1

Identification of four SUMO paralogs in the medaka fish, Oryzias latipes, and their2classification into two subfamilies.

3

Daisuke Seki1, 2, Shinpei Obata1, Tsuyoshi Shirozu1, Takeshi Kitano1 and Hisato

4

Saitoh1, 2, 3

5

Department of Biological Sciences, Graduate School of Science and Technology,

1

6
7

and
Global COE (Centers of Excellence) Program, Global Initiative Center for Pulsed

28Power Engineering, Kumamoto University, Kumamoto, 2-39-1 Kurokami,

9

Kumamoto 860-8555, Japan

10
11

To Whom correspondence should be addressed:

3

12

Email: hisa@kumamoto-u.ac.jp,

13

Full Postal Address: Department of Biological Sciences, Graduate School of

14

Science and Technology,

15

Kumamoto University, Kumamoto, 2-39-1 Kurokami, Kumamoto 860-8555, Japan

16

Phone: +81-96-342-3450, Fax: +81-96-342-3450

1

17

Abstract

18

exist in humans, but there is limited information about SUMO paralogs from other19

vertebrate species. We isolated the four cDNA encoding proteins, similar to human

20

SUMOs, from the medaka fish, Oryzias latipes: OlSUMO-1, OlSUMO-2, OlSUMO-3,

21

and OlSUMO-4. The amino acid sequences of OlSUMO-2, OlSUMO-3 and OlSUMO-4

22

are 89‒94% identical, but they share only 45% identity with OlSUMO-1. Phylogenetic

23

analysis, transient expression of OlSUMOs in cultured cells and in vitro binding of

24

OlSUMOs with two different SUMO-interacting proteins demonstrated that the medaka

25

SUMO paralogs can be grouped into two subfamilies, OlSUMO-1 and OlSUMO-2/3/4,

26

respectively. Furthermore, this is the first report of all four OlSUMO transcripts being

27

expressed in medaka embryos, implying that they have a role in fish development. The

28

study will improve understanding of the relationship between structural and functional

29

diversity of SUMO paralogs during vertebrate evolution.

At least four paralogs of the small ubiquitin-related modifier (SUMO)30
31

Keywords

32

medaka fish

Posttranslational modification; Small ubiquitin-related modifier (SUMO);

2

33

Introduction

34

Post-translational modifications are an important mechanism by which

35

structures and functions of cellular proteins are controlled. Among post-translational

36

protein modifications, sumoylation is a unique type in which the small ubiquitin-related

37

modifiers (SUMOs) are covalently conjugated to lysine residues in a wide variety of38

target proteins in eukaryotic cells. Sumoylation is important in regulating numerous

39

cellular processes, including transcription, epigenetic gene control, genomic instability,40

and protein degradation (Geiss-Friedlander and Melchior 2007; Wilson and Heaton

41

2008; Wang and Dasso 2009). The SUMO modification pathway is regulated markedly

42

not only by multiple enzymes involving SUMO proteases (SENPs), SUMO-activation

43

E1 enzyme (Aos1/Uba2), SUMO-conjugation E2 enzyme (Ubc9) and SUMO-E3

44

ligases, such as the protein inhibitor of the activated STAT (PIAS) family of proteins45

and Ran-binding protein 2/nucleoporin 358kDa (RanBP2/Nup358), but also by diverse

46

SUMO-interacting

proteins

that

recognize

conjugated47

SUMO-interacting

motifs(SIMs),

also

as

48

(Geiss-Friedlander and Melchior 2007; Wilson and Heaton 2008; Wang and Dasso492009).

