Báo cáo khoa học: Identification of substrates for transglutaminase in Physarum polycephalum, an acellular slime mold, upon cellular mechanical damage ppt

We observed transglutimase reaction products at injured sites in Physarum macroplasmodia upon mechanical damage.. Abbreviations ANT, adenine nucleotide translocator; Bio-Cd, biotinylated

in Physarum polycephalum, an acellular slime mold,

upon cellular mechanical damage

Fumitaka Wada1,*, Hiroki Hasegawa1, Akio Nakamura2, Yoshiaki Sugimura1, Yoshiki Kawai1,

Narie Sasaki3, Hideki Shibata1, Masatoshi Maki1and Kiyotaka Hitomi1

1 Department of Applied Molecular Biosciences, Graduate School of Bioagricultural Sciences, Nagoya University, Japan

2 Department of Molecular and Cellular Pharmacology, Faculty of Medicine, Gunma University Graduate School of Medicine,

Maebashi, Japan

3 Graduate Division of Life Science, Graduate School of Humanities and Sciences, Ochanomizu University, Tokyo, Japan

The transglutaminase (TGase; EC 2.3.2.13) enzyme

family catalyzes the Ca2+-dependent crosslinking of

the c-carboxyamide group of glutamine residues and

the e-amino group of lysine residues or primary amines [1,2] This reaction results in the formation of an iso-peptide bond between two proteins and the covalent

Keywords

adenine nucleotide translocator; calcium;

mechanical damage; Physarum

polycephalum; transglutaminase

Correspondence

K Hitomi, Department of Applied Molecular

Biosciences, Graduate School of

Bioagricultural Sciences, Nagoya University,

Chikusa, Nagoya, 464-8601, Japan

Fax: +81 52 789 5542

Tel: +81 52 789 5541

E-mail: hitomi@agr.nagoya-u.ac.jp

*Present address

RIKEN Brain Science Institute, Hirosawa,

Wako-shi, Saitama, Japan

Database

The nucleotide sequence of the Physarum

polycephalum adenine nucleotide

transloca-tor is available in the DDBJ ⁄ EMBL ⁄

Gen-Bank database under accession number

AB259838

(Received 2 August 2006, revised 17 March

2007, accepted 26 March 2007)

doi:10.1111/j.1742-4658.2007.05810.x

Transglutaminases are Ca2+-dependent enzymes that post-translationally modify proteins by crosslinking or polyamination at specific polypeptide-bound glutamine residues Physarum polycephalum, an acellular slime mold,

is the evolutionarily lowest organism expressing a transglutimase whose primary structure is similar to that of mammalian transglutimases We observed transglutimase reaction products at injured sites in Physarum macroplasmodia upon mechanical damage With use of a biotin-labeled primary amine, three major proteins constituting possible transglutimase substrates were affinity-purified from the damaged slime mold The purified proteins were Physarum actin, a 40 kDa Ca2+-binding protein with four EF-hand motifs (CBP40), and a novel 33 kDa protein highly homologous

to the eukaryotic adenine nucleotide translocator, which is expressed in mitochondria Immunochemical analysis of extracts from the damaged macroplasmodia indicated that CBP40 is partly dimerized, whereas the other proteins migrated as monomers on SDS⁄ PAGE Of the three pro-teins, CBP40 accumulated most significantly around injured areas, as observed by immunofluoresence These results suggested that transgluti-mase reactions function in the response to mechanical injury

Abbreviations

ANT, adenine nucleotide translocator; Bio-Cd, biotinylated cadaverine; CBB, Coomassie Brilliant Blue R250; CBP40, 40 kDa Ca 2+ -binding protein; DAPI, 4¢,6-diamidino-2-phenylindole; F-Cd, fluorescein cadaverine; HMC, Hepes-based magnesium and calcium buffer; KLH, keyhole limpet hemocyanin; PpANT, adenine nucleotide translocator from Physarum polycephalum; PpTGase, transglutaminase from Physarum polycephalum; PVDF, poly(vinylidene difluoride); TGase, transglutaminase.

incorporation of polyamines into proteins In

mam-mals, the crosslinking activity of several TGase

iso-zymes functions in blood coagulation, stabilization of

extracellular matrix, apoptosis, and skin barrier

forma-tion [3–7]

Similar crosslinking reactions are observed in

var-ious organisms, from microorganisms to animals

TGases with papain-like characteristics, such as Ca2+

-dependency and an active-center Cys residue, have

been identified in vertebrates and arthropods [1,2,8,9]

