Jpn. J. Protozool. Vol. 43, No. 1. (2010)19

Short Communication

Identification of the Dictyostelium discoideum gene for a protein showing
sequence similarity to iron superoxide dismutase
Kosuke NOMOTO, Yosuke UMEDA,
KATAYAMA and Hiro YASUKAWA*

Jin-ichiTANIGUCHI,

Takahiro

Graduate School of Science and Engineering, University of Toyama, Toyama 930-8555,
Japan.
Key words: Dictyostelium discoideum, iron superoxide dismutase

SUMMARY
Some protozoan parasites encode iron superoxide dismutase (FeSOD), but mammalians
do not, and FeSOD is therefore an attractive target for drug therapy. Database screening revealed
that the social amoeba Dictyostelium discoideum
encodes a protein similar to FeSOD. Since D.
discoideum is non-pathogenic and easy to maintain in laboratory conditions, analysis of the protein would contribute to drug design and chemotherapy for infectious diseases. RT-PCR analysis
showed that the gene for the protein was expressed at a high level in growing cells and atdecreased levels in subsequent developmental
stages. Fluorescence of green fluorescent protein
fused with the N-terminal region (Met1 - Leu35)of the D. discoideum protein was detected in mi*Corresponding author
Tel/Fax: 81-76-445-6875
E-mail: hiro@eng.u-toyama.ac.jp
Received: 23 April 2009; Accepted: 14 July 2009.

tochondria, indicating that the protein is localized
in mitochondria.
Oxidative stress that results from the generation of reactive oxygen species (ROS) is a significant source of cellular and DNA damage. Organisms have various enzymes such as superoxide
dismutase (SOD), catalase and peroxidase for detoxifying ROS. SOD converts superoxide anions toH2O2, which is then disproportionated to water bycatalase or peroxidase. Because of its importantrole, SOD is conserved widely in organisms. According to the metal ion cofactors identified in theactive sites, SODs of eukaryotes are classified ascopper/zinc SOD (Cu/ZnSOD), which are present
in bacteria, fungi, animals and plants; manganese
SOD (MnSOD), which have been found in bacteria and mitochondria; and iron SOD (FeSOD),
which have been detected in bacteria, chloroplasts
and protozoa (Fridovich, 1995; Miller, 2004).
Mammalians, including humans, generally possess
both Cu/ZnSOD and MnSOD but lack FeSOD,
whereas some protozoan parasites encode FeSOD.

20D. discoideum protein similar to FeSOD

Therefore, FeSOD is an attractive target for drugtherapy.
The social amoeba Dictyostelium discoideum isa model organism suitable for studying the molecular basis of cell biology. Previous studies andscreening in a database for D. discoideum (http://www.dictybase.org/) have shown that D. discoideum encodes one MnSOD designated SodM
(Akaza et al., 2006) and six Cu/ZnSODs designated SodA (Garcia et al., 2000), SodB (Tsuji etal., 2002), SodC (Tsuji et al., 2003), SodD (Akaza,et al., 2002), SodE (found in the database) andSodF (found in the database). Further screening inthe database revealed that D. discoideum encodes aprotein similar to FeSOD of Trichomonas
vaginalis. T. vaginalis infection is one of the mostcommon sexually transmitted human infections.
Metronidazole has been used for treatment oftrichomonosis, but clinical resistance to the agent
has frequently been reported since 1962
(Robinson, 1962; Sobel et al., 1999). Therefore, itis necessary to develop novel anti-trichomonads
agents. In this report, results obtained from analysis of the D. discoideum protein designated
DDB0217949 are described.
The full length of the coding sequence for theprotein was amplified from RNA of Ax2, an axenic strain of D. discoideum, by RT-PCR and thencloned and sequenced (759 bp). Two independent
clones were sequenced. Comparison of the genomic sequence found in the database and the nucleotide sequence determined in this study revealed that the open reading frame is interrupted
by three introns (Fig. 1A). The gene encodes apolypeptide consisting of 252 amino acids (Fig.
1B). Four amino acid residues responsible forFig. 1. Sequence analysis of DDB0217949. (A) Physical map of the gene for DDB0217949. Exons are indicated byboxes. Primers used in this study are indicated by arrowheads. Restriction sites for EcoRV (RV), HindIII (Hd), MfeI(Mf), NcoI (Nc) and SwaI (Sw) are indicated. (B) Nucleotide sequence of the gene and deduced amino acid sequenceof the gene product, DDB0217949, are presented. Coding region of the gene for DDB0217949 was amplified fromtotal RNA by the use of the primers 5’-GCATATGAATAGATATATTGTTAGTAAAACAATGGCATTGGTAC and5’-GCGGCCGCTTAATTATTTGATTGTGAATCAAGAAATTGTTCAAATGC. The reaction products were inserted into the EcoRV site of pT7Blue-2 (Merck, Darmstadt, Germany). Two independent clones were analyzed todetermine nucleotide sequence. Residues that function as ligands for the metal ion are shown in white letters.

