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

Identification of mammalian-type transglutaminase in
Physarum
polycephalum
Evidence from the cDNA sequence and involvement of GTP in the regulation
of transamidating activity
Fumitaka Wada
1
, Akio Nakamura
2
, Tomohiro Masutani
1
, Koji Ikura
3
, Masatoshi Maki
1
and Kiyotaka Hitomi
1
1
Department of Applied Biological Sciences, Graduate School of Bioagricultural Sciences, Nagoya University, Chikusa, Nagoya,
Japan;
2
Department of Pharmacology, Gunma University School of Medicine, Gunma, Japan;
3
Department of Applied Biology,
Faculty of Textile, Kyoto Institute of Technology, Kyoto, Japan
Transglutaminase (TGase) catalyses the post-translational
modification of proteins by transamidation of available
glutamine residues. While several TGase genes of fish and
arthropods have been cloned and appear to have similar
structures to those of mammals, no homologous gene has
been found in lower eukaryotes. We have cloned the acel-

lular slime mold Physarum polycephalum TGase cDNA
using RT-PCR with degenerated primers, based on the
partial amino acid sequence of the purified enzyme. The
cDNA contained a 2565-bp ORF encoding a 855-residue
polypeptide. By Northern blotting, an mRNA of  2600
bases was detected. In comparison with primary sequences
of mammalian TGases, surprisingly, significant similarity
was observed including catalytic triad residues (Cys, His,
Asn) and a GTP-binding region. The alignment of sequences
and a phylogenetic tree also demonstrated that the structure
of P. polycephalum TGase is similar to that of TGases of
vertebrates. Furthermore, we observed that the purified
TGase had GTP-hydrolysing activity and that GTP inhib-
ited its transamidating activity, as in the case of mammalian
tissue-type TGase (TGase 2).
Keywords:GTP;GTPase;Physarum polycephalum;trans-
glutaminase.
Transglutaminase (TGase; EC 2.3.2.13) catalyses cross-
linking between the c-carboxyamide of glutamine residues
and the e-amino group of lysine residues or other primary
amine. The reaction leads to the formation of an isopeptide
bond between two proteins and the covalent incorporation
of polyamine into proteins [1,2]. In mammals, TGases have
a wide distribution in various organs, tissues, and body
fluids, suggesting that they participate in a vast array of
physiological processes. Cross-linking reactions are involved
in clot formation, apoptosis, embryogenesis, angiogenesis,
and skin formation [3–8]. Similar cross-linking has also been
found in invertebrates, plants, unicellular eukaryotes, and
bacteria [9,10]. In vertebrates and some invertebrates, Ca

2+
is required for the enzymatic reaction by exposing a cysteine
residue in the active site domains, while the bacterial enzyme
is not Ca
2+
dependent [11]. This suggests that there are
structural differences responsible for the catalytic reactions
in different organisms. In humans, nine isozymes of TGase
have been found, and they form a large protein family [12].
In other mammals, several isozymes have also been found,
and the primary sequences appear to be significantly similar,
suggesting that these TGases evolved from a common
ancestor gene.
Among these TGases, tissue-type TGase (TGase 2),
which is distributed ubiquitously, has been studied exten-
sively [13–15]. In addition to its protein cross-linking
activity, TGase 2 appears to have other functions. While
GTP inhibits transamidating activity, TGase 2 also shares
GTP-hydrolysing activity [16–19]. TGase 2 has been shown
to function as a signal-transducing GTP-binding protein
that couples activated receptors, resulting in stimulation of
the effector enzyme [20,21]. Furthermore, TGase 2 was
found to be to localized at the cell surface and to mediate the
interaction of integrin with fibronectin [22,23]. The physio-
logical significance of these multifunctional roles of TGase 2
is currently under investigation.
TGase cDNAs have been isolated from other lower
vertebrates, such as fish, and the genes have been found to
have structural similarity with those of mammalian genes
[24,25]. TGase cDNAs of a few invertebrates, such as

ascidians [26], grasshopper (annulin) [27], and limulus [28],
have also been cloned. While the structures of these genes
have been shown to be homologous to those of the
mammalian gene, a TGase with a structure similar to a
mammalian-type has not been found in lower invertebrates
such as Caenorhabditis elegans. On the whole, protein
Correspondence to K. Hitomi, Department of Applied
Biological Sciences, Graduate School of Bioagricultural Sciences,
Nagoya University, Chikusa, Nagoya, 464-8601, Japan.
Fax: + 81 52 789 5542, Tel.: + 81 52 789 5541,
E-mail:
Abbreviations: TGase, transglutaminase; PpTGase, Physarum
polycephalum transglutaminase; PLC, phospholipase; AMV,
avian myeloblastosis virus.
Enzyme: transglutaminase (EC 2.3.2.13).
Note: The nucleotide sequence of Physarum polycephalum TGase
in this paper has been submitted to the DDBJ/EMBL/GenBank
under accession number AB076663.
(Received 4 February 2002, revised 24 May 2002,
accepted 29 May 2002)
Eur. J. Biochem. 269, 3451–3460 (2002) Ó FEBS 2002 doi:10.1046/j.1432-1033.2002.03026.x
disulfide isomerase has been reported to play a role in
transamidating activity in C. elegans and phylarial parasites
[29–31]. TGase genes in bacteria have also been cloned, but
the sequences were found to be completely different from
those of mammalian genes [32–34]. In Escherichia coli,
cytotoxic necrotizing factor 1 possesses TGase activity to
deamidate Rho A [35]. The results of those studies suggest
that the lower eukaryotes and bacterial enzymes evolved as
a separate lineage from the mammalian TGases. The