In bacteria, yeasts, and lower invertebrates such as

nematodes, genes encoding homologous proteins have

not been found [2,9,10] We, however, have reported

that Physarum polycephalum, an acellular slime mold,

is the evolutionarily lowest organism with a TGase

that has a primary structure similar to that of TGases

in mammals [11,12]

Physarum polycephalum, which belongs to the

My-cetozoa, is a model eukaryote with a unique life

cycle characterized by spores, amoebae,

macro-plasmodia, and microplasmodia The plasmodium,

used in this study, is a giant and multinucleated cell

with a veined structure and no internal cell walls So

far, Physarum has been used mainly in studies on

the cell cycle, inheritance of mitochondrial DNA,

and cytoplasmic streaming [13–18] Physarum is also

an appropriate model organism for studies on

responses to environmental stress For example, in

response to heat stress, Physarum enhances

glycosyla-tion of membrane sterol to induce its signal

trans-duction system to synthesize heat shock proteins

[19] Also, Physarum TGase activity is induced upon

exposure to ethanol or detergent, resulting in

tran-samidation of proteins [20]

In mammals, there are several reports that TGase

is activated in protective responses to environmental

stimuli and contributes to wound healing in various

cells [21–26] In some of these events, remodeling and

stabilization of extracellular matrix proteins by TGase

resulted in repair of chemical and mechanical injury

However, TGase substrates and their potential roles

in repair of damage in unicellular organism are

unknown

In this study, we further investigated the role of

P polycephalum TGase (PpTGase) in response to

mechanical damage Following mechanical damage, we

observed TGase reaction products around the

mechan-ically injured area On the basis of these observations,

we identified and characterized three preferred

gluta-mine-donor TGase substrates: 40 kDa Ca2+-binding

protein (CBP40) [27,28], Physarum actin [29], and a

novel protein with high structural similarity to

eukary-ote adenine nucleotide translocator (ANT)

Results Detection of TGase reaction products around injured areas

To investigate whether PpTGase is involved in the response to mechanical damage, we examined in situ enzymatic reactions in slime mold macroplasmodia fol-lowing injury As shown in Fig 1, after cells were stabbed with a toothpick, fixed proteins into which flu-orescein cadaverine (F-Cd) was incorporated by TGase catalysis were observed around the injured area This reaction was completely blocked by several inhibitors

of TGase, such as L-682.777, cystamine, and cadaver-ine These results indicate that labeled primary amine was incorporated into several glutamine-donor sub-strates by activated TGase upon mechanical damage

Purification of potential PpTGase substrates upon mechanical damage

Next, we identified the glutamine-donor substrate pro-teins that incorporated primary amines in response to damage in macroplasmodia Total cellular lysates were prepared from macroplasmodia damaged in the pres-ence of biotinylated cadaverine (Bio-Cd) Depending

on the time after injury, Bio-Cd was incorporated into several proteins (Fig 2) In control cells with no dam-age (both at 10 s and 180 s), only nonspecific bands (marked at the right with asterisks) were observed; those bands probably represent endogenous biotin-conjugating and biotin-binding proteins Furthermore,

no specific incorporation was observed in the copres-ence of several inhibitors or in the abscopres-ence of Bio-Cd During the assay period, levels of expressed PpTGase remained equivalent, as indicated by immunoblotting (Fig 2, lower panel) These results indicated that PpTGase catalyzed transamidation of several proteins acting as preferred glutamine-donor substrates when activated upon mechanical injury

Next, we purified these candidate substrates As they are likely to be attached to the plasma membrane, a soluble membrane fraction obtained by Triton X-100 treatment was subjected to purification As shown in Fig 3, three major proteins (p44, p40, and p33) were eluted as potential substrates, and these proteins were not obtained with the same procedure in the absence

of Bio-Cd (lane 7) Using peroxidase-conjugated streptavidin, the eluted proteins were detected as bio-tin-incorporated proteins (Fig 3B) In this fraction, there were other minor proteins as possible substrates, the amounts of which were not sufficient for the fol-lowing analysis The proteins in the gel were subjected

to trypsinization and then to TOF MS analysis On the basis of data in the database of molecular masses

of fragmented proteins, p40 and p44 were identified as CBP40 [27,28] and Physarum actin [29,30], respectively, whereas p33 was a novel protein not found in the database

Purification and molecular cloning of a novel

33 kDa substrate protein

In order to identify p33, we purified the protein by affinity chromatography and SDS⁄ PAGE Because the N-terminus of the protein was blocked, purified p33 was treated with cyanogen bromide, and the resul-ting fragments were subjected to amino acid sequence analysis

