.5% acryla- mide gels followed by staining with Coomassie brilliant blue. Using the antiserum, Western blotting was performed by the standard method. Immuno-signals were detected by colour development methods using diaminobenzidine as described before [43]. Assay for TGase activity TGase activity was determined in a microtiter plate assay essentially as described by Slaughter et al. [43,44]. Briefly, each microtiter well was coated with 1% dimethylcasein at 37 °C for 1 h and uncoated sites were blocked with skim milk. A premixed reaction mixture (180 lL) containing 100 m M Tris/HCl (pH 8.0), 10 m M dithiothreitol, 0.5 m M 5-(biotinamido)pentylamine (Pierce Chemical Co., Rock- ford, USA), and CaCl 2 (0.5 m M to 2 m M ) was added to the wells. The reaction was started by adding 20 lLofTGases solution to the premixed solution and then incubating at 37 °C for 1 h. TGase-catalysed conjugation of 5-(biotinam- ido)pentylamine into dimethylcasein was measured by streptavidin-peroxidase, H 2 O 2 , and o-phenylenediamine. An equal volume of 2 M H 2 SO 4 was added, and the absorbance at 450 nm was measured. Ó FEBS 2002 Mammalian-type transglutaminase in Physarum (Eur. J. Biochem. 269) 3453 Effects of nucleotides on the TGase activity GTP solution was added to the TGase solution at a concentration of 25–500 l M . GDP, GMP, and ATP were added at the concentration of 500 l M . These mixtures were preincubated at 0 °C for 1 h in the absence of CaCl 2 and added to the reaction mixture. The TGase activity was measured by the microtiter assay. Assays for GTP hydrolysis by TGase 2, TGase 3, and PpTGase GTP-hydrolysing activity was measured as described pre- viously [18,45]. The guinea pig TGase 2 and the recombin- ant mouse TGase 3 were purified from guinea pig liver and baculovirus-infected insect cells, respectively [13,46]. TGase 3 was proteolysed by dispase to activate the proen- zyme. Two micrograms of TGase 2, 1 lg TGase 3, and 2 lg purified PpTGase were mixed with 15 lCi [c- 32 P]GTP (2 l M ) in a reaction mixture containing 20 m M Tris/HCl (pH 7.5), 5 m M MgCl 2 ,1m M dithiothreitol, 1 m M EDTA. The reaction mixtures were incubated at 37 °Cforthe indicated periods of time, and the reaction was stopped by addition of 7 vol. 5% (w/v) charcoal in 50 m M NaH 2 PO 4 . The mixture was centrifuged at 12 000 g for 7 min. The amount of 32 P released from [c- 32 P]GTP was measured by scintillation counting of clear supernatant solution. RESULTS Purification of PpTGase TGase from P. polycephalum plasmodia cultured as migra- ting sheets was purified on the basis of enzymatic activity (Fig. 1A). After streptomycin sulfate precipitation, cellular protein was applied to an anion-exchange column and the unbound proteins were loaded onto a phenyl–Sepharose column in the presence of Ca 2+ . Almost homogeneous 100-kDa protein was obtained in the fraction eluted with EDTA from the phenyl-sepharose column. This result agreed well with the result reported previously by Mottahedeh & Marsh [39]. During all of the procedures, no other fractions with apparent TGase activities were observed, suggesting that the purified protein is the major TGase in Physarum plasmodia. To obtain highly purified TGase, the contaminating proteins were excluded by gel filtration chromatography using Superdex 200. The estima- ted molecular mass was 130–150 kDa, suggesting that this protein was in a monomeric form. Initially, determination of the amino-terminal amino acid sequence of purified protein was attempted, but no infor- mation was obtained, probably due to the protein modifi- cation. Therefore, the purified protein was treated with CNBr to cleave at the methionine residue. As shown in Fig. 1B, two major fragments, of  60 and  40 kDa, were obtained and subjected to sequencing. A 15-amino acid sequence (GPVPISAVKVGEKGI) was revealed in respect to the 40-kDa fragment, while the 60-kDa fragment provided no result. cDNA cloning and sequence Based on the amino acid sequence of the 40-kDa protein, degenerated primers corresponding to the sequence were designed for 3¢ RACE. cDNA was synthesized using an oligo dT adaptor primer, and then PCR amplification was performed using both M4 and the degenerated primer. A