50

known

SUMO

moieties

SUMO-bindingvia

domains

SUMOs are highly conserved from yeast to humans. At least three paralogs have

51 been reported in human and mice: SUMO-1/SMT3C, SUMO-2/SMT3A and
52 SUMO-3/SMT3B. SUMO-2 and SUMO-3 are more closely related to each other (95%
53 amino acid identity) than they are to SUMO-1 (≈50% identity). Although SUMO-1 and
54 SUMO-2/3 can be equally conjugated to a subset of proteins, several lines of evidence
55 indicate that SUMO-1 and SUMO-2/3 are conjugated to different proteins, and represent
56 unique signals regulating different cellular functions (Saitoh and Hinchey 2000; Tatham
57 et al. 2001; Rosas-Acosta et al. 2005; Vertegaal et al. 2006). Intriguingly, in humans but
58 not in mice, there is another SUMO paralog, designated as SUMO-4, which differs from59 SUMO-1/2/3 in that it not only seems to be expressed mainly in the kidney, lymph node
60 and spleen, but is also unable to form covalent modification with substrates because of a
61 unique proline residue at position 90 (Pro-90) (Guo et al. 2004; Owerbach et al. 2005). To
62 date, there is limited information on a SUMO paralog that is similar to human SUMO-4
63 in other vertebrate species, and when the structural and functional diversification of
64 SUMO paralogs occurred during vertebrate evolution remains uncertain. Thus, it is
65 important to identify and investigate other examples of vertebrate SUMOs.
3

66

Here we report the isolation of four cDNAs of medaka SUMO paralogs, termed:

67

OlSUMO-1, OlSUMO-2, OlSUMO-3 and OlSUMO-4. Medaka, Oryzias latipes, is a68

small egg-laying freshwater teleost fish with several advantages for biological69

experiments (Ozato et al. 1986; Wada et al. 1995; Ishikawa et al. 2000; Loosli et al.

70

2000; Kasahara et al. 2007; Shiraishi et al. 2008). Our data, including sequence71

comparison and quantitative analysis of transcripts during medaka embryogenesis,

72

protein expression and subcellular localization in cultured cells, and comparison of73

binding affinities to paralog-specific SUMO-binding proteins, suggest a possible

74

classification of medaka SUMOs into two subfamilies, OlSUMO-1 and OlSUMO-2/3/4,

75

and imply the absence of a functionally important proline residue in medaka SUMO

76

paralogs which corresponds to the Pro-90 in human SUMO-4, arguing the divergence77

and/or specialization of structure and function of human SUMO-4 during vertebrate

78

evolution.

79
80

Materials and Methods

81

Fish samples

82

The orange-red variety of medaka, the FLFII strain, was selected for the experiments.

83

Fish embryos were maintained in ERM (17 mM NaCl, 0.4 mM KCl, 0.27 mM

84

CaCl22H2O, 0.66 mM MgSO4, pH 7.0) at 26℃under a 14 hours light and 10 hours dark85

cycle. Developmental stages of the embryos were determined according to the86

description by Iwamatsu (Iwamatsu et al. 1994).

87
88

Database analysis and construction of the phylogenic tree

89

BLAST searches for DNA and protein identities were conducted using the medaka

90

transcription

91

www.ensembl.org/Oryzias_latipes/index.html) and the Medaka Genome Initiative

92(www.park.itc.u-tokyo.ac.jp/K-medaka/MGI2/MGI.html). Analyses of the predicated93

protein sequences were conducted using BLAST. Phylogenic tree are generated using

94

ClustalW server at the DDBJ (clustalw.ddbj.nig.ac.jp/top-j.html) with standard setting.

95

The GeneBank protein sequences and accession numbers used in these analyses were as

96

follows: human SUMO-1, P63165; human SUMO-2, P61956; human SUMO-3,

97

P55854; human SUMO-4, BAH05006; human ubiquitin, P62988; human Aos1,

98

AAD23902; human Uba2, CAG33037; human Ubc9, CAA05359; human PIAS1,

database(www.blast.ddbj.nig.ac.jp/top-j.html

4

and

99

O75925; human PIAS2, O75928; human PIAS4, AAH04389; human RanBP2/Nup358,

100

P49792; human SENP1, Q9P0U3; human SENP3, Q9H4L4; human SENP5, Q96HI0;101

human SENP6, Q9GZR1; human SERNP7, Q9BQF6; human Ran-binding protein

102

2/Nucleoporin

103

chromatin-associated factor 1 (MCAF1), Q6VMQ6; human Ring finger protein 4

104(RNF4), P78317; human thymine DNA glycosylase (TDG), Q13569; human histone105

H3-K9 methyltransferase SETDB1, Q15047.