On the basis of the partial amino acid sequence of one fragment, a cDNA clone encoding p33 was obtained by 3¢-RACE using degenerate primers: 5¢-RACE resulted

in 5¢-nucleotide sequences that probably include the ini-tiation codon ATG (Fig 4) The complete sequence shows an ORF of 936 bp encoding 312 amino acids with

a calculated molecular mass of 33 622 Da The amino acid sequence deduced from the nucleotide sequence was highly homologous to that of the ANT seen in sev-eral eukaryotes, and we therefore designated the protein

no damage

(10 s

)

10 s 30

s

60 s

180 s no damage

(18

0s)

+ cystamin e

- Bio-C d + L-682

.77 7

+ cadaveri ne

(kDa)

PpTGase

*

97

66

45

30

*

Fig 2 Detection of total cellular proteins that incorporated Bio-Cd

upon mechanical damage At time 0 s, growing macroplasmodia on

an agar plate were injured in the presence of Bio-Cd Total cellular

extracts of macroplasmodia were prepared at the indicated periods.

Samples were subjected to 10% SDS ⁄ PAGE and transferred to

PVDF membranes Top: Proteins incorporating Bio-Cd were

detec-ted using peroxidase-conjugadetec-ted streptavidin Samples from cells

without damage (10 s and 180 s) and from damaged cells (180 s)

in the presence of L-682.777 (40 l M ), cystamine (20 m M ) or

cada-verine (20 m M ), or in the absence of Bio-Cd, were prepared in

par-allel The asterisks indicate no specific signals Bottom: All samples

were subjected to immunoblotting using a monoclonal antibody to

PpTGase.

+cystamine

DIC

200 µm F-Cd

Fig 1 Incorporation of F-Cd into glutamine-donor substrates at injured sites in macroplasmodia Macroplasmodia grown on a PVDF mem-brane were injured in the presence of F-Cd After 3 min, the cells were fixed, and differential interference images (DIC) and fluorescent ima-ges (F-Cd) of the cells were obtained The same experiment was performed in the copresence of 40 l M L-682.777, 20 m M cystamine or

20 m M cadaverine in F-Cd solution The bar represents 200 lm.

(kDa) 97 66 45 30

p44 p40 p33

A

p44 p40 p33

97 66 45 30

B

(kDa)

Fig 3 Purification of proteins incorporating Bio-Cd from damaged slime mold The total cellular extract, cytosolic fraction and Triton X-100 soluble membrane fraction were prepared from Physarum macroplasmodia injured in the presence of Bio-Cd From the membrane fraction, proteins incorporating Bio-Cd were affinity-purified with streptavidin-sepharose To compare them with nonspecifically bound proteins, the same procedure without addition of Bio-Cd was also performed (A) CBB staining (B) Detection of biotinylated proteins by peroxidase-conju-gated streptavidin In both panels, lanes are as follows: lane 1, total cellular extract; lane 2, cytosolic fraction; lane 3, Triton X-100 soluble fraction; lane 4, dialyzed Triton X-100 soluble fraction (applied sample); lane 5, unbound fraction; lanes 6 and 7, eluted fractions from extracts prepared in the presence and absence of Bio-Cd, respectively.

Fig 4 Nucleotide and deduced amino acid

sequences of PpANT The complete amino

acid sequence of PpANT was deduced from

the nucleotide sequence The numbers of

nucleotide and amino acid residues are

shown on the left and right sides,

respect-ively The gray background indicates the

fragment cleaved by cyanogen bromide

treatment of the purified protein.

as PpANT (for P polycephalum ANT) The amino acid

sequence of PpANT was 50–77% identical to those of

human (ANT1, NP_001142; ANT2, NP_001143;

ANT3, NP_001627), mouse (ANT1, NP_031476;

ANT2, NP_0031477), bovine (NP_777083),

Caenorhab-ditis elegans(NP_001022799), Dictyostelium discoideum

(XP_647166), Arabidopsis thaliana (NP_850541), Zea

mays (CAA40781) and Saccharomyces cerevsiae

(NP_009523) homologs (Fig 5) From the PpANT

pri-mary structure, six possible membrane-spanning regions

were deduced from the distribution of hydrophobic

regions, as is observed in ANTs of other species

Although the initiation codon (ATG) was deduced from

the alignment, recombinant protein produced from

expression of the full-length cDNA in bacteria was of

the predicted size (data not shown)

