Báo cáo khoa học: Identification of substrates for transglutaminase in Physarum polycephalum, an acellular slime mold, upon cellular mechanical damage ppt

Identification of substrates for transglutaminase in Physarum polycephalum, an acellular slime mold, upon cellular mechanical damage Fumitaka Wada 1, *, Hiroki Hasegawa 1 , Akio Nakamura 2 , Yoshiaki Sugimura 1 , Yoshiki Kawai 1 , Narie Sasaki 3 , Hideki Shibata 1 , Masatoshi Maki 1 and Kiyotaka Hitomi 1 1 Department of Applied Molecular Biosciences, Graduate School of Bioagricultural Sciences, Nagoya University, Japan 2 Department of Molecular and Cellular Pharmacology, Faculty of Medicine, Gunma University Graduate School of Medicine, Maebashi, Japan 3 Graduate Division of Life Science, Graduate School of Humanities and Sciences, Ochanomizu University, Tokyo, Japan The transglutaminase (TGase; EC 2.3.2.13) enzyme family catalyzes the Ca 2+ -dependent crosslinking of the c-carboxyamide group of glutamine residues and the e-amino group of lysine residues or primary amines [1,2]. This reaction results in the formation of an iso- peptide bond between two proteins and the covalent Keywords adenine nucleotide translocator; calcium; mechanical damage; Physarum polycephalum; transglutaminase Correspondence K. Hitomi, Department of Applied Molecular Biosciences, 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 *Present address RIKEN Brain Science Institute, Hirosawa, Wako-shi, Saitama, Japan Database The nucleotide sequence of the Physarum polycephalum adenine nucleotide transloca- tor is available in the DDBJ ⁄ EMBL ⁄ Gen- Bank database under accession number AB259838 (Received 2 August 2006, revised 17 March 2007, accepted 26 March 2007) doi:10.1111/j.1742-4658.2007.05810.x Transglutaminases are Ca 2+ -dependent enzymes that post-translationally modify proteins by crosslinking or polyamination at specific polypeptide- bound glutamine residues. Physarum polycephalum, an acellular slime mold, is the evolutionarily lowest organism expressing a transglutimase whose primary structure is similar to that of mammalian transglutimases. We observed transglutimase reaction products at injured sites in Physarum macroplasmodia upon mechanical damage. With use of a biotin-labeled primary amine, three major proteins constituting possible transglutimase substrates were affinity-purified from the damaged slime mold. The purified proteins were Physarum actin, a 40 kDa Ca 2+ -binding protein with four EF-hand motifs (CBP40), and a novel 33 kDa protein highly homologous to the eukaryotic adenine nucleotide translocator, which is expressed in mitochondria. Immunochemical analysis of extracts from the damaged macroplasmodia indicated that CBP40 is partly dimerized, whereas the other proteins migrated as monomers on SDS ⁄ PAGE. Of the three pro- teins, CBP40 accumulated most significantly around injured areas, as observed by immunofluoresence. These results suggested that transgluti- mase reactions function in the response to mechanical injury. Abbreviations ANT, adenine nucleotide translocator; Bio-Cd, biotinylated cadaverine; CBB, Coomassie Brilliant Blue R250; CBP40, 40 kDa Ca 2+ -binding protein; DAPI, 4¢,6-diamidino-2-phenylindole; F-Cd, fluorescein cadaverine; HMC, Hepes-based magnesium and calcium buffer; KLH, keyhole limpet hemocyanin; PpANT, adenine nucleotide translocator from Physarum polycephalum; PpTGase, transglutaminase from Physarum polycephalum; PVDF, poly(vinylidene difluoride); TGase, transglutaminase. 2766 FEBS Journal 274 (2007) 2766–2777 ª 2007 The Authors Journal compilation ª 2007 FEBS incorporation of polyamines into proteins. In mam- mals, the crosslinking activity of several TGase iso- zymes functions in blood coagulation, stabilization of extracellular matrix, apoptosis, and skin barrier forma- tion [3–7]. Similar crosslinking reactions are observed in var- ious organisms, from microorganisms to animals. TGases with papain-like characteristics, such as Ca 2+ - dependency and an active-center Cys residue, have been identified in vertebrates and arthropods [1,2,8,9]. In bacteria, yeasts, and lower invertebrates such as nematodes, genes encoding homologous proteins have not been found [2,9,10]. We, however, have reported that Physarum polycephalum, an acellular slime mold, is the evolutionarily lowest organism with a TGase that has a primary structure similar to that of TGases in mammals [11,12]. Physarum polycephalum, which belongs to the My- cetozoa, is a model eukaryote with a unique life cycle characterized by spores, amoebae, macro- plasmodia, and microplasmodia. The plasmodium, used in this study, is