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Identification of mammalian-type transglutaminase in Physarumpolycephalum Evidence from the cDNA sequence and involvement of GTP in the regulation of transamidating activity Fumitaka Wad

Identification of mammalian-type transglutaminase in Physarum

polycephalum

Evidence from the cDNA sequence and involvement of GTP in the regulation

of transamidating activity

Fumitaka Wada1, Akio Nakamura2, Tomohiro Masutani1, Koji Ikura3, Masatoshi Maki1

and Kiyotaka Hitomi1

1

Department of Applied Biological Sciences, Graduate School of Bioagricultural Sciences, Nagoya University, Chikusa, Nagoya, Japan;2Department of Pharmacology, Gunma University School of Medicine, Gunma, Japan;3Department 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, Ca2+

is required for the enzymatic reaction by exposing a cysteine

residue in the active site domains,while the bacterial enzyme

is not Ca2+ 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: hitomi@agr.nagoya-u.ac.jp

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)

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 polycephalumis 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 purificaidentifica-tion 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

M A T E R I A L S A N D M E T H O D S

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 4C The plasmodia growing

as migrating sheets were collected and washed twice with a

solution of 0.4% glycerol,20 mM sodium citrate,10 mM

NaPO4(pH 5.0) After suspension in 2.5-pellet vols of TEN

buffer (20 mMTris/HCl,2 mMEDTA,80 mMNaCl,5 mM

2-mercaptoethanol,0.2 mM 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 mM NaCl,

10 mMTris/HCl,2.5 mM2-mercaptoethanol,pH 8.0) The column was washed with 1 column vol buffer A contain-ing 5% glycerol CaCl2 was added to the flow-through fraction to a final concentration of 1.2 mM,and this solution was passed through a phenyl–Sepharose column (Amersham Pharmacia Biotech) equilibrated with buffer A containing 0.5 mM CaCl2 The column was washed with 4 col vol equilibration buffer containing 10% glycerol followed by

4 col vol equilibration buffer containing 80 mMNaCl and 10% glycerol Bound proteins were eluted with 80 mMNaCl,

20 mMTris/HCl,2 mMdithiothreitol,2 mMMgCl2, 1 mM

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 PCRTMKit (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 lg total RNA in

a reaction mixture of 0.5 mM 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 94C for 0.5 min, 55 C for 1 min,and 72C 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 94C for 0.5 min,52C for 1 min, and 72 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 BigdyeTM terminator cycle sequencing ready reaction kit (PE Applied Biosystems)

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 30C 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 22C 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:

94C for 30 s,55 C for 30 s and 72 C for 1 min The

resulting PCR product was diluted 100-fold with sterile

H2O,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 Xprogram released from the European

Bioinfor-matics Institute [41],and phylogenetic trees were displayed

using the tree-viewing programNJPLOT[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 a32P-labelled

probe and washed at 65C for normal stringency

Expression of recombinant PpTGase inE 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 (His6)–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 NdeI and BamHI and then inserted 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 mM isopropyl thio-b-D-galactoside by cultivation at 37C for 3 h The His6–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 His6–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¢-ATAGTCGACTTAAACGACAATAACTTG-3¢ Next, the resulting DNA fragment was inserted into BamHI and SalI of pET-24d vector (Novagen),which was modified by attaching a His6 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

37C for 1 h and uncoated sites were blocked with skim milk A premixed reaction mixture (180 lL) containing

100 mM Tris/HCl (pH 8.0),10 mM dithiothreitol,0.5 mM

5-(biotinamido)pentylamine (Pierce Chemical Co.,Rock-ford,USA),and CaCl2(0.5 mMto 2 mM) was added to the wells The reaction was started by adding 20 lL of TGases solution to the premixed solution and then incubating at

37C for 1 h TGase-catalysed conjugation of 5-(biotinam-ido)pentylamine into dimethylcasein was measured by streptavidin-peroxidase,H2O2,and o-phenylenediamine

An equal volume of 2M H2SO4 was added,and the absorbance at 450 nm was measured

Effects of nucleotides on the TGase activity

GTP solution was added to the TGase solution at a

concentration of 25–500 lM GDP,GMP,and ATP were

added at the concentration of 500 lM These mixtures were

preincubated at 0C for 1 h in the absence of CaCl2and

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-32P]GTP

(2 lM) in a reaction mixture containing 20 mM Tris/HCl

(pH 7.5),5 mMMgCl2, 1 mMdithiothreitol,1 mMEDTA

The reaction mixtures were incubated at 37C for the

indicated periods of time,and the reaction was stopped by

addition of 7 vol 5% (w/v) charcoal in 50 mMNaH2PO4

The mixture was centrifuged at 12 000 g for 7 min The

amount of32P released from [c-32P]GTP was measured by

scintillation counting of clear supernatant solution

R E S U L T S

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 Ca2+ 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) was observed in the 3¢ 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.

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 Ca2+ 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 Ca2+-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, which is an essential amino acid residue for GTP

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 theCLUSTAL 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.

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-alumplasmodia (Fig 1,lane 6)

First,the inhibitory effect of GTP on enzymatic activity

was analysed with various concentrations of Ca2+,as

shown in Fig 6A At 0.5 mMCa2+,the enzymatic activity

was apparently decreased by the addition of 100–500 lM

GTP In the presence of 1 mMCa2+,an inhibitory effect

was observed only at a higher level of GTP In the case of

2 mMCa2+,inhibition by GTP was not observed These

results suggest that the TGase activity is regulated by the

presence of GTP and Ca2+ Additionally,in order to

confirm the specificity of the inhibition,other purine

nucleotides (GTP,GDP,GMP,and ATP) were examined

at 0.5 mM Ca2+ 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

32P-GTP,and then the release of32P 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

D I S C U S S I O N

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 Ca2+-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 melanogasterTGase (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 Ca2+-binding region reported in mammalian TGase 2 was also found [15] This is consistent with the finding that Ca2+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).

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 the full-length PpTGase cDNA as a probe (A) Lane 1, 5 lg; 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 Ca2+

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 GTP solution in an equal

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.

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

Arabidopsisand 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 Ca2+ 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

Ca2+[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 Ca2+ 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 of32P released

from [c- 32 P]GTP were determined.

using monodansylcadaverin as primary amine LAV1-2 has

recently been characterized as CBP40,which reversibly

forms large aggregates in a Ca2+-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

C O N C L U S I O N S

In summary,we have cloned TGase cDNA from P

poly-cephalumplasmodia 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

A C K N O W L E D G E M E N T S

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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