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:
*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. Cells were observed under an epifluo-
rescence microscope equipped with a phase-contrast
objective (Olympus, Tokyo, Japan).
Acknowledgements
This work was supported by a Grant-in-Aid for Scienti-
fic Research (C) no. 14560063 (to K. Hitomi), Young
Scientist Research grant no. 15000941 (to F. Wada),
and a TOYOAKI Science Foundation grant (to
K. Hitomi). We thank Dr K. Furuhashi (Shiga Univer-
sity, Japan) for providing us with antibody to Physarum
actin. F. Wada and Y Sugimura are Japanese Society
for the Promotion of Science (JSPS) Research Fellows.

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