a giant and multinucleated cell with a veined structure and no internal cell walls. So far, Physarum has been used mainly in studies on the cell cycle, inheritance of mitochondrial DNA, and cytoplasmic streaming [13–18]. Physarum is also an appropriate model organism for studies on responses to environmental stress. For example, in response to heat stress, Physarum enhances glycosyla- tion of membrane sterol to induce its signal trans- duction system to synthesize heat shock proteins [19]. Also, Physarum TGase activity is induced upon exposure to ethanol or detergent, resulting in tran- samidation of proteins [20]. In mammals, there are several reports that TGase is activated in protective responses to environmental stimuli and contributes to wound healing in various cells [21–26]. In some of these events, remodeling and stabilization of extracellular matrix proteins by TGase resulted in repair of chemical and mechanical injury. However, TGase substrates and their potential roles in repair of damage in unicellular organism are unknown. In this study, we further investigated the role of P. polycephalum TGase (PpTGase) in response to mechanical damage. Following mechanical damage, we observed TGase reaction products around the mechan- ically injured area. On the basis of these observations, we identified and characterized three preferred gluta- mine-donor TGase substrates: 40 kDa Ca 2+ -binding protein (CBP40) [27,28], Physarum actin [29], and a novel protein with high structural similarity to eukary- ote adenine nucleotide translocator (ANT). Results Detection of TGase reaction products around injured areas To investigate whether PpTGase is involved in the response to mechanical damage, we examined in situ enzymatic reactions in slime mold macroplasmodia fol- lowing injury. As shown in Fig. 1, after cells were stabbed with a toothpick, fixed proteins into which flu- orescein cadaverine (F-Cd) was incorporated by TGase catalysis were observed around the injured area. This reaction was completely blocked by several inhibitors of TGase, such as L-682.777, cystamine, and cadaver- ine. These results indicate that labeled primary amine was incorporated into several glutamine-donor sub- strates by activated TGase upon mechanical damage. Purification of potential PpTGase substrates upon mechanical damage Next, we identified the glutamine-donor substrate pro- teins that incorporated primary amines in response to damage in macroplasmodia. Total cellular lysates were prepared from macroplasmodia damaged in the pres- ence of biotinylated cadaverine (Bio-Cd). Depending on the time after injury, Bio-Cd was incorporated into several proteins (Fig. 2). In control cells with no dam- age (both at 10 s and 180 s), only nonspecific bands (marked at the right with asterisks) were observed; those bands probably represent endogenous biotin- conjugating and biotin-binding proteins. Furthermore, no specific incorporation was observed in the copres- ence of several inhibitors or in the absence of Bio-Cd. During the assay period, levels of expressed PpTGase remained equivalent, as indicated by immunoblotting (Fig. 2, lower panel). These results indicated that PpTGase catalyzed transamidation of several proteins acting as preferred glutamine-donor substrates when activated upon mechanical injury. Next, we purified these candidate substrates. As they are likely to be attached to the plasma membrane, a soluble membrane fraction obtained by Triton X-100 treatment was subjected to purification. As shown in Fig. 3, three major proteins (p44, p40, and p33) were eluted as potential substrates, and these proteins were not obtained with the same procedure in the absence of Bio-Cd (lane 7). Using peroxidase-conjugated streptavidin, the eluted proteins were detected as bio- tin-incorporated proteins (Fig. 3B). In this fraction, there were other minor proteins as possible substrates, the amounts of which were not sufficient for the fol- lowing analysis. The proteins in the gel were subjected F. Wada et al. Transglutaminase substrates in damaged Physarum FEBS Journal 274 (2007) 2766–2777 ª 2007 The Authors Journal compilation ª 2007 FEBS 2767 to trypsinization and then to TOF MS analysis. On the basis of data in the database of molecular masses of fragmented proteins, p40 and p44 were identified as CBP40 [27,28] and Physarum actin [29,30], respectively, whereas p33 was a novel protein not found in the database. Purification and molecular cloning of a novel 33 kDa substrate protein In order to identify p33, we purified the protein by affinity chromatography and SDS ⁄ PAGE. Because the N-terminus of the