358kDa(RanBP2/Nup358),

P49792;human

MBD1-containing

106
107

RNA extraction, cDNA cloning, and sequence analysis

108

Total RNA was extracted from 0-, 3-, 6- and 9-dpf embryos of FLFII medaka using

109

ISOGEN (Nippongene), and the first strand cDNA was synthesized from the total RNA

110

by oligo(dT) priming with SuperScript™ III First-Strand Synthesis SuperMix

111(Invitrogen). cDNAs encoding the open reading frames of OlSUMO-1, OlSUMO-2,

112

OlSUMO-3 and OlSUMO-4 were amplified by PCR using the following primers:

113

OlSUMO-1

114

5’-AAAACATCAGAAATTGTGGCT-3’,

115

5’-ACACTAGCCACAGCAGCAG-3’/rv 5’-AGGGATGTGGAAAGAAAACAGT-3’,

116

OlSUMO-3

117

5’-GGTCTGAAGGTGGTCACTTAAT-3’,

and

118

5’-AGCGCCAAAAGAGTGACG-3’/rv

5’-GCAGCATGTGTGGCTGA-3’,

119respectively.

forward(fw)

5’-CGCACACAGTCAGGATAAAC-3’/reverse
OlSUMO-2

fw

(rv)
fw

5’-TCCCGTCAATTCACCAGAC-3’/rv
OlSUMO-4

fw

120
121

Real-time reverse transcription-polymerase chain reaction (RT-PCR)

122

To quantitatively compare the amount of transcripts of medaka SUMO paralogs,

123

real-time RT-PCR was performed using LightCycler 350S (Roche) and LightCycler

124

FastStart DNA MasterPLUS SYBR Green I (Roche) according to the manufacturer’s

125

protocols. The sequences of the primer used in this assay were as follows: OlSUMO-1

126

fw 5'-GAGGCGACAAGAAAGATGGA-3'/rv 5'-TTTGGGGTTTGGTTATCTGC-3',

127

OlSUMO-2 fw; 5'-AAAACGAGCACATCAACCTG-3'/ rv

128

5'-GGCTGTCCATCAAATCGAAAT-3', OlSUMO-3 fw

129

5'-CAAGATGGGTCGGTGGTC-3'/rv 5'-CAAGCTGTGCAGGTGTATCC-3',

130

OlSUMO-4 fw 5'-TAGCAGGTCAGGATGGATCT-3'/rv

131

5'-GCAGGTGTGTCTGTCTCATTT-3', and medaka β-actin (the GeneBank accession5

132

number; S74868) fw 5'-TCCACCTTCCAGCAGATGTG-3'/ rv

133

5'-AGCATTTGCGGTGGACGAT-3'. We also used the transcripts derived from the134

GAPDH, RPL7 and 18srRNA genes as internal controls; the relative ratios of the135

SUMO paralogs were almost the same (data not shown).

136

Construction of expression plasmids

137

To generate pEGFP-OlSUMO-1/2/3/4gg and pEGFP-OlSUMO-1/2/3/4g mammalian138

expression plasmids, the DNA fragments of medaka SUMO paralogs were amplified by

139

PCR

140

5’-GAGAATTCATGTCAGACACGGAGAC-3’/rv

141

5’-ATGTCGACTTATCCGCCGGTCTGTTC-3’,

142

5’-GAGAATTCATGTCAGACACGGAGAC-3’/rv

143

5’-ATGTCGACTTAGCCGGTCTGTTCTTG-3’,

144

5’-GAAGAATTCATGGCAGACGAGACG-3’/rv

145

5’-TAGTCGACTTAACCTCCAGTCTGCTGTT-3’,

146

5’-GAAGAATTCATGGCAGACGAGACG-3’/rv

147

5’-TAGTCGACTTATCCAGTCTGCTGTTGGAA-3’,

148

5’-ATAGAATTCATGTCGGAGGAGAAGCCA-3’/rv

149

5’-ATGTCGACTTACCCTCCAGTCTGCTG-3’,

150

5’-ATAGAATTCATGTCGGAGGAGAAGCCA-3’/rv

151

5’-ATGTCGACTTATCCAGTCTGCTGCTG-3’,

152

5’-GAGGAATTCATGGCTGATGAAAAACCAAAG-3’/rv-5’TAGTCGACTTAGCCC

153

CCCGTCTG-3’,

154

5’-GAGGAATTCATGGCTGATGAAAAACCAAAG-3’ (the EcoRI and SalI sites are155

underlined for the fw and rv primers, respectively). PCR fragments were digested with