physiological roles of these invertebrate and bacterial
TGases also remain unclear.
Physarum polycephalum is a true slime mold and has been
used mainly in studies of cell motility [36,37]. This is one of
the lowest eukaryotes with a unique life cycle that is
characterized by spores, amoebae, and plasmodia. The
plasmodia are giant, multinuclear cells in which vigorous
cytoplasmic streaming is observed. Starvation of macro-
plasmodia causes differentiation into sporangia, which
undergo meiosis to form haploid spores. Germinating
spores form amoebae, which can fuse to produce diploid
plasmodia. Although there have been reports on identifica-
tion and purification of P. polycephalum TGase, no struc-
tural information has been presented [38,39].
To find out more about lower eukaryote TGases, their
physiological roles, and evolutionary relationship to other
TGases, we attempted the molecular cloning of P. poly-
cephalum TGase (PpTGase). In this study, based on the
partial amino acid sequences of the purified enzyme, a
cDNA clone encoding PpTGase was isolated. Unexpected-
ly, the primary structure deduced from its cDNA sequence
appeared to be significantly similar to those of mammalian
TGases. Furthermore, GTP inhibited the enzymatic activity
of PpTGase, which also displayed GTP-hydrolysing activ-
ity. We conclude that P. polycephalum is the lowest
organism that has characteristics of mammalian TGase 2.
MATERIALS AND METHODS
Culture of plasmodia
Plasmodia of P. polycephalum (strain Ng-1) were grown on
Quaker Oatmeal (Quaker Oats Company, Chicago, IL,

USA) in the dark [37]. The migrating sheets of plasmodia
were collected and used for experiments.
Purification of PpTGase
Purification was performed essentially as described by
Mottahedeh and Marsh with some modification [39]. All
procedures were performed at 4 °C. The plasmodia growing
as migrating sheets were collected and washed twice with a
solution of 0.4% glycerol, 20 m
M
sodium citrate, 10 m
M
NaPO
4
(pH 5.0). After suspension in 2.5-pellet vols of TEN
buffer (20 m
M
Tris/HCl, 2 m
M
EDTA, 80 m
M
NaCl, 5 m
M
2-mercaptoethanol, 0.2 m
M
phenylmethanesulfonyl fluor-
ide, pH 8.0), the cells were homogenized. The homogenate
was centrifuged at 10 000 g for 20 min, and the supernatant
was further centrifuged at 100 000 g for 40 min. Strepto-
mycin sulfate was slowly added to the resultant supernatant
to a final concentration of 2 mgÆmL

)1
, and the mixture was
placed on ice for 30 min. Insoluble material was removed by
centrifugation at 20 000 g for 15 min. The supernatant was
mixed with an equal volume of 10% glycerol and then
applied to a DEAE-cellulose column (Amersham Pharma-
cia Biotech) equilibrated with buffer A (40 m
M
NaCl,
10 m
M
Tris/HCl, 2.5 m
M
2-mercaptoethanol, pH 8.0). The
column was washed with  1 column vol. buffer A contain-
ing 5% glycerol. CaCl
2
was added to the flow-through
fraction to a final concentration of 1.2 m
M
, and this solution
was passed through a phenyl–Sepharose column (Amersham
Pharmacia Biotech) equilibrated with buffer A containing
0.5 m
M
CaCl
2
. The column was washed with 4 col. vol.
equilibration buffer containing 10% glycerol followed by
4 col. vol. equilibration buffer containing 80 m

M
NaCl and
10% glycerol. Bound proteins were eluted with 80 m
M
NaCl,
20 m
M
Tris/HCl, 2 m
M
dithiothreitol, 2 m
M
MgCl
2
,1m
M
EDTA, 10% glycerol, pH 8.0. The eluted solution was
concentrated using a Centricon-50 concentrator (Millipore)
and then fractionated by gel filtration using a Superdex-200
column (Amersham Pharmacia Biotech) for further purifi-
cation. The fractions with TGase activity were collected,
concentrated, and used for the experiments. Samples were
analysed by SDS/PAGE in a 7.5% acrylamide gel and
stained with Coomassie brilliant blue.
Cleavage of the PpTGase with CNBr and amino acid
sequencing
The purified TGase was dissolved in 70% formic acid and
treated with CNBr at room temperature for 24 h in the
dark. The reaction product was separated by SDS/PAGE in
a subjected to 12.5% acrylamide gel and transferred to
poly(vinylidene difluoride) membrane (Millipore). Protein