protein was blocked, purified p33 was treated with cyanogen bromide, and the resul- ting fragments were subjected to amino acid sequence analysis. On the basis of the partial amino acid sequence of one fragment, a cDNA clone encoding p33 was obtained by 3¢-RACE using degenerate primers: 5¢-RACE resulted in 5¢-nucleotide sequences that probably include the ini- tiation codon ATG (Fig. 4). The complete sequence shows an ORF of 936 bp encoding 312 amino acids with a calculated molecular mass of 33 622 Da. The amino acid sequence deduced from the nucleotide sequence was highly homologous to that of the ANT seen in sev- eral eukaryotes, and we therefore designated the protein no damage ( 10 s) 10 s 30 s 60 s 180 s no damage ( 18 0s ) + cystamin e - Bio-Cd + L-682 . 77 7 + cadaverine (kDa) PpTGase * 97 66 45 30 * Fig. 2. Detection of total cellular proteins that incorporated Bio-Cd upon mechanical damage. At time 0 s, growing macroplasmodia on an agar plate were injured in the presence of Bio-Cd. Total cellular extracts of macroplasmodia were prepared at the indicated periods. Samples were subjected to 10% SDS ⁄ PAGE and transferred to PVDF membranes. Top: Proteins incorporating Bio-Cd were detec- ted using peroxidase-conjugated streptavidin. Samples from cells without damage (10 s and 180 s) and from damaged cells (180 s) in the presence of L-682.777 (40 l M), cystamine (20 mM) or cada- verine (20 m M), or in the absence of Bio-Cd, were prepared in par- allel. The asterisks indicate no specific signals. Bottom: All samples were subjected to immunoblotting using a monoclonal antibody to PpTGase. +cystamineNo inhibitors + cadaverine+ L-682.777 DIC 200 µm F-Cd Fig. 1. Incorporation of F-Cd into glutamine-donor substrates at injured sites in macroplasmodia. Macroplasmodia grown on a PVDF mem- brane were injured in the presence of F-Cd. After 3 min, the cells were fixed, and differential interference images (DIC) and fluorescent ima- ges (F-Cd) of the cells were obtained. The same experiment was performed in the copresence of 40 l M L-682.777, 20 mM cystamine or 20 m M cadaverine in F-Cd solution. The bar represents 200 lm. Transglutaminase substrates in damaged Physarum F. Wada et al. 2768 FEBS Journal 274 (2007) 2766–2777 ª 2007 The Authors Journal compilation ª 2007 FEBS (kDa) 97 66 45 30 p44 p40 p33 A p44 p40 p33 97 66 45 30 B 1234567 1234567 (kDa) Fig. 3. Purification of proteins incorporating Bio-Cd from damaged slime mold. The total cellular extract, cytosolic fraction and Triton X-100 soluble membrane fraction were prepared from Physarum macroplasmodia injured in the presence of Bio-Cd. From the membrane fraction, proteins incorporating Bio-Cd were affinity-purified with streptavidin-sepharose. To compare them with nonspecifically bound proteins, the same procedure without addition of Bio-Cd was also performed. (A) CBB staining. (B) Detection of biotinylated proteins by peroxidase-conju- gated streptavidin. In both panels, lanes are as follows: lane 1, total cellular extract; lane 2, cytosolic fraction; lane 3, Triton X-100 soluble fraction; lane 4, dialyzed Triton X-100 soluble fraction (applied sample); lane 5, unbound fraction; lanes 6 and 7, eluted fractions from extracts prepared in the presence and absence of Bio-Cd, respectively. Fig. 4. Nucleotide and deduced amino acid sequences of PpANT. The complete amino acid sequence of PpANT was deduced from the nucleotide sequence. The numbers of nucleotide and amino acid residues are shown on the left and right sides, respect- ively. The gray background indicates the fragment cleaved by cyanogen bromide treatment of the purified protein. F. Wada et al. Transglutaminase substrates in damaged Physarum FEBS Journal 274 (2007) 2766–2777 ª 2007 The Authors Journal compilation ª 2007 FEBS 2769 as PpANT (for P. polycephalum ANT). The amino acid sequence of PpANT was 50–77% identical to those of human (ANT1, NP_001142; ANT2, NP_001143; ANT3, NP_001627), mouse (ANT1, NP_031476; ANT2, NP_0031477), bovine (NP_777083), Caenorhab- ditis elegans (NP_001022799), Dictyostelium discoideum (XP_647166), Arabidopsis thaliana (NP_850541), Zea mays (CAA40781) and Saccharomyces cerevsiae (NP_009523) homologs (Fig. 5). From the PpANT pri- mary structure, six possible membrane-spanning regions were deduced from the distribution of hydrophobic regions, as is observed in ANTs of other species. Although the initiation codon (ATG) was deduced from the alignment, recombinant protein produced from expression of the full-length cDNA in bacteria was of the predicted size (data not shown). It is known that eukaryote ANT is the most abun- dant