156

EcoRI-SalI and inserted into EcoRI-XhoI-digested pEGFP-C2 (Clontech) or pGEX-4T-1

157(Amersham Pharmacia Biotech). pEGFP-human SUMO-1 and SUMO-3 plasmid

158

constructs as described previously (Saitoh et al. 1998; Saitoh and Hinchey 2000;

159

Uchimura et al. 2006; Uwada et al. 2010).

using

the

following

oligonucleotide

primers:

OlSUMO-4g

OlSUMO-1gg

fw

OlSUMO-1g

fw

OlSUMO-2gg

fw

OlSUMO-2g

fw

OlSUMO-3gg

fw

OlSUMO-3g

fw

OlSUMO-4gg

fw
fw

160
161

Cell culture, transfection, and immunoblotting

162

Hela cells (maintained in Dulbecco’s modified Eagale’s medium containing 10% fetal

163

bovine serum and antibiotics at 37oC in a 5% CO2 incubator) were transfected with the164

appropriate pEGFP-expression plasmids using GeneJuice (TaKaRa). After 24 hours the6165

cells were washed with PBS and lysed in two pellet volumes of 3xSDS sample buffer.

166

Proteins were separated on SDS-PAGE followed by Western blotting using anti-GFP

167

antibody (Santa Cruz). For detection of GFP signals under the microscope, the cells

168transfected with GFP-constructs were grown on coverslips and fixed with 4%

169

paraformaldehyde. The cells were permeabilized with 0.2% Triton X-100, mounted

170

under coverslips, and analyzed using Biorevo BZ-9000 (Keyence).

171
172

Glutathione S-transferase(GST)-pulldown assay

173

pGEX, pGEX-human SUMO-1/3, pGEX-OlSUMO-1/2/3/4, pET28-RanBP2-IR and174

pET28-MCAF1 plasmids were introduced into E. coli BL21(DE3) and Rosetta(DE3)175

strains. The recombinant GST, GST-human SUMO-1/3, GST-OlSUMO-1/2/3/4,

176(His)6-RanBP2-IR and (His)6-MCAF1 fusion proteins were expressed as described

177

previously (Uchimura et al. 2006; Uwada et al. 2010). A GST-pulldown assay was178

carried out as described previously (Uchimura et al. 2006; Uwada et al., 2010). The

179

proteins were separated on SDS-PAGE followed by immunoblot analysis using

180

anti-(His)6-tag antibody (Roche) and anti-GST antibody (Santa Cruz).

181
182

Results

183

The SUMO pathway components in the medaka data base184

For a comprehensive analysis of the SUMO modification pathway in lower

185

vertebrate species, we used human genes encoding various components in the SUMO

186

pathway to search for orthologs in medaka protein and DNA databases. Our initial

187

search identified at least four distinct medaka SUMO genes: OlSUMO-1, OlSUMO-2,

188

OlSUMO-3 and OlSUMO-4, which contain multiple small introns (Fig.1A). In addition,

189

there were at least: one gene similar to the human Aos1 (SUMO-E1) gene, one gene190

similar to human Uba2 (SUMO-E1) gene, and a gene similar to the human Ubc9

191

(SUMO-E2) gene. At least five distinct genes encoding genes similar to human192

de-sumoylation protease SENPs, genes similar to several human SUMO-E3s, including

193

RanBP2/Nup358, PIAS1, PIAS2 and PIAS4, and genes similar to currently identified

194

human SUMO-interacting/-binding proteins, such as MCAF1, RNF4, TDG and

195

SETDB1 were also detected (Table 1). These results strongly suggest the existence of a

196

protein-conjugation pathway in medaka analogous to the human SUMO pathways.