major PCR product of 1200 bp, as an expected size, was obtained. Using the degenerated primers for nested PCR, a defined single 1200-bp DNA was produced. The deduced primary sequence from the nucleotide sequence of the amplified DNA is similar to that of the corresponding position in the mammalian TGase, thus providing evidence that the PCR product is TGase cDNA of P. polycephalum. To obtain a cDNA encoding the 5¢ portion of PpTGase, we carried out 5¢ RACE using specific primers based on the partial cDNA sequence obtained by 3¢ RACE. A single PCR product of 1600 bp was produced by two successive reactions. In the amino acid sequence deduced from the amplified cDNA sequence, a 15-amino acid sequence, which was determined by protein sequencing, was observed (Fig. 2, grey background). Although the predicted amino- acid sequence had similarity with the sequences of mam- malian TGase, the length of the cDNA was smaller than the length deduced from the molecular size of the purified protein. Furthermore, an initiation codon was not observed in the sequence obtained. Therefore, we performed a further 5¢ RACE in order to obtain a cDNA encoding the 5¢ upper region. The resulting product, which was 700 bp in length, revealed novel 83 bp sequences that included a putative initiation codon and part of the 5¢ untranslated region. Finally, a full-size composite cDNA sequence encoding PpTGase was obtained from the nucleotide sequences of the three RACE products. The full-length cDNA of PpTGase was 2624 bp long and contained 22- and 34-bp noncoding regions at the 5¢ and 3¢ ends, respectively. One polyadenylation signal (AATAAA) wasobservedinthe3¢ untranslated region. The complete sequence shows an ORF of 2565 bp corresponding to 855 amino acids with a molecular mass of 93 611 Da (Fig. 2). Fig. 1. Purification and cleavage of PpTGase. (A) Approximately 1–5 lg protein from each step in the purification procedure was separated by SDS/PAGE on 7.5% acrylamide gels followed by staining with Coomassie brilliant blue: molecular mass markers (lane M); total cellular extract (lane 1); soluble fraction (lane 2); supernatant fraction after streptomycin sulfate precipitation (lane 3); flow-through fraction of DEAE–Sephacel chromatography (lane 4); eluted fraction of phenyl sepharose chromatography (lane 5); and peak fraction from size separation (Superdex 200) (lane 6). The arrow indicates the position of PpTGase. (B) Purified PpTGase was treated with CNBr and separated by SDS/PAGE on 12.5% acrylamide gels. Arrows indicate the fragments analysed for amino-acid sequencing. 3454 F. Wada et al. (Eur. J. Biochem. 269) Ó FEBS 2002 That sequence included a Cys active site, and the other two critical residues for catalytic activity, His and Asp, were also observed. Both putative GTP-binding (Tyr345–Phe359) and Ca 2+ binding (Val613–Arg635) regions, which have been identified in human TGase 2, were found (Fig. 2). Next, we aligned the PpTGase sequence with other TGases with respect to the middle region around the GTP- binding region, active site, and Ca 2+ -binding region, which are highly homologous among many TGases (Fig. 3). The amino acid sequences of PpTGase were 40–50% identical to those of human TGase 2 [14] and TGases of red sea bream [24], ascidians [26], grasshopper [27], fruit fly, and limulus [28].Ser,whichisanessentialaminoacidresidueforGTP binding, is also conserved (region A in Fig. 3). In respect to the outside regions of A, B, and C, PpTGase showed low but significant similarity to human TGase 2 except for the presence, in the PpTGase, of a long amino-terminal region that is missing in the human enzyme. Furthermore, in order to clarify the molecular evolutionary relationship of PpTGase, we made a phylogenetic tree using the CLUSTAL X program based on the full-length amino-acid sequences (Fig. 4). When aligned according to the middle region with high homology among the various TGases (from the front of region A to the end of region C in Fig. 3), a similar phylogenetic tree was drawn (data not shown). Band 4.2, which is an enzymatically inactive TGase-like protein found in erythrocytes, located at a far position. PpTGase was situated closer to the other invertebrate TGases