protein in mitochondria [31]. We also investigated the cellular distribution of PpANT in Physarum macr- oplasmodia using a polyclonal antibody. The cell was counterstained with 4¢,6-diamidino-2-phenylindole (DAPI) to visualize both the nucleus (Fig. 6, arrow) and mitochondrial nucleoid (Fig. 6, arrowhead). By phase-contrast (Fig. 6A) and DAPI fluorescence micr- oscopy (Fig. 6B), mitochondria of macroplasmodia were observed as oval-shaped structures and each of them contained a rod-like mitochondrial nucleoid. Fluorescence immunostaining microscopy revealed that Fig. 5. Multiple alignment of PpANT with several eukaryotic ANTs. Amino acid sequences were aligned using the default setting of CLUSTAL X, a multiple sequence alignment program. Amino acid residues common to all sequences are denoted by an asterisk above the sequences, whereas conservative residues are indicated by a colon (: high) or a period (. low). A B C D 5 µm Fig. 6. Immunolocalization of PpANT in Physarum macroplasmodia. A growing macroplasmodum was fixed and reacted with polyclonal antibody to PpANT and then developed by an Alexa Fluor 488-con- jugated secondary antibody. Mitochondrial and nuclear DNAs were counterstained with DAPI. (A) Merged image of phase-contrast and DAPI staining. (B) DAPI staining image. (C) Immunostaining image obtained using antibody to PpANT. (D) Merged image of (B) and (C). The arrows and arrowheads indicate nucleus and mitochondria, respectively. Enlarged images are shown in the inset. The bar rep- resents 5 lm. Transglutaminase substrates in damaged Physarum F. Wada et al. 2770 FEBS Journal 274 (2007) 2766–2777 ª 2007 The Authors Journal compilation ª 2007 FEBS the staining patterns of PpANT coincided with the mitochondria, as was expected (Fig. 6C,D). Immunoblotting analysis of potential substrates upon mechanical damage In order to find how these possible substrates reacted with PpTGase upon cellular injury, we performed immunoblotting of total cellular extracts (Fig. 7, left). The first identified substrate, CBP40, migrated as a 40 kDa protein, but the levels of a higher molecular mass band (80 kDa), probably corresponding to a dimer, increased over time. The slight band (80 kDa) with no damage was produced during the preparation of extracts. This crosslinked product was not observed in the presence of cystamine, suggesting that CBP40 was dimerized by TGase in response to mechanical damage. PpANT and actin were detected at the pre- dicted monomeric size, without no possible dimer form. Affinity-purified proteins incorporating Bio-Cd were recognized by respective antibodies (Fig. 7, eluted frac- tion), confirming that proteins were transamidated upon injury. Taken together, these results suggested that CBP40, actin, and PpANT are enzymatically modified by PpTGase in mechanically damaged macro- plasmodia. Cellular analysis of potential substrates in injured macroplasmodia To investigate the localization of potential substrates around the injured area, each protein was analyzed by immunostaining in cells (Fig. 8). In the absence of cell no damage (10s) 10 s 30 s 60 s 180 s no damage (180 s) +cystamine (kDa) 97 66 45 30 97 66 45 30 97 66 45 30 eluted fraction (Fig. 3, lane 6) CBB anti-CBP40 anti-actin (Physarum) anti-PpANT 97 66 45 30 no damage (10s) 10 s 30 s 60 s 180 s no damage (180 s) + cystamine (kDa) (kDa) (kDa) eluted fraction (Fig. 3, lane 6) A B C Fig. 7. Immunoblot analysis of potential PpTGase substrates upon cellular injury. Total cellular extracts were prepared at the indicated times (10–180 s) from damaged macroplasmodia growing on a plate. Upon injury, HMC buffer was added to the plate, and cells were stabbed with toothpicks several times. As a control, cystamine was added to block the TGase reaction. The right lanes of all blots contains purified pro- tein, which incorporated Bio-Cd from injured macroplasmodia using streptavidin-sepharose chromatography (Fig. 3, lane 6). Samples were subjected to SDS ⁄ PAGE followed by CBB staining (right) and immunoblotting analysis using each polyclonal antibody (left): (A) anti-CBP40; (B) anti-Physarum actin; (C) anti-PpANT. The closed arrows indicate each protein. The open arrow indicates a possible CBP40 dimer. F. Wada et al. Transglutaminase substrates in damaged Physarum FEBS Journal 274 (2007) 2766–2777 ª 2007 The Authors Journal compilation ª 2007 FEBS 2771 damage (J, K, L), all proteins were stained uniformly. CBP40 protein was strongly stained around the injured area (D, G), suggesting that this protein accumulates or is aggregated upon damage. However, both Physa- rum actin and PpANT showed no apparent difference