197
7

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Isolation of four different SUMO transcripts: OlSUMO-1, OlSUMO-2, OlSUMO-3, and199

OlSUMO-4

200

To confirm that all four OlSUMO genes can be transcribed in medaka, we

201

designed primer pairs and used them to amplify the cDNA fragments using total RNA

202

prepared from fertilized medaka embryos (see Materials and methods section for

203

details). The cDNA fragments for all of the primer pairs, corresponding to full-length

204

OlSUMO-1/2/3/4, amplified efficiently; we then cloned them and determined their

205

DNA sequences. The GeneBank accession numbers of OlSUMO-1, OlSUMO-2,

206

OlSUMO-3 and OlSUMO-4 are as follows: GQ463435, GQ463436, GQ463438 and207

GQ463437, respectively. We found three sequences identical to GQ463435, GQ463437,

208

GQ463438

209(www.ensembl.org/Oryzias_latipes/index.html) and have deposited them as SUMO-1,

210

SUMO-2 and SUMO-4, respectively. We thus gave the nomenclatures for GQ463435,

211

GQ463436, GQ463438 and GQ463437 as OlSUMO-1, OlSUMO-2, OlSUMO-3 and212

OlSUMO-4, respectively. Although it appears that designation in the database does not213

consider any biological criteria such as homology search and phylogenetic alignment

214(see below), we followed the OlSUMO nomenclature (as above) to avoid confusion.

in

the

Ensemble’s

medakagene

database

215

The amino acid sequences deduced from each cDNA are shown in Fig. 1B.

216

Comparisons of the amino acid sequences showed that OlSUMOs are only 16–17%

217

similar to ubiquitin. The similarities of OlSUMO-1 versus OlSUMO-2, OlSUMO-1

218

versus OlSUMO-3, OlSUMO-1 versus OlSUMO-4, OlSUMO-2 versus OlSUMO-3,

219

OlSUMO-2 versus OlSUMO-4 and OlSUMO-3 versus OlSUMO-4 were 48, 46, 48, 92,220

89 and 94%, respectively. Thus, OlSUMO-2, OlSUMO-3 and OlSUMO-4 proteins are

221

highly homologous, and they are approximately 50% identical to OlSUMO-1.

222

Phylogenetic analysis showed that OlSUMO-1 had the highest similarity to human

223

SUMO-1 (HsSUMO-1), and OlSUMO-3 was closely related to human SUMO-3

224(HsSUMO-3); OlSUMO-2 and OlSUMO-4 were equally related to human SUMO-2

225(HsSUMO-2) (Fig. 1C). The relationship of human SUMO-4 to OlSUMO-2/4 is no226

closer than that of human SUMO-2 to OlSUMO-2/4. It should be noted that a proline at

227

90 amino acid residue in human SUMO-4, which appears critical for this paralog’s

228

function (Guo et al. 2004; Owerbach et al. 2005), is not conserved in either OlSUMO

229

paralog.

230
8

231

OlSUMO proteins have potential to serve in the SUMOylation pathway

232

To confirm that the proteins encoded by the four medaka SUMO genes attach to233

other proteins similarly to SUMO proteins from other organisms, cDNAs of the234

OlSUMOs were expressed in Hela cells to see whether OlSUMO proteins could

235

conjugate to cellular proteins. We first generated the constructs that lack the C-terminal

236

amino acids from the highly conserved di-glycine (gly-gly) residues of each OlSUMO

237(Fig. 1B). The generated fragments, OlSUMO-1gg, OlSUMO-2gg, OlSUMO-3gg and238

OlSUMO-4gg, were fused to the C-terminus of an enhanced green fluorescent protein,239

generating

240

EGFP-OlSUMO-4gg

241

constructs were then expressed in Hela cells, and total cell lysates prepared from the242transfected cells were analyzed by Western blot with anti-GFP antibody.