than to human and fish TGases. Among human TGases, however, TGase 4 was placed significantly close to PpTGase. Northern blotting We performed Northern blot analysis using total RNA prepared from plasmodia. As shown in Fig. 5A, a single band was observed at the size of  2600 nucleotides. This length agrees well with that of the PpTGase cDNA obtained. No other RNA hybridized even under lower stringency hybridization conditions, such as lower tempera- ture (data not shown). Western blotting To confirm that we had obtained the full-length cDNA, recombinant protein was produced in E. coli and analysed. As the polyclonal antibody had been raised against the C-terminal portion of the PpTGase, Western blotting analysis was performed in respect to the recombinant protein and PpTGase in the plasmodial lysate as well as the purified PpTGase (Fig. 5B and C). The recombinant PpTGase protein was successfully expressed at the molecu- lar weight of 100 kDa (Fig. 5C, lane 2). No difference in size was observed between the purified protein and PpTGase in plasmodia lysate, suggesting that PpTGase was not degraded during the purification procedure (Fig. 5C, lanes 3 and 4). The recombinant protein and the PpTGase protein from Fig. 2. cDNA and deduced amino-acid sequences of PpTGase. A complete amino-acid sequence of PpTGase was deduced from the nucleotide sequence. The numbers of nucleo- tides and amino-acid residues are shown on the left and right sides, respectively. The grey background indicates the amino acid sequence determined from the CNBr fragment. The asterisk indicates the stop codon. Three amino acid residues of the catalytic triad are boxed. The single and double lines indicate the putative Ca 2+ - and GTP-binding sites, respectively. Ó FEBS 2002 Mammalian-type transglutaminase in Physarum (Eur. J. Biochem. 269) 3455 plasmodia migrated to similar positions probably because of the attachment of the hexahistidine, the recombinant PpTGase protein appeared to be slightly larger. These results indicate that the cDNA obtained covered the entire coding region. Involvement of GTP in the regulation of TGase function In the deduced primary sequence, we found a putative GTP-binding site [21]. This prompted us to investigate the relationship of nucleotides to regulation of the tran- samidating activity, which has been extensively studied in respect to TGase 2. As we have not yet been able to produce a soluble recombinant protein, experiments were performed using completely purified TGase protein from P. polyceph- alum plasmodia (Fig. 1, lane 6). First, the inhibitory effect of GTP on enzymatic activity was analysed with various concentrations of Ca 2+ , as shown in Fig. 6A. At 0.5 m M Ca 2+ , the enzymatic activity was apparently decreased by the addition of 100–500 l M GTP. In the presence of 1 m M Ca 2+ , an inhibitory effect was observed only at a higher level of GTP. In the case of 2m M Ca 2+ , inhibition by GTP was not observed. These results suggest that the TGase activity is regulated by the presence of GTP and Ca 2+ . Additionally, in order to confirm the specificity of the inhibition, other purine nucleotides (GTP, GDP, GMP, and ATP) were examined at 0.5 m M Ca 2+ . Fig. 6B shows the relative enzymatic activity in the presence of the nucleotides. GTP and ATP clearly inhibited the activity, while GDP showed weak inhibition. GMP did not block the enzymatic activity. Next, GTP-hydrolysing activity of the purified PpTGase was investigated. Mammalian TGase 2 has both transam- idating and GTP-hydrolysing activities, whereas TGase 3 has no GTPase activity. These proteins were incubated with 32 P-GTP, and then the release of 32 P was measured. The amount of radioactivity released by guinea pig TGase 2 and PpTGase increased up to 60 min in a time-dependent fashion (Fig. 7) and also depended on the amounts of the proteins (data not shown). Although the hydrolysing activity was weaker than that of TGase 2, PpTGase had an apparent GTP-hydrolysing activity. DISCUSSION cDNA sequence of PpTGase Although the overall identity with the mammalian TGase primary sequence is low in the deduced sequence of the Physarum TGase, the middle region of the sequence is significantly conserved. The