bands of interest were excised and sequenced by automated
Edman degradation.
3¢ RACE
3¢ RACE was performed using an RNA LA PCR
TM
Kit
(AMV) Verson 1.1 (TAKARA Biomedicals, Tokyo,
Japan). Total RNA from plasmodia was obtained by the
acid guanidium/phenol/chloroform (AGPC) method. The
first-strand cDNA was synthesized using 1 lgtotalRNAin
a reaction mixture of 0.5 m
M
dNTPs, 40 U RNasin, 4 U
avian myeloblastosis virus (AMV) reverse transcriptase, and
an oligo dT-adaptor primer in the buffer supplied. The
resulting cDNAs were subjected to PCR with M13 primer
M4 and a degenerated primer, 5¢-GTTCCTATCACC
GCCGT(A/T/G/C)AA(A/G)GT(A/T/G/C)GG(A/T/G/C)
GA(A/G)AA-3¢, which was designed on the basis of amino
acid sequence, VPISAVKV GEK. Amplification conditions
were as follows: 30 cycles at 94 °Cfor0.5min,55°C
for 1 min, and 72 °C for 1 min. Using the reaction products
as a template, nested PCR was performed with M4
and another degenerated primer, 5¢-AA(A/G)GT(A/T/
G/C)GG(A/C/T)GA(A/G)AA(A/G)GG(A/T/G/C)AT-3¢,
designed from the amino acid sequence, KVGEKGI. The
amplification conditions were as follows: 30 cycle at 94 °C
for 0.5 min, 52 °Cfor1min,and72°C for 1 min. The
PCR products obtained from 3¢ RACE was cloned into a
TA-cloning vector pCR-TOPO (Invitrogen, USA) accord-

ing to the manufacturer’s instructions. The nucleotide
sequences of the isolated clones were determined with an
automated fluorescent sequencer, ABI PRISM 310 (PE
Applied Biosystems), using a Bigdye
TM
terminator cycle
sequencing ready reaction kit (PE Applied Biosystems).
3452 F. Wada et al. (Eur. J. Biochem. 269) Ó FEBS 2002
5¢ RACE
5¢ RACE was performed using reverse transcriptase and
RNA ligase, according to the manufacturer’s protocols
(5¢-Full RACE Core Set, TAKARA Biomedicals, Tokyo,
Japan) [40]. First-strand cDNA was synthesized from
0.35 lg poly(A)
+
RNA and purified with an oligo(dT)
cellulose column using an AMV reverse transcriptase XL
with a specific primer, 5¢-GCGAGCATTGGTGCCTACA
G-3¢ (antisense, nucleotide sequence positions 1857–1876),
which was phosphorylated by T4 polynucleotide kinase.
After degradation of the template poly(A)
+
RNA with
RNase H at 30 °C for 1 h, the resulting single-stranded
cDNA was precipitated with ethanol and dissolved in 40 lL
of a reaction mixture containing 20% poly(ethylene glycol)
6000, RNA ligation buffer, and 1 U T4 RNA ligase. To
change the cDNAs to circular and/or concatamer cDNAs,
the reaction solution was incubated at 22 °C for 16 h. The
cDNAs were used directly as a template for the first PCR

amplification with primers 5¢-GGCGGATATAGACTTG
TCAGG-3¢ (sense, 1796–1816) and 5¢-CTCGTCAGCATT
CACTTCCG-3¢ (antisense, 1752–1771), which correspond
to the cDNA sequence obtained by 3¢ RACE. The reaction
was carried out for 25 cycles under the following conditions:
94 °C for 30 s, 55 °C for 30 s and 72 °C for 1 min. The
resulting PCR product was diluted 100-fold with sterile
H
2
O, and a 1-lL aliquot was used as a template for the
second nested PCR amplification with primers 5¢-GGA
CAATTACAGATTCAGTGGGAAAG-3¢ (sense, 1815–
1840) and 5¢-CGAGTATACGAAATCGATGTCGTAG-
3¢ (anti sense, 1729–1753) under the same conditions. In the
second 5¢ RACE, first-strand cDNA was synthesized with
another oligonucleotide primer, 5¢-CCCCTCCTAATAGC
GAAGAA-3¢ (antisense, 692–711). The first PCR amplifi-
cation was performed with gene-specific primers: 5¢-GGTC
ATTCAGTCGATCGATTTAC-3¢ (sense, 616–638) and
5¢-TGGAACAACTGGAACGGGTGCTG-3¢ (antisense,
522–544). For the nested PCR, the primers 5¢-CAAGTCG
AGAAGAATAGAGC-3¢ (sense, 638–657) and 5¢-CGAG
TAAAGGTTTGGTGCCTGTT-3¢ (antisense, 485–507)
were used. Cloning and nucleotide sequencing were carried
out as described for the 3¢ RACE.
Computer analyses
Multiple sequence alignment was performed using the
CLUSTAL X
program released from the European Bioinfor-
matics Institute [41], and phylogenetic trees were displayed

using the tree-viewing program
NJPLOT
[42].
Northern blot analysis
Total RNA extracted from plasmodia was electrophoresed
in a 1% agarose-formaldehyde gel and transferred to a
Hybond N
+
nylon membrane (Amersham Pharmacia
Biotech). The membrane was hybridized with a
32
P-labelled
probe and washed at 65 °C for normal stringency.
Expression of recombinant PpTGase in
E. coli
Recombinant proteins were produced using partial
C-terminal region (PpTGase-C: corresponding to
554Gly)855Val) and full-length cDNA for preparation of
antiserum and analysis of PpTGase, respectively.
To prepare antiserum against PpTGase-C, we generated
a hexahistidine (His
6
)–PpTGase-C fusion protein as an
antigen. PCR was performed using the PpTGase cDNA as
a template with primers 5¢-CGGGATCCATATGGGACC
CGTGCCTATTTCTGCT-3¢ and 5¢-CCGGATCCTTAA
ACGACAATAACTTGGGCTTG-3¢. The PCR product
was digested with NdeIandBamHIandtheninserted
between the same sites of the pET19b vector (Novagen,
Madison, USA). E. coli host strain BL21(DE3) trans-