It is known that eukaryote ANT is the most

abun-dant protein in mitochondria [31] We also investigated

the cellular distribution of PpANT in Physarum

macr-oplasmodia using a polyclonal antibody The cell was

counterstained with 4¢,6-diamidino-2-phenylindole

(DAPI) to visualize both the nucleus (Fig 6, arrow)

and mitochondrial nucleoid (Fig 6, arrowhead) By

phase-contrast (Fig 6A) and DAPI fluorescence

micr-oscopy (Fig 6B), mitochondria of macroplasmodia

were observed as oval-shaped structures and each of

them contained a rod-like mitochondrial nucleoid

Fluorescence immunostaining microscopy revealed that

Fig 5 Multiple alignment of PpANT with several eukaryotic ANTs Amino acid sequences were aligned using the default setting of CLUSTAL X ,

a multiple sequence alignment program Amino acid residues common to all sequences are denoted by an asterisk above the sequences, whereas conservative residues are indicated by a colon (: high) or a period ( low).

5 µm

Fig 6 Immunolocalization of PpANT in Physarum macroplasmodia.

A growing macroplasmodum was fixed and reacted with polyclonal antibody to PpANT and then developed by an Alexa Fluor 488-con-jugated secondary antibody Mitochondrial and nuclear DNAs were counterstained with DAPI (A) Merged image of phase-contrast and DAPI staining (B) DAPI staining image (C) Immunostaining image obtained using antibody to PpANT (D) Merged image of (B) and (C) The arrows and arrowheads indicate nucleus and mitochondria, respectively Enlarged images are shown in the inset The bar rep-resents 5 lm.

the staining patterns of PpANT coincided with the

mitochondria, as was expected (Fig 6C,D)

Immunoblotting analysis of potential substrates

upon mechanical damage

In order to find how these possible substrates reacted

with PpTGase upon cellular injury, we performed

immunoblotting of total cellular extracts (Fig 7, left)

The first identified substrate, CBP40, migrated as a

40 kDa protein, but the levels of a higher molecular

mass band (80 kDa), probably corresponding to a

dimer, increased over time The slight band (80 kDa)

with no damage was produced during the preparation

of extracts This crosslinked product was not observed

in the presence of cystamine, suggesting that CBP40

was dimerized by TGase in response to mechanical

damage PpANT and actin were detected at the pre-dicted monomeric size, without no possible dimer form

Affinity-purified proteins incorporating Bio-Cd were recognized by respective antibodies (Fig 7, eluted frac-tion), confirming that proteins were transamidated upon injury Taken together, these results suggested that CBP40, actin, and PpANT are enzymatically modified by PpTGase in mechanically damaged macro-plasmodia

Cellular analysis of potential substrates in injured macroplasmodia

To investigate the localization of potential substrates around the injured area, each protein was analyzed by immunostaining in cells (Fig 8) In the absence of cell

no damage (10s) 10 s 30 s 60 s 180 s no damage (180 s)+cystamine (kDa)

97 66 45 30

97 66 45 30

97 66 45 30

eluted fraction(Fig 3, lane 6)

CBB

anti-CBP40

anti-actin

(Physarum)

anti-PpANT

97 66 45 30

no damage (10s) 10 s 30 s 60 s 180 s no damage (180 s)

+ cystamine (kDa)

(kDa)

(kDa)

eluted fraction(Fig 3, lane 6)

A

B

C

Fig 7 Immunoblot analysis of potential PpTGase substrates upon cellular injury Total cellular extracts were prepared at the indicated times (10–180 s) from damaged macroplasmodia growing on a plate Upon injury, HMC buffer was added to the plate, and cells were stabbed with toothpicks several times As a control, cystamine was added to block the TGase reaction The right lanes of all blots contains purified pro-tein, which incorporated Bio-Cd from injured macroplasmodia using streptavidin-sepharose chromatography (Fig 3, lane 6) Samples were subjected to SDS ⁄ PAGE followed by CBB staining (right) and immunoblotting analysis using each polyclonal antibody (left): (A) anti-CBP40; (B) anti-Physarum actin; (C) anti-PpANT The closed arrows indicate each protein The open arrow indicates a possible CBP40 dimer.

damage (J, K, L), all proteins were stained uniformly.