sequences around the GTP- binding region, catalytic site, and Ca 2+ -binding region are highly homologous to the corresponding regions of the human TGase 2 and the other invertebrate TGases (Fig. 3). The eight amino-acid residues surrounding the active site Cys (region B in Fig. 3) except those of Drosophila melanogaster TGase (the sequence of which was predicted from the database; accession number AAF52590), are identical. In addition to this catalytic Cys site, His and Asp, which comprise a catalytic triad with Cys, are also conserved. Furthermore, a putative Ca 2+ -binding region reported in mammalian TGase 2 was also found [15]. This is consistent with the finding that Ca 2+ was required for the enzymatic activity of PpTGase. These findings suggest that an acyl-transfer reaction identical to that of mammalian TGases is executed in the catalytic reaction of PpTGase. Compared with those of human TGase 2, an additional region exists at the amino terminus of PpTGase, which is not highly conserved. Among human TGases, keratino- cyte-type TGase (TGase 1) contains such a longer amino Fig. 3. Alignment of highly similar regions of PpTGase with various eukaryote TGases. In the upper panel, regions of human TGase 2 and PpTGase that are very similar are shaded. Alignment was performed with respect to the selected sequences around the following regions: A, GTP-binding region; B, catalytic site; C, Ca 2+ -binding region. The amino-acid sequences were aligned by using the CLUSTAL X program. Gaps indicated by hyphens have been introduced to improve the sequence alignments. Conserved amino acid residues are shaded. The dark-shaded S (region A) and C (region B) indicate essential amino acid residues for GTP binding and catalytic reaction, respectively. The numbers represent the amino acid residue numbers of the TGases: human TGase 2, red sea bream (Pagrus major), ascidians (Ciona intestinalis), grasshopper (Schistocerca americana), fruit flies (D. melanogaster), limuli (Tachypleus tridentatus), and slime mold (P. polycephalum). With respect to the corresponding sequence to the Drosophila TGase, cDNA sequence was searched from database with the TBLASTN search engine to identify cDNA with homology to vertebrate TGases (accession no. AAF52590). 3456 F. Wada et al. (Eur. J. Biochem. 269) Ó FEBS 2002 terminus that is required for binding to the plasma membrane, thereby being involved in the formation of the corneum [47]. Additional amino-terminal residues of TGase 1 include the sequences that are post-translationally modified by fatty acid chains conferring membrane associ- ation, however, we could not find such a primary sequence. Similar long amino-terminal sequences are also found in ascidian, grasshopper, and Limulus TGases, suggesting a common characteristic of nonmammalian TGases [26–28]. Several gene structures responsible for enzymatic activity have been reported in various organisms. TGases with homologous primary structure have been cloned in fish and some invertebrates such as red sea bream [24], salmon [25], zebrafish (C. Rodolfo et al. Abstracts in the 6th Interna- tional Conference on Transglutaminase and Protein Cross- linking Reactions, Lyon, France, 2000), ascidians [26], Fig. 5. Northern and Western blot analysis of PpTGase. Northern blot analysis of total RNA from Physarum plasmodia was performed using thefull-lengthPpTGasecDNAasaprobe(A).Lane1,5lg; lane 2, 10 lg. The arrow indicates the transcripts of PpTGase. Mouse ribo- somal RNA was used as a size marker. Analyses of the recombinant and the plasmodial PpTGases were performed by SDS/PAGE on 7.5% acrylamide gels (B) and Western blotting (C). Lane 1, cellular protein of E. coli transformed with a control vector; lane 2, cellular protein of E. coli transformed with the vector harbouring PpTGase cDNA; lane 3, cellular protein of Physarum plasmodia; lane 4, purified PpTGase from Physarum plasmodia. Lane M, molecular mass marker. In lane 2, to reduce the recombinant PpTGase proteins in the E. coli lysate sample the lysate of E. coli expressing PpTGase ( 5% of the total cell protein) was diluted 50-fold with that of E. coli harbouring pET-24d (negative control, lane 1). In lane 4 of (C), the sample in (B) was diluted 20-fold with SDS buffer. The arrows in (B) and (C) indicate the positions of PpTGase. Fig. 6. Effects of purine nucleotides on the inhibition of PpTGase activity at various Ca 2+ concentrations. The activities of TGase were measured as described in Materials and methods. (A) The cross-linking activities of PpTGase in the presence of 0.5 m M (d), 1 m M (m), or 2 m M CaCl 2 (j) with 0–500 l M GTP are shown. The purified enzyme (0.5 lg) was tested using 5 m M GTPsolutioninanequal volume. (B) The enzymatic activities of PpTGase in the presence of 0.5 m M CaCl 2 with 500 l M nucleotides are shown. Data represent the mean of triplicate assays. Fig. 4. Phylogenetic tree of the full-length amino acid sequences of several TGases. The full-length amino acid sequences of several eukaryote TGases, including the human TGase family (human TGase 1, TGase 2, TGase 3, TGase 4, TGase 5, TGase 7, Factor XIII, and band 4.2.), were aligned by using the CLUSTAL X program, and a bootstrap tree file was created. The phylogenetic tree was drawn with the provided tree-viewing program NJPLOT . The values indicate the number of times that branches are clustered together out of 100 bootstrap trials (values > 50 are labelled.). Horizontal branch lengths are drawn to scale with the bar indicating 0.05 amino-acid replacement per site. Ó FEBS 2002 Mammalian-type transglutaminase in Physarum (Eur. J. Biochem. 269) 3457 grasshoppers [27], and limuli [28]. In lower eukaryotes, however, homologous genes have not been reported so far. Although there are reports of proteins with transamidating activities and their substrates in C. elegans, no similar TGase protein has been discovered yet [29,48]. In C. elegans and filariae, protein disulfide isomerase plays a role in transamidating activity, although the specific activity is comparatively low [30,31]. In the genome database of Arabidopsis and yeast, no gene with a structure similar to that of mammalian TGase genes has been discovered. As an acellular slime mold Physarum belongs to the Mycetozoa, which has been placed as an outgroup of animal–fungi clades in phylogenetic analyses of various genes [49]. Therefore, it is a noteworthy finding that Physarum has a TGase gene with a structure homologous to that of mammalian TGase genes. Our results also indicate the possibility that homologous genes could exist in other lower eukaryotes. In microorganisms, several genes responsible for TGase activity have been cloned and characterized [32–35]. The structures of these genes were found to be different from those of mammals, although a slight similarity between the TGase family and a cysteine protease family, including those in vertebrates, invertebrates, and microorganisms has been shown [9]. In the deduced primary sequence of PpTGase, we could not find any region homologous with those of microbial TGase DNA. In the phylogenetic tree PpTGase belongs to the inver- tebrate TGases as a predictable result (Fig. 4). Unexpect- edly, TGase 4 is located at a position close to PpTGase among human TGases. TGase 4 is produced in the prostate and is responsible for formation of copulatory plugs in rodents, but its actual physiological significance in humans is unknown [50]. TGase 4 is also a unique enzyme as a glycosylated and secreted protein. Although these charac- teristics are not observed in PpTGase, there might be functional similarity between mammalian TGase 4 and PpTGase. Involvement of GTP in the function of PpTGase In the case of both TGase 2 and TGase 3, GTP inhibits the enzymatic activity, while Ca 2+ is known to prevent the inhibition [16,45]. The binding of GTP caused a conform- ational change that reduced the affinity of TGase 2 for Ca 2+ [17]. In this study, a similar inhibitory effect was also observed in PpTGase, and this inhibition was blocked in the presence of a high concentration of Ca 2+ . ATP also slightly inhibited the enzymatic activity of PpTGase, while this nucleotide had no inhibitory effect on either TGase 2 or TGase 3. A recent study has shown that the enzymatic activity of TGase 4 was inhibited by the presence of GTP or ATP, as in the case of PpTGase [51]. These results suggest that the mechanism by which nucleotides