formed with the expression plasmid was grown in Luria–
Bertani medium to an optical density of 0.5 at 600 nm, and
the expression was induced with 1 m
M
isopropyl thio-
b-
D
-galactoside by cultivation at 37 °C for 3 h. The
His
6
–PpTGase-C fusion protein was purified from E. coli
according to the manufacturer’s instructions. Antisera were
produced in two rabbits by immunization with an emulsion
containing approximately 1 mg His
6
–PpTGase-C protein in
Freund’s complete adjuvant. Rabbits were inoculated by
subcutaneous injection into the shaven back. One mg
purified protein in Freund’s incomplete adjuvant was used
for subsequent boosts. Three booster injections were given
at 2-week intervals after the primary injection. Two weeks
after the last immunization, blood was collected from the
heart.
For expression of the full-length cDNA in E. coli, the
same system was used except for the vector. PCR was
performed to insert the restriction enzyme sites (BamHI and
SalI) at the termini of the amplified DNA with primers
5¢-GTGGATCCTATGACTACCGTATTCTTT-3¢ and
5¢-ATAGTCGACTTAAACGACAATAACTTG-3¢.Next,
the resulting DNA fragment was inserted into BamHI and

SalI of pET-24d vector (Novagen), which was modified by
attaching a His
6
tag at the N-terminus of the expressed
protein. For analysis of the expressed protein, harvested
cells were washed and lysed in SDS sample buffer. The
sample was treated with sonication and heated for 3 min.
The sample was analysed by SDS/PAGE on 7.5% acryla-
mide gels followed by staining with Coomassie brilliant blue.
Using the antiserum, Western blotting was performed by
the standard method. Immuno-signals were detected by
colour development methods using diaminobenzidine as
described before [43].
Assay for TGase activity
TGase activity was determined in a microtiter plate assay
essentially as described by Slaughter et al. [43,44]. Briefly,
each microtiter well was coated with 1% dimethylcasein at
37 °C for 1 h and uncoated sites were blocked with skim
milk. A premixed reaction mixture (180 lL) containing
100 m
M
Tris/HCl (pH 8.0), 10 m
M
dithiothreitol, 0.5 m
M
5-(biotinamido)pentylamine (Pierce Chemical Co., Rock-
ford, USA), and CaCl
2
(0.5 m
M

to 2 m
M
) was added to the
wells. The reaction was started by adding 20 lLofTGases
solution to the premixed solution and then incubating at
37 °C for 1 h. TGase-catalysed conjugation of 5-(biotinam-
ido)pentylamine into dimethylcasein was measured by
streptavidin-peroxidase, H
2
O
2
, and o-phenylenediamine.
An equal volume of 2
M
H
2
SO
4
was added, and the
absorbance at 450 nm was measured.
Ó FEBS 2002 Mammalian-type transglutaminase in Physarum (Eur. J. Biochem. 269) 3453
Effects of nucleotides on the TGase activity
GTP solution was added to the TGase solution at a
concentration of 25–500 l
M
. GDP, GMP, and ATP were
added at the concentration of 500 l
M
. These mixtures were
preincubated at 0 °C for 1 h in the absence of CaCl

2
and
added to the reaction mixture. The TGase activity was
measured by the microtiter assay.
Assays for GTP hydrolysis by TGase 2, TGase 3,
and PpTGase
GTP-hydrolysing activity was measured as described pre-
viously [18,45]. The guinea pig TGase 2 and the recombin-
ant mouse TGase 3 were purified from guinea pig liver
and baculovirus-infected insect cells, respectively [13,46].
TGase 3 was proteolysed by dispase to activate the proen-
zyme. Two micrograms of TGase 2, 1 lg TGase 3, and
2 lg purified PpTGase were mixed with 15 lCi [c-
32
P]GTP
(2 l
M
) in a reaction mixture containing 20 m
M
Tris/HCl
(pH 7.5), 5 m
M
MgCl
2
,1m
M
dithiothreitol, 1 m
M
EDTA.
The reaction mixtures were incubated at 37 °Cforthe

indicated periods of time, and the reaction was stopped by
addition of 7 vol. 5% (w/v) charcoal in 50 m
M
NaH
2
PO
4
.
The mixture was centrifuged at 12 000 g for 7 min. The
amount of
32
P released from [c-
32
P]GTP was measured by
scintillation counting of clear supernatant solution.
RESULTS
Purification of PpTGase
TGase from P. polycephalum plasmodia cultured as migra-
ting sheets was purified on the basis of enzymatic activity
(Fig. 1A). After streptomycin sulfate precipitation, cellular
protein was applied to an anion-exchange column and the
unbound proteins were loaded onto a phenyl–Sepharose
column in the presence of Ca
2+
. Almost homogeneous
100-kDa protein was obtained in the fraction eluted with
EDTA from the phenyl-sepharose column. This result
agreed well with the result reported previously by
Mottahedeh & Marsh [39]. During all of the procedures,
no other fractions with apparent TGase activities were