CBP40 protein was strongly stained around the injured

area (D, G), suggesting that this protein accumulates

or is aggregated upon damage However, both

Physa-rum actin and PpANT showed no apparent difference

in staining pattern in injured versus noninjured areas

(E, F, H, I)

Discussion

Although most eukaryotic cells and tissues exhibit

pro-tective elements, cells can suffer damage following

environmental insult To respond to mechanical

dam-age, adaptive systems have been developed not only at

the tissue level but also at the cellular level Membrane

resealing, for example, triggered by Ca2+ entry upon

disruption, is a membrane-repair process allowing

cells to survive [32,33] Although it is likely that

various molecules and mechanisms participate in

responses to mechanical challenge, the process is not

well understood

TGases are Ca2+-dependent crosslinking enzymes,

and are thus likely to function in such mechanisms

[1,2] Indeed, in mammals, it has been shown that

TGases respond to environmental attack by

participa-ting in wound healing [21–26] In fibroblasts, for

exam-ple, TGase maintains tissue integrity by formation of

an SDS-insoluble shell-like structure following rapid

loss of Ca2+homeostasis [23]

We have focused on the physiologic significance of

TGase in Physarum, as this is the lowest known

organism exhibiting a TGase similar to that expressed

in mammals [11,12] Upon mechanical damage,

Physarum displayed TGase-dependent incorporation

of a fluorescent-labeled primary amine into gluta-mine-donor substrate protein(s) (Fig 1) The product was observed around the mechanically injured area, suggesting that Ca2+ influx activated a latent form of intracellular TGase since PpTGase is Ca2+-dependent

as in the case for mammalian TGase [11] The sub-strate proteins might localize around the membrane that activated TGase can access Based on time-dependent transamidation, as shown in Fig 2, several proteins underwent modification without change in the amount of PpTGase, indicating that endogenous TGase activity was stimulated by damage Although unidentified minor proteins in the purified fraction may also be substrates, the further analyzed gluta-mine-donor substrates consisted of mainly three pro-teins: actin, CBP40, and PpANT This observation is consistent with the fact that chemical damage of Physarum microplasmodia by treatment with ethanol

or detergent results in transamidation of actin and CBP40 [20]

Mechanical damage also resulted in the crosslinking

of CBP40 to form a covalently bound dimeric form, and enhanced its levels around the injured area CBP40, which has four EF-hand motifs in the C-termi-nus and a putative a-helix domain in the N-termiC-termi-nus, aggregates reversibly in a Ca2+-dependent manner via the N-terminus in vitro [27] TGase may contribute to self-assembly of CBP40, where the crosslinked dimer form acts as core to initiate further assembly Although CBP40 orthologs in other organisms have not been reported, such a crosslinking reaction is remi-niscent of clot formation in vertebrates

injured area

uninjured area DIC

anti-CBP40

anti-actin

(Physarum)

anti-PpANT

100 m

100 m

injured area A

B

C

D

E

F

G

H

I

J

K

L

Fig 8 Immunostaining of potential PpTGase substrates in macroplasmodia A macro-plasmodium grown on a PVDF membrane was injured by a toothpick (A–C; DIC, differ-ential interference images) Then, fixed and permeabilized cells were immunostained with respective antibodies against CBP40 (D,

G, J), Physarum actin (E, H, K), and PpANT (F, I, L) using an Alexa Fluor 488-conjugated secondary antibody The indicated injured region is enlarged (G–I; box in panels D–F) Immunostaining analyses for uninjured areas are shown at the same scale in parallel (J–L) All immunostained signals are shown as stacked images in the vertical direction The bars represent 10 lm and 100 lm.

Both actin and PpANT, identified as potential

sub-strates, also incorporated Bio-Cd by transamidation

upon mechanical damage, although significant

aggre-gation or accumulation was not observed by

immuno-staining In western blot analysis, actin and PpANT

did not show apparent changes in molecular size

following damage, suggesting that they are modified

by transamidation or deamidation, as reported for

several substrates [34–36] Physarum actin, which is

highly homologous to mammalian actin, is implicated

as a force-generating system in actomyosin fibrils

[29] In mammals, actin, as both G-actin and F-actin

is a favorable TGase 2 substrate in vitro [37,38] In

this study, the distribution of actin was not affected

by injury in the presence or absence of a TGase

inhibitor (Fig 6, and data not shown) In Physarum,

monomer actin might not be affected even after

modification

PpANT, another potential TGase substrate, was

cloned for the first time in this study On the basis of

its considerable homology to ANTs in other

eukaryo-tes and observation of its exclusive localization in

mitochondria, it is likely that PpANT functions as an

antiporter mediating ADP⁄ ATP exchange in the slime

mold Although we could not show the localization of

PpTGase in mitochondria, TGase activity was detected

in the purified mitochondrial fraction in mammalian

liver and brain [40] Additionally, in

TGase2-over-expressing cells, TGase2 has been reported to localize

to mitochondria upon induction of apoptosis [41]