243

Non-conjugated forms of EGFP-OlSUMOgg proteins that migrate at around 45 kDa

244(EGFP~27kDa+SUMO~18kDa) were clearly identified (arrowheads in Fig. 2). In

245

addition, all EGFP-OlSUMOgg proteins form a number of conjugates that migrate at246

higher molecular mass than the 45 kDa-monomer band. It should be noted that the247

signal intensities at higher molecular mass varied among the cells expressing different

248

constructs, suggesting functional heterogeneity among OlSUMO paralogs. Removal of249

the C-terminal invariant gly residues from the EGFP-OlSUMOgg constructs, termed

250

EGFP-OlSUMO-1g, EGFP-OlSUMO-2g, EGFP-OlSUMO-3g and EGFP-OlSUMO-4g,

251

completely removed the high molecular mass bands. Thus these data indicate that all252

OlSUMO proteins have the potential to conjugate to other proteins, and suggest that253

activation of the gly residue at the C-terminal end is critical for transfer of SUMO to254

target proteins, arguing that all OlSUMO paralogs are logically active in mammalian255

cultured cells.

EGFP-OlSUMO1gg,
fusion

EGFP-OlSUMO2gg,

proteins,

respectively.

EGFP-OlSUMO-3gg
EGFP-tagged

and

OlSUMOgg

256
257

Biochemical distinctions among OlSUMOs

258

To improve understanding of biochemical and physiological properties of259

OlSUMO paralogs, we tested subcellular localization of four OlSUMO paralogs. This260

was carried out by transient transfection assay using EGFP-OlSUMOs. A marked

261

concentration of green fluorescence at the nuclear rim was observed in many of the262

EGFP-OlSUMO-1-transfected Hela cells shown (Fig. 3A). In contrast, a mild

263

concentration of the signal at the nuclear rim in the EGFP-OlSUMO-2/3/4-transfected9264Hela cells implies that OlSUMO-1 and OlSUMO-2/3/4 have different requirements in

265

their subcellular localization at the nuclear rim. It should be noted that we observed

266

some punctuate concentrations in the nucleus of both the EGFP-OlSUMO-1 and267

EGFP-OlSUMO-2/3/4 transfected cells, indicating the possibility of colocalization of

268

OlSUMO-1 and OlSUMO-2/3/4 in the nuclear punctuate structures in interphase cells.

269

Next, we conducted a GST-pulldown assay to compare the affinity of two types

270

of SIM-containing SUMO-interacting proteins, RanBP2 and MCAF1, to the OlSUMO

271

paralogs. RanBP2, a SIM-containing protein localized at the nuclear pore, binds more272

readily to human SUMO-1 than it does to SUMO-2/3 (Saitoh et al. 1998; Song et al.

273

2004; Hecker et al. 2006), and MCAF1, a SIM-containing protein involved in274

nuclear/chromatin function, binds more readily to human SUMO-2/3 than it does to275

SUMO-1 (Hecker et al. 2006; Uchimura et al. 2006; Sekiyama et al. 2008; Uwada et al.

2762010). The (His)6-RanBP2-IR protein was detected in the GST-OlSUMO-1-pulldown

277

fraction, but not in GST-OlSUMO2/3/4-pulldown fractions (Fig. 3B). In contrast, the278(His)6-MCAF1 protein was detected in the GST-OlSUMO-2/3/4-pulldown fractions,279

but not in the GST-OlSUMO-1-pulldown fraction. These results indicate that280

OlSUMO1and OlSUMO2/3/4 paralogs can be distinguished by their subcellular281

localization,

282

SUMO-2/3-specific SIM-containing proteins, supporting the notion that OlSUMO

283

paralogs can be grouped into two subfamilies.

anddifferentiatedbiochemicallyby

SUMO-1-specific

and

284
285

Quantitative analysis of transcripts of OlSUMOs during medaka embryogenesis286

To elucidate functional heterogeneity or differential regulation among OlSUMO

287

paralogs, or both, we assessed the relative amounts of OlSUMO transcripts in medaka

288

embryos at different developmental stages using real-time RT-PCR. All OlSUMO

289

transcripts were reasonably abundant in 0-dpf embryos (stage 8~9), 3-dpf embryos

290(stage 29), 6-dpf embryos (stage 36), and 9-dpf embryos (stage 39) (Fig. 4). While the291

expression levels of OlSUMO-2/4 in 0-dpf embryos and OlSUMO-2/3/4 in 3-dpf

292

embryos were significantly higher than they were in the others, all of the OlSUMO