observed, suggesting that the purified protein is the major
TGase in Physarum plasmodia. To obtain highly purified
TGase, the contaminating proteins were excluded by gel
filtration chromatography using Superdex 200. The estima-
ted molecular mass was 130–150 kDa, suggesting that this
protein was in a monomeric form.
Initially, determination of the amino-terminal amino acid
sequence of purified protein was attempted, but no infor-
mation was obtained, probably due to the protein modifi-
cation. Therefore, the purified protein was treated with
CNBr to cleave at the methionine residue. As shown in
Fig. 1B, two major fragments, of  60 and  40 kDa, were
obtained and subjected to sequencing. A 15-amino acid
sequence (GPVPISAVKVGEKGI) was revealed in respect
to the 40-kDa fragment, while the 60-kDa fragment
provided no result.
cDNA cloning and sequence
Based on the amino acid sequence of the 40-kDa protein,
degenerated primers corresponding to the sequence were
designed for 3¢ RACE. cDNA was synthesized using an
oligo dT adaptor primer, and then PCR amplification was
performed using both M4 and the degenerated primer. A
major PCR product of 1200 bp, as an expected size, was
obtained. Using the degenerated primers for nested PCR, a
defined single 1200-bp DNA was produced. The deduced
primary sequence from the nucleotide sequence of the
amplified DNA is similar to that of the corresponding
position in the mammalian TGase, thus providing evidence
that the PCR product is TGase cDNA of P. polycephalum.
To obtain a cDNA encoding the 5¢ portion of PpTGase,

we carried out 5¢ RACE using specific primers based on the
partial cDNA sequence obtained by 3¢ RACE. A single
PCR product of 1600 bp was produced by two successive
reactions. In the amino acid sequence deduced from the
amplified cDNA sequence, a 15-amino acid sequence, which
was determined by protein sequencing, was observed
(Fig. 2, grey background). Although the predicted amino-
acid sequence had similarity with the sequences of mam-
malian TGase, the length of the cDNA was smaller than the
length deduced from the molecular size of the purified
protein. Furthermore, an initiation codon was not observed
in the sequence obtained. Therefore, we performed a further
5¢ RACE in order to obtain a cDNA encoding the 5¢ upper
region. The resulting product, which was 700 bp in length,
revealed novel 83 bp sequences that included a putative
initiation codon and part of the 5¢ untranslated region.
Finally, a full-size composite cDNA sequence encoding
PpTGase was obtained from the nucleotide sequences of the
three RACE products.
The full-length cDNA of PpTGase was 2624 bp long and
contained 22- and 34-bp noncoding regions at the 5¢ and 3¢
ends, respectively. One polyadenylation signal (AATAAA)
wasobservedinthe3¢ untranslated region. The complete
sequence shows an ORF of 2565 bp corresponding to 855
amino acids with a molecular mass of 93 611 Da (Fig. 2).
Fig. 1. Purification and cleavage of PpTGase. (A) Approximately
1–5 lg protein from each step in the purification procedure was
separated by SDS/PAGE on 7.5% acrylamide gels followed by
staining with Coomassie brilliant blue: molecular mass markers (lane
M); total cellular extract (lane 1); soluble fraction (lane 2); supernatant

fraction after streptomycin sulfate precipitation (lane 3); flow-through
fraction of DEAE–Sephacel chromatography (lane 4); eluted fraction
of phenyl sepharose chromatography (lane 5); and peak fraction from
size separation (Superdex 200) (lane 6). The arrow indicates the
position of PpTGase. (B) Purified PpTGase was treated with CNBr
and separated by SDS/PAGE on 12.5% acrylamide gels. Arrows
indicate the fragments analysed for amino-acid sequencing.
3454 F. Wada et al. (Eur. J. Biochem. 269) Ó FEBS 2002
That sequence included a Cys active site, and the other two
critical residues for catalytic activity, His and Asp, were also
observed. Both putative GTP-binding (Tyr345–Phe359)
and Ca
2+
binding (Val613–Arg635) regions, which have
been identified in human TGase 2, were found (Fig. 2).
Next, we aligned the PpTGase sequence with other
TGases with respect to the middle region around the GTP-
binding region, active site, and Ca
2+
-binding region, which
are highly homologous among many TGases (Fig. 3). The
amino acid sequences of PpTGase were 40–50% identical to
those of human TGase 2 [14] and TGases of red sea bream
[24], ascidians [26], grasshopper [27], fruit fly, and limulus
[28].Ser,whichisanessentialaminoacidresidueforGTP
binding, is also conserved (region A in Fig. 3). In respect to
the outside regions of A, B, and C, PpTGase showed low
but significant similarity to human TGase 2 except for the
presence, in the PpTGase, of a long amino-terminal region
that is missing in the human enzyme. Furthermore, in

order to clarify the molecular evolutionary relationship of
PpTGase, we made a phylogenetic tree using the
CLUSTAL X
program based on the full-length amino-acid sequences
(Fig. 4). When aligned according to the middle region with
high homology among the various TGases (from the front
of region A to the end of region C in Fig. 3), a similar
phylogenetic tree was drawn (data not shown). Band 4.2,
which is an enzymatically inactive TGase-like protein found
in erythrocytes, located at a far position. PpTGase was
situated closer to the other invertebrate TGases than to
human and fish TGases. Among human TGases, however,
TGase 4 was placed significantly close to PpTGase.
Northern blotting
We performed Northern blot analysis using total RNA
prepared from plasmodia. As shown in Fig. 5A, a single
band was observed at the size of  2600 nucleotides. This
length agrees well with that of the PpTGase cDNA
obtained. No other RNA hybridized even under lower
stringency hybridization conditions, such as lower tempera-
ture (data not shown).
Western blotting
To confirm that we had obtained the full-length cDNA,
recombinant protein was produced in E. coli and analysed.
As the polyclonal antibody had been raised against the
C-terminal portion of the PpTGase, Western blotting
analysis was performed in respect to the recombinant
protein and PpTGase in the plasmodial lysate as well as the
purified PpTGase (Fig. 5B and C). The recombinant
PpTGase protein was successfully expressed at the molecu-