Jpn. J. Protozool. Vol. 43, No. 1. (2010)

binding to metal ion cofactor (Edwards et al.,1998) are conserved in DDB0217949 (His51,His102, Asp184 and His188). Analysis of the aminoacid sequence by TargetP 1.1 (http://www.
cbs.dtu.dk/services/TargetP/) and WoLF PSORT(http://wolfpsort.org/) predicted that DDB0217949
is localized in mitochondria. Sequences responsible for mitochondria localization predicted by thesoftwares are Met1 - Tyr27 (TargetP 1.1) and Met1 Lys23 (WoLF PSORT).
The identity between the proteins of D. discoideum
and T. vaginalis is 31%. Amino acid sequences
around the conserved residues are aligned (Fig. 2).
The multiple sequence alignment showed homology between the proteins.
Next, experiments were performed to analyze
the expression pattern of the gene for
DDB0217949 throughout the life cycle of D. discoideum. D. discoideum amoebae grow as unicellular organisms in the presence of nutrients but initiate a developmental process to form a multicellular21

structure under a starved condition. D. discoideum
amoebae aggregate into groups of 105 cells, which
are called mounds. Each mound organizes into a
structure called a slug, and then the slug changes
its form to a fruiting body consisting of a stalk andsporangium (Loomis, 1975). RNA samples were
purified from growing amoebae and developing
cells for RT-PCR analysis. The expression patternof rnlA (Ogawa et al., 2000) was analyzed as acontrol. The results showed that the gene for
DDB0217949 was expressed at a high level ingrowing cells and at decreased levels in subsequent stages (Fig. 3).
Subcellular localization of FeSOD has beenstudied in some protozoan parasites. For instance,localization of FeSOD in Acanthamoeba castellanii was determined by western blots and enzymeassays following the separation of cells into threeparts, a cytoplasmic fraction, a detergentextractable fraction (membrane fraction) and aninsoluble fraction. The experimental results indi-Fig. 2. Alignment of FeSODs. The amino acid sequences around the residues responsible for iron binding are aligned.
The accession numbers of SODs are AAT91955 (Acanthamoeba castellanii SODI), XP_665591 (Cryptosporidium
hominis), DQ156546 (C. parvum Cp-mtSOD), CAA50204 (Entamoeba histolytica), AF003964 (Leishmania chagasiFeSODA), AF003963 (L. chagasi FeSODB1), AF312581 (L. chagasi FeSODB2), AAQ14562 (Leishmania donovaniFeSODA), AAQ14560 (L. donovani FeSODB1), AAQ14557 (L. donovani FeSODB2), AF113157 (Plasmodiumfalciparum PfFeSOD1), AY586514 (P. falciparum PfFeSOD2), AF022423 (Trichomonas vaginalis TvSOD6),
AAX77683 (Trypanosoma brucei SODA), AAX77680 (T. brucei SODB1), AAX77681 (T. brucei SODB2),
AAX77682 (T. brucei SODC), AY864902 (Trypanosoma cruzi FeSODA) and AAC47549 (T. cruzi FeSODB). Sequence analysis was performed by the use of the program ClustalW (http://clustalw.ddbj.nig.ac.jp/top-j.html). Residues that function as ligands for the metal ion are shown in white letters.

22

D. discoideum protein similar to FeSOD

Fig. 3. Expression pattern of the gene for DDB0217949.
Expression levels of the gene in D. discoideum wereanalyzed by RT-PCR. Conditions for cell culture, development, RNA preparation and reaction are the same as
those described in a previous report (Yasukawa, 2009).
Primers used are 5’-GCATATGGCCTCAGAAATGCAAAAAAGATTTATATACACACTCCCAG and5'-GCGGCCGCTTAATTATTTGATTGTGAATCAAGA
AATTGTTCAAATGC. Primers used to amplify rnlA, aninternal marker, are 5’-TTACATTTATTAGACCCGAA
ACCAAGCG and 5’-TTCCCTTTAGACCTATGGACCTTAGCG.

cated that FeSOD of A. castellanii was present in
the cytoplasmic and membrane fractions (Choi etal., 2000). In Trypanosoma brucei, which encodes
four FeSODs, localization of the enzymes wasexamined by expressing c-myc-tagged or GFPtagged FeSODs. The experimental results revealed
that two FeSODs are localized in the glycosome
and that the other two are localized in the mitochondrion (Wilkinson et al., 2006).
To determine the subcellular localization ofDDB0217949, D. discoideum Ax2 was transformed by a plasmid carrying a gene for Nterminal 35 residues of DDB0217949 (Met1 Leu35) fused with GFP. Fluorescence was detectedin mitochondria by microscopic analysis, indicating that DDB0217949 is localized in mitochondria(Fig. 4).
A previous study has shown that SodM is also
localized in D. discoideum mitochondria(Yasukawa, 2009). However, it is not clear at present whether DDB0217949 co-localizes withSodM in mitochondria. RT-PCR analysis showed adifference in the expression pattern between SodM
and DDB0217949. Maximum level of expressionof the gene for SodM was observed at the aggrega-Fig. 4. Cellular localization of DDB0217949 in D. discoideum. A HindIII - SwaI fragment of the plasmid constructed for sequencing was inserted into the HindIII BamHI site of pEGFP-N1 (Clontech, CA, USA) with alinker
preparedfromoligonucleotides(5’GACTCTAGAG and 5’-GATCCTCTAGAGTC). This
procedure was performed to fuse the N-terminal 35 residues of DDB0217949 and the full length of GFP. Thenthe fusion gene was placed downstream of the actin15
promoter on pTIKL-Bsr-Exp, an E. coli - D. discoideum
shuttle vector (Larochelle et al., 1997). D. discoideum
Ax2 (A - C) and Ax2 transformed by the plasmid constructed in this study (D - F) were photographed under amicroscope. A and D: Visible light. B and E: Fluorescence of Mitored. C and F: Fluorescence of GFP.

tion stage (Yasukawa, 2009), while that of thegene for DDB0217949 was observed at the growing stage. These observations suggest that theyfunction at different stages in the life cycle of D.
discoideum.
As described here, D. discoideum expresses a
mitochondria-localizing protein showing sequencesimilarity to FeSOD. D. discoideum is a nonpathogenic eukaryotic microorganism and is easy
to maintain in laboratory conditions. Analysis ofDDB0217949 would contribute to drug design andchemotherapy for infectious diseases. Purificationand characterization of this protein are in progress.

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