Determining whether transamidation by PpANT

regu-lates ATP-translocating activity or induces apoptosis

will require further study

As shown in Fig 3B, there were minor

biotin-incor-porating proteins present upon injury As recovery

from cellular damage might require more than three

major substrates, further investigation of unidentified

substrates and crosslinking reactions would be

neces-sary We have recently established a system to identify

the TGase preferred substrate sequence with respect to

mammalian TGases [42] Applying this system to the

identification of PpTGase preferred substrates should

reveal other substrates and potentially define a

net-work of substrates Additionally, knockdown analyses

of TGases and their substrates by an RNA interference

method that has recently been established in this

organism might be also useful [43]

Although little is known about the physiologic

func-tions of TGases in nonmammalian species, there are

several reports of TGases being essential for defense

against environmental factors [8,44,45] Cellular

responses to mechanical damage are required for

euk-aryotes to maintain their homeostasis In the horseshoe

crab, for example, TGase is implicated in the forma-tion of coagulin polymers upon aggregaforma-tion of hemo-cytes, and it also crosslinks several chitin-binding proteins in the cuticle [8,46] As evolutionarily lower organisms do not possess an acquired immune system, TGase activity may be particularly important in defending these organisms against environmental challenges Further investigation of possible TGase substrates in the slime mold should provide insights into the responses of eukaryotic cells to mechanical damage

Experimental procedures Cell culture

Physarum macroplasmodia were basically grown on 1.5% agar plates containing MEA medium consisting of 0.165% mycological peptone (Oxoid, Basingstoke, UK), 1% malt extract (Oxoid), and 5 lgÆmL)1 hemin (ICN Biomedicals Inc., Irvine, CA) [13,14] In the case of observation of the fixed macroplasmodia, cells were grown on a poly(vinylid-ene difluoride) (PVDF) membranes (Millipore, Bedford, MA) located on the agar plate Both cultures were grown

in complete darkness at 25C

Incorporation of F-Cd into PpTGase substrate

in the damaged slime mold

Macroplasmodia cells grown on a PVDF membrane were transferred to a 35 mm dish containing Hepes-based mag-nesium and calcium buffer (HMC; 20 mm Hepes⁄ NaOH,

pH 7.4, 10 mm NaCl, 40 mm KCl, 2 mm CaCl2, 7 mm MgCl2) F-Cd (Invitrogen, Carlsbad, CA) was added to a final concentration of 0.1 mm, and then the cells were injured by stabbing them with a toothpick After 3 min, cells on a PVDF membrane were washed with HMC buf-fer and then fixed at room temperature for 15 min in a solution of 10% trichloroacetic acid The cells were then washed with NaCl⁄ Pi buffer (10 mm sodium phosphate,

pH 7.4, 150 mm NaCl) three times, and incubated in NaCl⁄ Pi buffer containing 1.0% Triton X-100 for 30 min

at room temperature Cells were removed from the mem-brane, and located on a coverslip coated with 0.01% poly(l-lysine) After drying, these samples were mounted

on a glass slide with antifading solution containing Mowiol 4-88 (Calbiochem, Darmstadt, Germany) and glycerol Samples were analyzed under a confocal laser-scanning microscope (LSM5 PASCAL; Zeiss, Gottingen, Germany)

Cystamine (Sigma, St Louis, MO), cadaverine (Sigma), and L-682.777 (N-Zyme, product name: 1,3,4,5-tetrameth-yl-2-[(2-oxopropyl)thio]imidazolium chloride) were used to inhibit the enzymatic reaction by PpTGase

Detection and purification of TGase substrates

upon cellular damage to macroplasmodia

HMC buffer containing Bio-Cd at a final concentration

of 0.2 mm was added to macroplasmodia growing on

MEA agar plates The cells were injured with a bundle

of toothpicks several times, as described above After

var-ious periods, the TGase reaction was halted by the

addi-tion of cystamine From the cells homogenized with lysis

buffer (20 mm Tris⁄ Cl, pH 7.5, 100 mm NaCl, 2 mm

2-mercaptoethanol, 20 mm cystamine, 1 mm

phenyl-methylsulfonylfluoride, 25 ngÆlL)1 leupeptin, and 1 lm

pepstatin), total cell extract was prepared by

solubiliza-tion with SDS-dye buffer and boiled For detecsolubiliza-tion of

Bio-Cd incorporated into cellular proteins, the proteins

were subjected to SDS⁄ PAGE and blotted onto a PVDF

membrane, which was then developed by

peroxidase-con-jugated streptavidin (Rockland, Gilbertsville, PA) and the

chemiluminescent method using the Super Signal West

Pico chemiluminescent substrate detection kit (Pierce,

Rockland, IL)