in staining pattern in injured versus noninjured areas (E, F, H, I). Discussion Although most eukaryotic cells and tissues exhibit pro- tective elements, cells can suffer damage following environmental insult. To respond to mechanical dam- age, adaptive systems have been developed not only at the tissue level but also at the cellular level. Membrane resealing, for example, triggered by Ca 2+ entry upon disruption, is a membrane-repair process allowing cells to survive [32,33]. Although it is likely that various molecules and mechanisms participate in responses to mechanical challenge, the process is not well understood. TGases are Ca 2+ -dependent crosslinking enzymes, and are thus likely to function in such mechanisms [1,2]. Indeed, in mammals, it has been shown that TGases respond to environmental attack by participa- ting in wound healing [21–26]. In fibroblasts, for exam- ple, TGase maintains tissue integrity by formation of an SDS-insoluble shell-like structure following rapid loss of Ca 2+ homeostasis [23]. We have focused on the physiologic significance of TGase in Physarum, as this is the lowest known organism exhibiting a TGase similar to that expressed in mammals [11,12]. Upon mechanical damage, Physarum displayed TGase-dependent incorporation of a fluorescent-labeled primary amine into gluta- mine-donor substrate protein(s) (Fig. 1). The product was observed around the mechanically injured area, suggesting that Ca 2+ influx activated a latent form of intracellular TGase since PpTGase is Ca 2+ -dependent as in the case for mammalian TGase [11]. The sub- strate proteins might localize around the membrane that activated TGase can access. Based on time- dependent transamidation, as shown in Fig. 2, several proteins underwent modification without change in the amount of PpTGase, indicating that endogenous TGase activity was stimulated by damage. Although unidentified minor proteins in the purified fraction may also be substrates, the further analyzed gluta- mine-donor substrates consisted of mainly three pro- teins: actin, CBP40, and PpANT. This observation is consistent with the fact that chemical damage of Physarum microplasmodia by treatment with ethanol or detergent results in transamidation of actin and CBP40 [20]. Mechanical damage also resulted in the crosslinking of CBP40 to form a covalently bound dimeric form, and enhanced its levels around the injured area. CBP40, which has four EF-hand motifs in the C-termi- nus and a putative a-helix domain in the N-terminus, aggregates reversibly in a Ca 2+ -dependent manner via the N-terminus in vitro [27]. TGase may contribute to self-assembly of CBP40, where the crosslinked dimer form acts as core to initiate further assembly. Although CBP40 orthologs in other organisms have not been reported, such a crosslinking reaction is remi- niscent of clot formation in vertebrates. injured area uninjured area DIC anti-CBP40 anti-actin (Physarum) anti-PpANT 100 m 100 m injured area A B C D E F G H I J K L Fig. 8. Immunostaining of potential PpTGase substrates in macroplasmodia. A macro- plasmodium grown on a PVDF membrane was injured by a toothpick (A–C; DIC, differ- ential interference images). Then, fixed and permeabilized cells were immunostained with respective antibodies against CBP40 (D, G, J), Physarum actin (E, H, K), and PpANT (F, I, L) using an Alexa Fluor 488-conjugated secondary antibody. The indicated injured region is enlarged (G–I; box in panels D–F). Immunostaining analyses for uninjured areas are shown at the same scale in parallel (J–L). All immunostained signals are shown as stacked images in the vertical direction. The bars represent 10 lm and 100 lm. Transglutaminase substrates in damaged Physarum F. Wada et al. 2772 FEBS Journal 274 (2007) 2766–2777 ª 2007 The Authors Journal compilation ª 2007 FEBS Both actin and PpANT, identified as potential sub- strates, also incorporated Bio-Cd by transamidation upon mechanical damage, although significant aggre- gation or accumulation was not observed by immuno- staining. In western blot analysis, actin and PpANT did not show apparent changes in molecular size following damage, suggesting that they are modified by transamidation or deamidation, as reported for several substrates [34–36]. Physarum actin, which is highly homologous to mammalian actin, is implicated as a force-generating system in actomyosin fibrils [29]. In mammals, actin, as both G-actin and F-actin is a favorable TGase 2 substrate in vitro [37,38]. In this study, the distribution of actin was not affected by injury in the presence or absence of a TGase inhibitor (Fig. 6, and data not