lar weight of 100 kDa (Fig. 5C, lane 2). No difference in size
was observed between the purified protein and PpTGase in
plasmodia lysate, suggesting that PpTGase was not degraded
during the purification procedure (Fig. 5C, lanes 3 and 4).
The recombinant protein and the PpTGase protein from
Fig. 2. cDNA and deduced amino-acid
sequences of PpTGase. A complete amino-acid
sequence of PpTGase was deduced from the
nucleotide sequence. The numbers of nucleo-
tides and amino-acid residues are shown on
the left and right sides, respectively. The grey
background indicates the amino acid sequence
determined from the CNBr fragment. The
asterisk indicates the stop codon. Three amino
acid residues of the catalytic triad are boxed.
The single and double lines indicate the
putative Ca
2+
- and GTP-binding sites,
respectively.
Ó FEBS 2002 Mammalian-type transglutaminase in Physarum (Eur. J. Biochem. 269) 3455
plasmodia migrated to similar positions probably because
of the attachment of the hexahistidine, the recombinant
PpTGase protein appeared to be slightly larger. These
results indicate that the cDNA obtained covered the entire
coding region.
Involvement of GTP in the regulation of TGase function
In the deduced primary sequence, we found a putative
GTP-binding site [21]. This prompted us to investigate the
relationship of nucleotides to regulation of the tran-

samidating activity, which has been extensively studied in
respect to TGase 2. As we have not yet been able to produce
a soluble recombinant protein, experiments were performed
using completely purified TGase protein from P. polyceph-
alum plasmodia (Fig. 1, lane 6).
First, the inhibitory effect of GTP on enzymatic activity
was analysed with various concentrations of Ca
2+
, as
shown in Fig. 6A. At 0.5 m
M
Ca
2+
, the enzymatic activity
was apparently decreased by the addition of 100–500 l
M
GTP. In the presence of 1 m
M
Ca
2+
, an inhibitory effect
was observed only at a higher level of GTP. In the case of
2m
M
Ca
2+
, inhibition by GTP was not observed. These
results suggest that the TGase activity is regulated by the
presence of GTP and Ca
2+

. Additionally, in order to
confirm the specificity of the inhibition, other purine
nucleotides (GTP, GDP, GMP, and ATP) were examined
at 0.5 m
M
Ca
2+
. Fig. 6B shows the relative enzymatic
activity in the presence of the nucleotides. GTP and ATP
clearly inhibited the activity, while GDP showed weak
inhibition. GMP did not block the enzymatic activity.
Next, GTP-hydrolysing activity of the purified PpTGase
was investigated. Mammalian TGase 2 has both transam-
idating and GTP-hydrolysing activities, whereas TGase 3
has no GTPase activity. These proteins were incubated with
32
P-GTP, and then the release of
32
P was measured. The
amount of radioactivity released by guinea pig TGase 2 and
PpTGase increased up to 60 min in a time-dependent
fashion (Fig. 7) and also depended on the amounts of the
proteins (data not shown). Although the hydrolysing
activity was weaker than that of TGase 2, PpTGase had
an apparent GTP-hydrolysing activity.
DISCUSSION
cDNA sequence of PpTGase
Although the overall identity with the mammalian TGase
primary sequence is low in the deduced sequence of the
Physarum TGase, the middle region of the sequence is

significantly conserved. The sequences around the GTP-
binding region, catalytic site, and Ca
2+
-binding region are
highly homologous to the corresponding regions of the
human TGase 2 and the other invertebrate TGases (Fig. 3).
The eight amino-acid residues surrounding the active site
Cys (region B in Fig. 3) except those of Drosophila
melanogaster TGase (the sequence of which was predicted
from the database; accession number AAF52590), are
identical. In addition to this catalytic Cys site, His and Asp,
which comprise a catalytic triad with Cys, are also
conserved. Furthermore, a putative Ca
2+
-binding region
reported in mammalian TGase 2 was also found [15]. This is
consistent with the finding that Ca
2+
was required for the
enzymatic activity of PpTGase. These findings suggest that
an acyl-transfer reaction identical to that of mammalian
TGases is executed in the catalytic reaction of PpTGase.
Compared with those of human TGase 2, an additional
region exists at the amino terminus of PpTGase, which is
not highly conserved. Among human TGases, keratino-
cyte-type TGase (TGase 1) contains such a longer amino
Fig. 3. Alignment of highly similar regions of PpTGase with various eukaryote TGases. In the upper panel, regions of human TGase 2 and PpTGase
that are very similar are shaded. Alignment was performed with respect to the selected sequences around the following regions: A, GTP-binding
region; B, catalytic site; C, Ca
2+