For purification of potential TGase substrates, the

dam-aged slime mold in the presence of Bio-Cd was harvested

after 3 min The cells were washed and suspended by lysis

buffer The harvested cells were homogenized and

centri-fuged at 10 000 g for 10 min using a SRX-4 centrifuge

(TOMY) and TA-4 rotor The unsolubilized fraction was

treated with the TNE buffer (20 mm Tris⁄ HCl, pH 7.5,

100 mm NaCl, 5 mm EDTA, 2 mm 2-mercaptoethanol)

containing 2% Triton X-100, 20 mm cystamine and

prote-ase inhibitors for 1 h at 4C The membrane fraction was

obtained as a supernatant by centrifugation [10 000 g for

20 min using a SRX-4 centrifuge (TOMY) and TA-4 rotor,

and 100 000 g for 30 min using TL100 centrifuge

(Beck-man) and TLA100.3 rotor] The supernatant was dialyzed

against TNE buffer overnight to remove unincorporated

Bio-Cd, and then applied to a streptavidin-conjugated

col-umn previously equilibrated with the same buffer After

several washings with TNE buffer, the bound proteins were

eluted with 1 mm Tris⁄ Cl buffer (pH 8.0) containing 4%

SDS buffer The eluate was concentrated and subjected to

SDS⁄ PAGE following by Coomassie Brilliant Blue (CBB)

staining The protein bands of interest were excised and

further analyzed by using standard MALDI-TOF MS

methodology

To identify p33 protein, the protein was excised from

12.5% SDS⁄ PAGE gel and then subjected to

carbamidome-thylation using iodoacetoamide The protein concentrated

by acetone precipitation was dissolved in 70% formic acid,

and treated with cyanogen bromide at room temperature

for 24 h in the dark The reaction product was separated

on a 15% SDS⁄ PAGE gel and transferred to a PVDF

membrane The cleaved protein bands were excised and

sequenced by automated Edman degradation

Molecular cloning of a novel 33 kDa protein

3¢-RACE was performed with the RNA LA PCR Kit Ver.1.1 (TAKARA Biomedicals, Japan) Total RNA from macroplasmodia was obtained by the acid guanidium phe-nol chloroform method The first-strand cDNA was syn-thesized using 1 lg of total RNA in a reaction mixture of 1.0 mm dNTPs, 16 U of RNasin, 14 U of AMV reverse transcriptase, and oligo dT-M4 adaptor primer in the sup-plied buffer The resulting cDNAs were subjected to PCR with M13 primer M4 and the degenerate primer 5¢-GCT GGAGCTGCT(A⁄ T)(C ⁄ G)(A ⁄ T ⁄ G ⁄ C) (C ⁄ T)T(A ⁄ T ⁄ G ⁄ C) AC(A⁄ T ⁄ G ⁄ C)TTTGT-3¢, which was designed on the basis

of the amino acid sequence AGAASLTFVY Amplification conditions were as follows: 30 cycles at 95C for 30 s,

51C for 30 s, and 72 C for 90 s

The PCR products obtained from 3¢-RACE was cloned into a TA-cloning vector, pCR 2.1-TOPO (Invitrogen, Car-lsbad, CA), according to the manufacturer’s instructions The nucleotide sequences of the isolated clones were deter-mined with an automated fluorescent sequencer, ABI PRISM 310 (PE Applied Biosystems, Foster City, CA), using a Bigdye terminator cycle sequencing ready reaction kit (PE Applied Biosystems)

In order to obtain 5¢-terminal cDNA, 5¢-RACE was per-formed using reverse transcriptase and RNA ligase, accord-ing to the manufacturer’s protocols (5¢-Full RACE Core Set; TAKARA Biomedicals) First-strand cDNA was syn-thesized from 1 lg of the poly(A)+RNA, purified with an oligo(dT) cellulose column, using AMV reverse transcrip-tase XL with a specific primer, 5¢- TAGAGACCAGTGA TACCATC-3¢ (antisense, nucleotide sequence number 577–596), and then phosphorylated by T4 polynucleotide kinase After degradation of the template poly(A)+RNA with RNaseH at 30C for 1 h, the resulting single-strand cDNA was precipitated with ethanol and dissolved in

40 lL of a reaction mixture containing 20% poly(ethylene glycol) #4000, RNA ligation buffer, and 1 U of T4 RNA ligase To change the cDNAs to circular and⁄ or concatemer cDNAs, the reaction solution was incubated at 15C for