293

transcripts seemed to undergo small changes in abundance through embryogenesis. To

294

compare the relative amounts of the subfamilies at each embryonic stage, we integrated295

the values of the OlSUMO-2/3/4 transcripts and compared them with those of296

OlSUMO-1. The levels of the transcripts from the OlSUMO-2/3/4 subfamily were
10

297

constantly higher than those of OlSUMO-1 at any of embryonic stages (Fig. 4 and data298

not shown). The OlSUMO-2/3/4 transcripts were approximately 8-fold more abundant

299

than the OlSUMO-1 transcript, especially at 3-dpf. These data indicate that transcripts

300

of all four SUMO paralogs exist during medaka embryogenesis, and that the amount of301

OlSUMO-2/3/4 transcript is greater than that of OlSUMO-1 at any stage of302

development.

303
304

Discussion

305

By searching the medaka genome and EST databases, we discovered gene loci306

and cDNA sequences for multiple components of the SUMO modification system, four

307

SUMO paralogs: OlSUMO-1, OlSUMO-2, OlSUMO-3 and OlSUMO-4; one E1 (one308

OlAos1 and one OlUba2); one E2 (OlUbc9); three OlPIAS families of E3s;

309

OlRanBP2/Nup358; another class of E3; and a five-gene family encoding putative

310

SUMO proteases (OlSENPs). We also found several mammalian orthologs of311

downstream effector proteins from the SUMO modification pathway, including MCAF1

312(Uchimira et al. 2006), TDG (Baba et al. 2005), SETDB1 (Ivanov et al. 2007), RNF4

313

(Häkli et al. 2005), and others (data not shown). These SUMO pathway components in314

the medaka database suggest that there is a SUMO modification pathway in medaka.

315

Given that we and others also found multiple SUMO paralogs and SUMO modification

316

enzymes in the zebrafish (Danio reio) database (data not shown; Nowak and317

Hammerschmidt 2006; Yuan et al. 2009), our search implies sumoylation as an

318

important signaling mechanism in fishes.

319

To date, in humans, four SUMO paralogs, HsSUMO-1 to HsSUMO-4, have320

been identified; HsSUMO-1, HsSUMO-2 and HsSUMO-3 can act as protein modifiers,

321

whereas SUMO-4 seems to be expressed only in restricted tissues and may not have the322

ability to be conjugated to other proteins (Guo et al. 2004; Owerbach et al. 2005;

323

Geiss-Friedlander and Melchior 2007; Wilson and Heaton 2008), indicating that324

HsSUMO-4 constitutes a subgroup that is distinct from HsSUMO-1/2/3. Our

325

biochemical studies using SIM-containing proteins, RanBP2 and MCAF1, support the326

notion of two SUMO subfamilies in medaka (Fig. 3B). With regard to their subcellular

327

localization, we also found subfamily-specific properties (Fig. 3A). These findings

328

suggest that OlSUMOs can be grouped into two subfamilies; OlSUMO-1 and329

OlSUMO-2/3/4 subfamilies. In addition, all OlSUMOs are expressed throughout
11

330

medaka embryogenesis (Fig. 4). When transiently expressed in Hela cells, all331

OlSUMOs were incorporated into higher molecular mass regions, suggesting that they332

all have ability to be conjugate to cellular proteins in vivo (Fig. 2). An amino acid333

sequence alignment experiment suggested that all medaka SUMO paralogs do not334

contain a unique proline residue located at position 90 in HsSUMO-4 (Fig.1). Thus we

335

suppose that all OlSUMOs appear distinct from HsSUMO-4 and suggest the emergence336

of human SUMO-4 paralog after mammalian evolution.

337

The SUMO modification system is essential in most organisms including S.

338

cerevisiae, C. elegans, Arabidopsis thaliana and mice (Geiss-Friedlander and Melchior3392007), and may play a critical role in some parasitic diseases (Cabral et al. 2008).