inhibit the enzymatic activity of PpTGase might be similar to that by which they inhibit the enzymatic activity of TGase 4. Hydrolysing activity of GTP was also found in the purified PpTGase protein as in the case of TGase 2. Mammalian TGase 2 has been shown to contribute to molecular events underlying signalling mediated by the a-adrenergic receptor, although this function is not related to TGase activity [52]. After stimulation by epinephrine, the adrenoreceptor recruits a GTP-binding protein, Gh, which is identical to TGase 2 [20]. The GTP-bound form of Gh then interacts and activates phospholipase C (PLC), which in turn modulates various processes such as blood pressure. The regions critical for GTP/ATP-hydrolytic activity (1–185 amino acids in guinea pig liver TGase 2) and also for interaction with the PLC (665–672 amino acids in human TGase 2) have been identified [53,54]. Although significant sequence similarity was found in PpTGase with respect to the region for hydrolytic activity, no region homologous with the PLC-interacting region has been found. Whether the hydrolysing activity of GTP of PpTGase is related to certain cellular signalling in the slime mold remains to be determined. As the production of soluble recombinant protein for PpTGase will help to clarify, works in this area are in progress. More recently, based on the X-ray structure of human TGase 2, other GTP-binding sites were shown [55] rather than those reported previously [21]. The residues are not identical to those in PpTGase, suggesting the possibility that a somewhat different binding motif might be related. Possible role of PpTGase There have been reports on purification of TGase from P. polycephalum [38,39]. Mottahedeh & Marsh reported the purification of TGase with a molecular mass of 101 kDa from liquid-cultured plasmodia as a major protein respon- sible for cross-linking activity. Although we cultured plasmodia growing as migrating sheets for purification, our purified protein was probably identical to the 101-kDa protein reported by Mottahedeh & Marsh. No other fractions showing TGase activities were found by the purification procedure used in this study, suggesting that the purified PpTGase is responsible for the major cross-linking reaction in P. polycephalum.Further- more, the result of the Northern blotting indicates that a single species of transcript arises from the genomic locus corresponding to PpTGase. Mottahedeh & Marsh also reported an increase in TGase activity following cellular damage, and they suggested that the enzyme is involved in coagulation of damaged areas [39]. LAV1-2, which is a major calcium-binding protein in P. polycephalum, was shown to be a substrate of PpTGase Fig. 7. Time-courses of GTP hydrolysis by TGase 2, TGase 3, and PpTGase. GTP-hydrolysing activities of guinea pig TGase 2, mouse TGase 3, and PpTGase were determined using 2 lg, 1 lg, and 2 lg proteins, respectively. The reaction mixtures were incubated at 37 °C for the indicated periods of time, and then amounts of 32 P released from [c- 32 P]GTP were determined. 3458 F. Wada et al. (Eur. J. Biochem. 269) Ó FEBS 2002 using monodansylcadaverin as primary amine. LAV1-2 has recently been characterized as CBP40, which reversibly forms large aggregates in a Ca 2+ -dependent manner [56]. Upon cellular damage, the level of CBP40 increases and it localizes to the cellular membrane (A. Nakamura, N. Miki, S. Ogihara, F. Wada, K. Hitomi, M. Maki, Y. Hanyuda & K. Kohama, unpublished data). Therefore, the cross-linked form of CBP40 might be involved in recovery from cellular damage. Although the regulatory mechanisms of PpTGase gene expression remain unclear, the cDNA obtained and the antibodies can be developed into powerful tools for such studies. CONCLUSIONS In summary, we have cloned TGase cDNA from P.poly- cephalum plasmodia. 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Identification of mammalian-type transglutaminase in Physarum polycephalum Evidence from the cDNA sequence and involvement of GTP in the regulation of transamidating. that by which they inhibit the enzymatic activity of TGase 4. Hydrolysing activity of GTP was also found in the purified PpTGase protein as in the case of