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-

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

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,

)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

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-

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

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

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

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

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

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-

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+

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+

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

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-

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

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. This is the lowest organism in which
mammalian type-TGase has so far been found. Although
the result of a gene-disruption study on mammalian
TGase 2 have recently been reported, the apparent pheno-
type has not been described in knockout mice [57]. Perhaps
because of the presence of various isozymes, it may be
difficult to observe noticeable phenomena in animals by
gene disruption. In lower organisms such as slime molds,
however, phenomena could be observed by the method of
gain- or loss-of-function, and such experiments might reveal
novel physiological functions of TGase.
ACKNOWLEDGEMENTS
We thank Dr H. Shibata, T. Nakayama, and N. Ikeda for technical
assistance and helpful discussion. This work was supported by a Grant-

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