16 h The cDNAs were directly used as a template for the first PCR amplification with primers 5¢-GGTGAACGCCA GTTCAATGGC-3¢ (S1, sense, 523–543) and 5¢-CGGACT TGTTGTCGTTAGCCAAAC-3¢ (A1, antisense, 485–508), which correspond to the cDNA sequence obtained by 3¢-RACE The reaction was carried out for 30 cycles with the following conditions: 94C for 30 s, 53 C for 30 s, and 72C for 90 s The resulting PCR product was diluted 1000-fold with sterile H2O, and a 1 lL aliquot was used as

a template for the second nested PCR amplification with primers 5¢-GCTTGCTGGATGTCTACAGAAAGACC-3¢ (S2, sense, 542–567) and 5¢-ACGAGTACGGGCGTAGTC GAG-3¢ (A2, antisense, 466–486) under the same condi-tions Additional 5¢-RACE reactions using other primers

[5¢-AGCAGCGATGTTG-3¢ (antisense, 411–423), 5¢-GAA

TGTTCGCTGTCCCCAAG-3¢ (sense, 362–381), 5¢-GTCC

TTGAAGGCGAAGTTGAG-3¢ (antisense, 331–351),

5¢-GCCTCCTACGGAAAGAAGTTC-3¢ (sense, 385–405)

and 5¢-CTTGGGTGGGGAAGTAACGG-3¢ (antisense,

309–328)] produced putative full-length cDNA Cloning

and nucleotide sequencing were carried out as described for

3¢-RACE

Finally, after completion of cloning of putative

full-length cDNA, oligonucleotides encoding 5¢- and 3¢-ends

were prepared (5¢-CTGGATCCCGAGAAGAAGAACGA

CCTCAG-3¢ and 5¢-GATGCTCGAGTTATCCACCTCCG

CCAGAG-3¢), and used for PCR reaction to obtain

directly full-length cDNA

Polyclonal antibodies

Polyclonal antibody against Physarum actin was kindly

pro-vided by K Furuhashi (Shiga University, Japan) [30]

Anti-bodies against PpTGase [12], and CBP40 [27] were

prepared as described previously Polyclonal antibody

against PpANT was prepared by immunization of peptide

conjugated with keyhole limpet hemocyanin (KLH; Sigma)

On the basis of the deduced amino acid sequence, a peptide

(YDSLKPALSPLENNPVALGC) corresponding to the

amino acid sequence of region 199–217 with an additional

Cys residue at the C-terminus was synthesized Then, the

Cys residue of the peptides was covalently crosslinked with

KLH using m-maleimidobenzoil-N-hydroxysuccinimide

ester, and used as immunogen to raise antibody in rabbit

By subcutaneous immunization of the peptide–KLH six

times, antiserum was prepared The antibody was

affinity-purified from antisera using a column that immobilized the

peptide

Immunologic analysis of potential substrates

from total cellular lysates

For western blotting, total cellular extracts were prepared

from the injured macroplasmodia by stabbing with

tooth-picks as described above The harvested cells were

homo-genized with lysis buffer, and then solubilized directly in

SDS sample buffer Next, the samples were subjected to

SDS⁄ PAGE and western blotting using PVDF membranes

Antibodies were reacted by standard methods, and

immuno-signals were detected by the chemiluminescent method as

described above

Immunostaining analysis

Macroplasmodia cells grown on a PVDF membrane were

damaged and fixed as described above After being washed

with NaCl⁄ Pi, cells were incubated in NaCl⁄ Pi containing

1% BSA to prevent nonspecific binding for 1 h at 37C

Then, the cells fixed by trichloroacetic acid solution were incubated in the presence of each polyclonal antibody Sub-sequently, cells were incubated with Alexa Fluor 488-conju-gated goat anti-rabbit serum (Molecular Probes) Samples were analyzed with a confocal laser-scanning microscope (Zeiss) as described above, using 488 nm and 505–530 nm filters The software used was lsm image browser (Zeiss)

In the case of counterstaining of DNA (Fig 6), cells were fixed by 3.7% formaldehyde for 15 min, and then subjected

to the immunostaining reaction as described above Before mounting of samples on a glass slide, DNA was counter-stained with DAPI Cells were observed under an epifluo-rescence microscope equipped with a phase-contrast objective (Olympus, Tokyo, Japan)

Acknowledgements This work was supported by a Grant-in-Aid for

Scienti-fic Research (C) no 14560063 (to K Hitomi), Young Scientist Research grant no 15000941 (to F Wada), and a TOYOAKI Science Foundation grant (to

K Hitomi) We thank Dr K Furuhashi (Shiga Univer-sity, Japan) for providing us with antibody to Physarum actin F Wada and Y Sugimura are Japanese Society for the Promotion of Science (JSPS) Research Fellows

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