shown). In Physarum, monomer actin might not be affected even after modification. PpANT, another potential TGase substrate, was cloned for the first time in this study. On the basis of its considerable homology to ANTs in other eukaryo- tes and observation of its exclusive localization in mitochondria, it is likely that PpANT functions as an antiporter mediating ADP ⁄ ATP exchange in the slime mold. Although we could not show the localization of PpTGase in mitochondria, TGase activity was detected in the purified mitochondrial fraction in mammalian liver and brain [40]. Additionally, in TGase2-over- expressing cells, TGase2 has been reported to localize to mitochondria upon induction of apoptosis [41]. Determining whether transamidation by PpANT regu- lates ATP-translocating activity or induces apoptosis will require further study. As shown in Fig. 3B, there were minor biotin-incor- porating proteins present upon injury. As recovery from cellular damage might require more than three major substrates, further investigation of unidentified substrates and crosslinking reactions would be neces- sary. We have recently established a system to identify the TGase preferred substrate sequence with respect to mammalian TGases [42]. Applying this system to the identification of PpTGase preferred substrates should reveal other substrates and potentially define a net- work of substrates. Additionally, knockdown analyses of TGases and their substrates by an RNA interference method that has recently been established in this organism might be also useful [43]. Although little is known about the physiologic func- tions of TGases in nonmammalian species, there are several reports of TGases being essential for defense against environmental factors [8,44,45]. Cellular responses to mechanical damage are required for euk- aryotes to maintain their homeostasis. In the horseshoe crab, for example, TGase is implicated in the forma- tion of coagulin polymers upon aggregation of hemo- cytes, and it also crosslinks several chitin-binding proteins in the cuticle [8,46]. As evolutionarily lower organisms do not possess an acquired immune system, TGase activity may be particularly important in defending these organisms against environmental challenges. Further investigation of possible TGase substrates in the slime mold should provide insights into the responses of eukaryotic cells to mechanical damage. Experimental procedures Cell culture Physarum macroplasmodia were basically grown on 1.5% agar plates containing MEA medium consisting of 0.165% mycological peptone (Oxoid, Basingstoke, UK), 1% malt extract (Oxoid), and 5 lgÆmL )1 hemin (ICN Biomedicals Inc., Irvine, CA) [13,14]. In the case of observation of the fixed macroplasmodia, cells were grown on a poly(vinylid- ene difluoride) (PVDF) membranes (Millipore, Bedford, MA) located on the agar plate. Both cultures were grown in complete darkness at 25 °C. Incorporation of F-Cd into PpTGase substrate in the damaged slime mold Macroplasmodia cells grown on a PVDF membrane were transferred to a 35 mm dish containing Hepes-based mag- nesium and calcium buffer (HMC; 20 mm Hepes ⁄ NaOH, pH 7.4, 10 mm NaCl, 40 mm KCl, 2 m m CaCl 2 ,7mm MgCl 2 ). F-Cd (Invitrogen, Carlsbad, CA) was added to a final concentration of 0.1 mm, and then the cells were injured by stabbing them with a toothpick. After 3 min, cells on a PVDF membrane were washed with HMC buf- fer and then fixed at room temperature for 15 min in a solution of 10% trichloroacetic acid. The cells were then washed with NaCl ⁄ P i buffer (10 mm sodium phosphate, pH 7.4, 150 mm NaCl) three times, and incubated in NaCl ⁄ P i buffer containing 1.0% Triton X-100 for 30 min at room temperature. Cells were removed from the mem- brane, and located on a coverslip coated with 0.01% poly(l-lysine). After drying, these samples were mounted on a glass slide with antifading solution containing Mowiol 4-88 (Calbiochem, Darmstadt, Germany) and glycerol. Samples were analyzed under a confocal laser- scanning microscope (LSM5 PASCAL; Zeiss, Gottingen, Germany). Cystamine (Sigma, St Louis, MO), cadaverine (Sigma), and L-682.777 (N-Zyme, product name: 1,3,4,5-tetrameth- yl-2-[(2-oxopropyl)thio]imidazolium chloride) were used to inhibit the enzymatic reaction by PpTGase. F. Wada et al. Transglutaminase substrates in damaged Physarum FEBS Journal 274 (2007) 2766–2777 ª 2007 The Authors Journal compilation ª 2007 FEBS 2773 Detection and purification of TGase substrates upon cellular damage to macroplasmodia HMC buffer containing Bio-Cd at a final concentration of 0.2 mm was added to macroplasmodia growing on MEA