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 ª2007 The Authors Journal compilation ª2007 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 ª2007 The Authors Journal compilation ª2007 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 (180s
)
+ cystamine
- Bio-Cd
+ L-682.777
+ 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 lM), cystamine (20 mM) or cada-
verine (20 mM), 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 lML-682.777, 20 mMcystamine or
20 mMcadaverine 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 ª2007 The Authors Journal compilation ª2007 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 ª2007 The Authors Journal compilation ª2007 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).
AB
CD
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 ª2007 The Authors Journal compilation ª2007 FEBS