-binding region. The amino-acid sequences were aligned by using the
CLUSTAL X
program. Gaps indicated by
hyphens have been introduced to improve the sequence alignments. Conserved amino acid residues are shaded. The dark-shaded S (region A) and C
(region B) indicate essential amino acid residues for GTP binding and catalytic reaction, respectively. The numbers represent the amino acid residue
numbers of the TGases: human TGase 2, red sea bream (Pagrus major), ascidians (Ciona intestinalis), grasshopper (Schistocerca americana), fruit
flies (D. melanogaster), limuli (Tachypleus tridentatus), and slime mold (P. polycephalum). With respect to the corresponding sequence to the
Drosophila TGase, cDNA sequence was searched from database with the
TBLASTN
search engine to identify cDNA with homology to vertebrate
TGases (accession no. AAF52590).
3456 F. Wada et al. (Eur. J. Biochem. 269) Ó FEBS 2002
terminus that is required for binding to the plasma
membrane, thereby being involved in the formation of
the corneum [47]. Additional amino-terminal residues of
TGase 1 include the sequences that are post-translationally
modified by fatty acid chains conferring membrane associ-
ation, however, we could not find such a primary sequence.
Similar long amino-terminal sequences are also found in
ascidian, grasshopper, and Limulus TGases, suggesting a
common characteristic of nonmammalian TGases [26–28].
Several gene structures responsible for enzymatic activity
have been reported in various organisms. TGases with
homologous primary structure have been cloned in fish and
some invertebrates such as red sea bream [24], salmon [25],
zebrafish (C. Rodolfo et al. Abstracts in the 6th Interna-
tional Conference on Transglutaminase and Protein Cross-
linking Reactions, Lyon, France, 2000), ascidians [26],
Fig. 5. Northern and Western blot analysis of PpTGase. Northern blot
analysis of total RNA from Physarum plasmodia was performed using

thefull-lengthPpTGasecDNAasaprobe(A).Lane1,5lg; lane 2,
10 lg. The arrow indicates the transcripts of PpTGase. Mouse ribo-
somal RNA was used as a size marker. Analyses of the recombinant
and the plasmodial PpTGases were performed by SDS/PAGE on
7.5% acrylamide gels (B) and Western blotting (C). Lane 1, cellular
protein of E. coli transformed with a control vector; lane 2, cellular
protein of E. coli transformed with the vector harbouring PpTGase
cDNA; lane 3, cellular protein of Physarum plasmodia; lane 4, purified
PpTGase from Physarum plasmodia. Lane M, molecular mass marker.
In lane 2, to reduce the recombinant PpTGase proteins in the E. coli
lysate sample the lysate of E. coli expressing PpTGase ( 5% of the
total cell protein) was diluted 50-fold with that of E. coli harbouring
pET-24d (negative control, lane 1). In lane 4 of (C), the sample in (B)
was diluted 20-fold with SDS buffer. The arrows in (B) and (C) indicate
the positions of PpTGase.
Fig. 6. Effects of purine nucleotides on the
inhibition of PpTGase activity at various Ca
2+
concentrations. The activities of TGase were
measured as described in Materials and
methods. (A) The cross-linking activities of
PpTGase in the presence of 0.5 m
M
(d), 1 m
M
(m), or 2 m
M
CaCl
2
(j) with 0–500 l

M
GTP
are shown. The purified enzyme (0.5 lg) was
tested using 5 m
M
GTPsolutioninanequal
volume. (B) The enzymatic activities of
PpTGase in the presence of 0.5 m
M
CaCl
2
with 500 l
M
nucleotides are shown. Data
represent the mean of triplicate assays.
Fig. 4. Phylogenetic tree of the full-length amino acid sequences of
several TGases. The full-length amino acid sequences of several
eukaryote TGases, including the human TGase family (human
TGase 1, TGase 2, TGase 3, TGase 4, TGase 5, TGase 7, Factor
XIII, and band 4.2.), were aligned by using the
CLUSTAL X
program,
and a bootstrap tree file was created. The phylogenetic tree was drawn
with the provided tree-viewing program
NJPLOT
. The values indicate
the number of times that branches are clustered together out of 100
bootstrap trials (values > 50 are labelled.). Horizontal branch lengths
are drawn to scale with the bar indicating 0.05 amino-acid replacement
per site.

Ó FEBS 2002 Mammalian-type transglutaminase in Physarum (Eur. J. Biochem. 269) 3457
grasshoppers [27], and limuli [28]. In lower eukaryotes,
however, homologous genes have not been reported so far.
Although there are reports of proteins with transamidating
activities and their substrates in C. elegans, no similar
TGase protein has been discovered yet [29,48]. In C. elegans
and filariae, protein disulfide isomerase plays a role in
transamidating activity, although the specific activity is
comparatively low [30,31]. In the genome database of
Arabidopsis and yeast, no gene with a structure similar to
that of mammalian TGase genes has been discovered. As an
acellular slime mold Physarum belongs to the Mycetozoa,
which has been placed as an outgroup of animal–fungi
clades in phylogenetic analyses of various genes [49].
Therefore, it is a noteworthy finding that Physarum has a
TGase gene with a structure homologous to that of
mammalian TGase genes. Our results also indicate the
possibility that homologous genes could exist in other lower
eukaryotes.
In microorganisms, several genes responsible for TGase
activity have been cloned and characterized [32–35]. The
structures of these genes were found to be different from
those of mammals, although a slight similarity between the
TGase family and a cysteine protease family, including
those in vertebrates, invertebrates, and microorganisms has
been shown [9]. In the deduced primary sequence of
PpTGase, we could not find any region homologous with
those of microbial TGase DNA.
In the phylogenetic tree PpTGase belongs to the inver-
tebrate TGases as a predictable result (Fig. 4). Unexpect-