agar plates. The cells were injured with a bundle of toothpicks several times, as described above. After var- ious periods, the TGase reaction was halted by the addi- tion of cystamine. From the cells homogenized with lysis buffer (20 mm Tris ⁄ Cl, pH 7.5, 100 mm NaCl, 2 mm 2- mercaptoethanol, 20 mm cystamine, 1 mm phenyl- methylsulfonylfluoride, 25 ngÆlL )1 leupeptin, and 1 lm pepstatin), total cell extract was prepared by solubiliza- tion with SDS-dye buffer and boiled. For detection of Bio-Cd incorporated into cellular proteins, the proteins were subjected to SDS ⁄ PAGE and blotted onto a PVDF membrane, which was then developed by peroxidase-con- jugated streptavidin (Rockland, Gilbertsville, PA) and the chemiluminescent method using the Super Signal West Pico chemiluminescent substrate detection kit (Pierce, Rockland, IL). For purification of potential TGase substrates, the dam- aged slime mold in the presence of Bio-Cd was harvested after 3 min. The cells were washed and suspended by lysis buffer. The harvested cells were homogenized and centri- fuged at 10 000 g for 10 min using a SRX-4 centrifuge (TOMY) and TA-4 rotor. The unsolubilized fraction was treated with the TNE buffer (20 mm Tris ⁄ HCl, pH 7.5, 100 mm NaCl, 5 mm EDTA, 2 mm 2-mercaptoethanol) containing 2% Triton X-100, 20 m m cystamine and prote- ase inhibitors for 1 h at 4 °C. The membrane fraction was obtained as a supernatant by centrifugation [10 000 g for 20 min using a SRX-4 centrifuge (TOMY) and TA-4 rotor, and 100 000 g for 30 min using TL100 centrifuge (Beck- man) and TLA100.3 rotor]. The supernatant was dialyzed against TNE buffer overnight to remove unincorporated Bio-Cd, and then applied to a streptavidin-conjugated col- umn previously equilibrated with the same buffer. After several washings with TNE buffer, the bound proteins were eluted with 1 mm Tris ⁄ Cl buffer (pH 8.0) containing 4% SDS buffer. The eluate was concentrated and subjected to SDS ⁄ PAGE following by Coomassie Brilliant Blue (CBB) staining. The protein bands of interest were excised and further analyzed by using standard MALDI-TOF MS methodology. To identify p33 protein, the protein was excised from 12.5% SDS ⁄ PAGE gel and then subjected to carbamidome- thylation using iodoacetoamide. The protein concentrated by acetone precipitation was dissolved in 70% formic acid, and treated with cyanogen bromide at room temperature for 24 h in the dark. The reaction product was separated on a 15% SDS ⁄ PAGE gel and transferred to a PVDF membrane. The cleaved protein bands were excised and sequenced by automated Edman degradation. Molecular cloning of a novel 33 kDa protein 3¢-RACE was performed with the RNA LA PCR Kit Ver.1.1 (TAKARA Biomedicals, Japan). Total RNA from macroplasmodia was obtained by the acid guanidium phe- nol chloroform method. The first-strand cDNA was syn- thesized using 1 lg of total RNA in a reaction mixture of 1.0 mm dNTPs, 16 U of RNasin, 14 U of AMV reverse transcriptase, and oligo dT-M4 adaptor primer in the sup- plied buffer. The resulting cDNAs were subjected to PCR with M13 primer M4 and the degenerate primer 5¢-GCT GGAGCTGCT(A ⁄ T)(C ⁄ G)(A ⁄ T ⁄ G ⁄ C) (C ⁄ T)T(A ⁄ T ⁄ G ⁄ C) AC(A ⁄ T ⁄ G ⁄ C)TTTGT-3¢, which was designed on the basis of the amino acid sequence AGAASLTFVY. Amplification conditions were as follows: 30 cycles at 95 °C for 30 s, 51 °C for 30 s, and 72 °C for 90 s. The PCR products obtained from 3¢-RACE was cloned into a TA-cloning vector, pCR 2.1-TOPO (Invitrogen, Car- lsbad, CA), according to the manufacturer’s instructions. The nucleotide sequences of the isolated clones were deter- mined with an automated fluorescent sequencer, ABI PRISM 310 (PE Applied Biosystems, Foster City, CA), using a Bigdye terminator cycle sequencing ready reaction kit (PE Applied Biosystems). In order to obtain 5¢-terminal cDNA, 5¢-RACE was per- formed using reverse transcriptase and RNA ligase, accord- ing to the manufacturer’s protocols (5¢-Full RACE Core Set; TAKARA Biomedicals). First-strand cDNA was syn- thesized from 1 lg of the poly(A) + RNA, purified with an oligo(dT) cellulose column, using AMV reverse transcrip- tase XL with a specific primer, 5¢- TAGAGACCAGTGA TACCATC-3¢ (antisense, nucleotide sequence number 577–596), and then phosphorylated by T4 polynucleotide kinase. After degradation of the template poly(A) + RNA with RNaseH at 30 °C for 1 h, the resulting single-strand cDNA was precipitated with ethanol and dissolved in 40 lL of a reaction mixture containing 20% poly(ethylene glycol) #4000, RNA ligation buffer, and 1 U of T4 RNA ligase. To change the cDNAs to circular and ⁄ or concatemer