edly, TGase 4 is located at a position close to PpTGase
among human TGases. TGase 4 is produced in the prostate
and is responsible for formation of copulatory plugs in
rodents, but its actual physiological significance in humans
is unknown [50]. TGase 4 is also a unique enzyme as a
glycosylated and secreted protein. Although these charac-
teristics are not observed in PpTGase, there might be
functional similarity between mammalian TGase 4 and
PpTGase.
Involvement of GTP in the function of PpTGase
In the case of both TGase 2 and TGase 3, GTP inhibits the
enzymatic activity, while Ca
2+
is known to prevent the
inhibition [16,45]. The binding of GTP caused a conform-
ational change that reduced the affinity of TGase 2 for
Ca
2+
[17]. In this study, a similar inhibitory effect was also
observed in PpTGase, and this inhibition was blocked in the
presence of a high concentration of Ca
2+
. ATP also slightly
inhibited the enzymatic activity of PpTGase, while this
nucleotide had no inhibitory effect on either TGase 2 or
TGase 3. A recent study has shown that the enzymatic
activity of TGase 4 was inhibited by the presence of GTP or
ATP, as in the case of PpTGase [51]. These results suggest
that the mechanism by which nucleotides inhibit the
enzymatic activity of PpTGase might be similar to that by

which they inhibit the enzymatic activity of TGase 4.
Hydrolysing activity of GTP was also found in the
purified PpTGase protein as in the case of TGase 2.
Mammalian TGase 2 has been shown to contribute to
molecular events underlying signalling mediated by the
a-adrenergic receptor, although this function is not related
to TGase activity [52]. After stimulation by epinephrine, the
adrenoreceptor recruits a GTP-binding protein, Gh, which
is identical to TGase 2 [20]. The GTP-bound form of Gh
then interacts and activates phospholipase C (PLC), which
in turn modulates various processes such as blood pressure.
The regions critical for GTP/ATP-hydrolytic activity (1–185
amino acids in guinea pig liver TGase 2) and also for
interaction with the PLC (665–672 amino acids in human
TGase 2) have been identified [53,54]. Although significant
sequence similarity was found in PpTGase with respect to
the region for hydrolytic activity, no region homologous
with the PLC-interacting region has been found. Whether
the hydrolysing activity of GTP of PpTGase is related to
certain cellular signalling in the slime mold remains to be
determined. As the production of soluble recombinant
protein for PpTGase will help to clarify, works in this area
are in progress.
More recently, based on the X-ray structure of human
TGase 2, other GTP-binding sites were shown [55] rather
than those reported previously [21]. The residues are not
identical to those in PpTGase, suggesting the possibility that
a somewhat different binding motif might be related.
Possible role of PpTGase
There have been reports on purification of TGase from

P. polycephalum [38,39]. Mottahedeh & Marsh reported the
purification of TGase with a molecular mass of 101 kDa
from liquid-cultured plasmodia as a major protein respon-
sible for cross-linking activity.
Although we cultured plasmodia growing as migrating
sheets for purification, our purified protein was probably
identical to the 101-kDa protein reported by Mottahedeh &
Marsh. No other fractions showing TGase activities were
found by the purification procedure used in this study,
suggesting that the purified PpTGase is responsible for the
major cross-linking reaction in P. polycephalum.Further-
more, the result of the Northern blotting indicates that a
single species of transcript arises from the genomic locus
corresponding to PpTGase.
Mottahedeh & Marsh also reported an increase in TGase
activity following cellular damage, and they suggested that
the enzyme is involved in coagulation of damaged areas [39].
LAV1-2, which is a major calcium-binding protein in
P. polycephalum, was shown to be a substrate of PpTGase
Fig. 7. Time-courses of GTP hydrolysis by TGase 2, TGase 3, and
PpTGase. GTP-hydrolysing activities of guinea pig TGase 2, mouse
TGase 3, and PpTGase were determined using 2 lg, 1 lg, and 2 lg
proteins, respectively. The reaction mixtures were incubated at 37 °C
for the indicated periods of time, and then amounts of
32
P released
from [c-
32
P]GTP were determined.
3458 F. Wada et al. (Eur. J. Biochem. 269) Ó FEBS 2002

using monodansylcadaverin as primary amine. LAV1-2 has
recently been characterized as CBP40, which reversibly
forms large aggregates in a Ca
2+
-dependent manner [56].
Upon cellular damage, the level of CBP40 increases and it
localizes to the cellular membrane (A. Nakamura, N. Miki,
S. Ogihara, F. Wada, K. Hitomi, M. Maki, Y. Hanyuda &
K. Kohama, unpublished data). Therefore, the cross-linked
form of CBP40 might be involved in recovery from cellular
damage. Although the regulatory mechanisms of PpTGase
gene expression remain unclear, the cDNA obtained and
the antibodies can be developed into powerful tools for such
studies.
CONCLUSIONS
In summary, we have cloned TGase cDNA from P.poly-
cephalum plasmodia. 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-

in-Aid for Scientific Research no. 12660074 from the Ministry of
Education, Science, Sports and Culture of Japan.
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