cDNAs, the reaction solution was incubated at 15 ° C for 16 h. The cDNAs were directly used as a template for the first PCR amplification with primers 5¢-GGTGAACGCCA GTTCAATGGC-3¢ (S1, sense, 523–543) and 5¢-CGGACT TGTTGTCGTTAGCCAAAC-3¢ (A1, antisense, 485–508), which correspond to the cDNA sequence obtained by 3¢-RACE. The reaction was carried out for 30 cycles with the following conditions: 94 °C for 30 s, 53 °C for 30 s, and 72 °C for 90 s. The resulting PCR product was diluted 1000-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¢-GCTTGCTGGATGTCTACAGAAAGACC-3¢ (S2, sense, 542–567) and 5¢-ACGAGTACGGGCGTAGTC GAG-3¢ (A2, antisense, 466–486) under the same condi- tions. Additional 5¢-RACE reactions using other primers Transglutaminase substrates in damaged Physarum F. Wada et al. 2774 FEBS Journal 274 (2007) 2766–2777 ª 2007 The Authors Journal compilation ª 2007 FEBS [5¢-AGCAGCGATGTTG-3¢ (antisense, 411–423), 5¢-GAA TGTTCGCTGTCCCCAAG-3¢ (sense, 362–381), 5¢-GTCC TTGAAGGCGAAGTTGAG-3¢ (antisense, 331–351), 5¢-GCCTCCTACGGAAAGAAGTTC-3¢ (sense, 385–405) and 5¢-CTTGGGTGGGGAAGTAACGG-3¢ (antisense, 309–328)] produced putative full-length cDNA. Cloning and nucleotide sequencing were carried out as described for 3¢-RACE. Finally, after completion of cloning of putative full- length cDNA, oligonucleotides encoding 5¢- and 3¢-ends were prepared (5¢-CTGGATCCCGAGAAGAAGAACGA CCTCAG-3¢ and 5¢-GATGCTCGAGTTATCCACCTCCG CCAGAG-3¢), and used for PCR reaction to obtain directly full-length cDNA. Polyclonal antibodies Polyclonal antibody against Physarum actin was kindly pro- vided by K. Furuhashi (Shiga University, Japan) [30]. Anti- bodies against PpTGase [12], and CBP40 [27] were prepared as described previously. Polyclonal antibody against PpANT was prepared by immunization of peptide conjugated with keyhole limpet hemocyanin (KLH; Sigma). On the basis of the deduced amino acid sequence, a peptide (YDSLKPALSPLENNPVALGC) corresponding to the amino acid sequence of region 199–217 with an additional Cys residue at the C-terminus was synthesized. Then, the Cys residue of the peptides was covalently crosslinked with KLH using m-maleimidobenzoil-N-hydroxysuccinimide ester, and used as immunogen to raise antibody in rabbit. By subcutaneous immunization of the peptide–KLH six times, antiserum was prepared. The antibody was affinity- purified from antisera using a column that immobilized the peptide. Immunologic analysis of potential substrates from total cellular lysates For western blotting, total cellular extracts were prepared from the injured macroplasmodia by stabbing with tooth- picks as described above. The harvested cells were homo- genized with lysis buffer, and then solubilized directly in SDS sample buffer. Next, the samples were subjected to SDS ⁄ PAGE and western blotting using PVDF membranes. Antibodies were reacted by standard methods, and immuno- signals were detected by the chemiluminescent method as described above. Immunostaining analysis Macroplasmodia cells grown on a PVDF membrane were damaged and fixed as described above. After being washed with NaCl ⁄ P i , cells were incubated in NaCl ⁄ P i containing 1% BSA to prevent nonspecific binding for 1 h at 37 °C. Then, the cells fixed by trichloroacetic acid solution were incubated in the presence of each polyclonal antibody. Sub- sequently, cells were incubated with Alexa Fluor 488-conju- gated goat anti-rabbit serum (Molecular Probes). Samples were analyzed with a confocal laser-scanning microscope (Zeiss) as described above, using 488 nm and 505–530 nm filters. The software used was lsm image browser (Zeiss). In the case of counterstaining of DNA (Fig. 6), cells were fixed by 3.7% formaldehyde for 15 min, and then subjected to the immunostaining reaction as described above. Before mounting of samples on a glass slide, DNA was counter- stained with DAPI. 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K-H, Cho S-Y, Kim C-W, Shin D-M, Kwon J-C, Song K-Y, Park S-C & Kim I-G (2003) Transglutaminase 2 inhibits Rb binding of human papillomavirus E7 by incorporating polyamine EMBO J 22, 5273–5282 Walther DJ, Peter J-U, Winter S, Holtje M, Paulmann N, Grohmann M, Vowinckel J, Alamo-Bethencourt V, Wilhelm CS, Ahnert-Hilger G et al (2003) Serotonylation of small GTPase is a signal transduction pathway that . Identification of substrates for transglutaminase in Physarum polycephalum, an acellular slime mold, upon cellular mechanical damage Fumitaka. TGase resulted in repair of chemical and mechanical injury. However, TGase substrates and their potential roles in repair of damage in unicellular organism are unknown. In