340

Whether individual SUMO proteins are essential in organisms that have multiple

341

SUMO paralogs remains unclear. Genetic studies have linked SUMO-1

342

haploinsufficiency to the cleft lip or palate condition in humans, indicating that

343

SUMO-1 and SUMO-2/3 play a role in development (Alkuraya et al. 2006; Pauws and344

Stanier 2007). On the other hand, mice lacking SUMO-1 develop without any apparent

345

abnormalities, implying that humans and mice may be different in their specific346

requirements for SUMO paralogs (Evdokimov et al. 2008; Zhang et al. 2008). In future347

experiments using the medaka system, it is important to investigate which348

developmental processes can be compensatory among SUMO paralogs, to elucidate349

whether more subtle phenotypic differences are involved in the development of tissues

350

and organs lacking either SUMO paralogs, and to identify targets for SUMOylation by351

different OlSUMO paralogs during embryogenesis and organogenesis. We believe our352

study provides a basis for such experiments.

353
354

Acknowledgments

355

We thank Ms. Maki Kuroki for help with extraction of RNA from medaka embryos. We

356

appreciate the helpful discussions with the members of the Saitoh Laboratory . This

357

work was supported by grants to Hisato Saitoh from the Naito Foundation, the Novartis

358

Foundation, the Ministry of Education, Culture, Sport, Science and Technology

359(MEXT), and the Japan Society for the Promotion of Science (JSPS). Daisuke Seki is a360

Junior Research Associate of the global COE Program at the Global Initiative Center for

361

Pulsed Power Engineering.

12

362

Table 1. Components of the medaka SUMO pathway.

363
364

a. Accession number, ensemble medaka database accession number.

365

b. Expression

366
367
368
369

confirmedbythepresence

of

EST

in

the

database(www.ensembl.org/Oryzias_latipes).
c. Expression confirmed by RT-PCR using total RNA from 9- days after
fertilization (daf) of the medaka embryo.
d. NT, not-tested by RT-PCR.
13

370

e. ND, not-determined.

371

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Figure Legends

496
497

Figure 1. Identification and characterization of the medaka OlSUMO gene family.

498

A, schematic representation of organization of the medaka OlSUMO genes. Black boxes499

and lines indicate exons and introns, respectively. The numbers of chromosome on

500

which OlSUMO-1, OlSUMO-2, OlSUMO-3 and OlSUMO-4 gene loci are located are

501

shown on the left side.

502

B, amino acid sequence alignment of medaka OlSUMO paralogs by the ClustalW503

method. The GeneBank accession numbers of OlSUMO-1, OlSUMO-2, OlSUMO-3 and504

OlSUMO-4 are as follows: GQ463435, GQ463436, GQ463438 and GQ463437,

505

respectively. Potential sumoylation sites (KXE) are boxed. The Ub domain present in506

all OlSUMO paralogs is outlined. The arrowhead represents the potential processing site

507

by SENPs that expose the highly-conserved gly-gly residues (reverse type) involved in

508

SUMO conjugation. The asterisk denotes a proline at 90 amino acid residue in human509

SUMO-4, which appears critical for paralog function (grey box).

510

C, phylogenic relationship among medaka OlSUMO-1/2/3/4, human SUMOs

511(HsSUMO-1/2/3/4), C. elegans SUMO (CeSUMO) and D. melanogaster SUMO512

(DrSUMO). Phylogenic tree are generated using ClustalW server at the DDBJ

513

(clustalw.ddbj.nig.ac.jp/top-j.html) with standard setting. Bootstrap values (1,000

514

replicates) are shown at the branches.

515
516

Figure 2. Ability of OlSUMO paralogs to conjugate to cellular proteins.

517

Hela cells were transfected with EGFP vector (lane 1), pEGFP-HsSUMO-1gg (lane 2),

518

pEGFP-HsSUMO-1g (lane 3), pEGFP-HsSUMO-3gg (lane 4), pEGFP-HsSUMO-3g

519

(lane

520

pEGFP-OlSUMO-2gg (lane 8), pEGFP-OlSUMO-2g (lane 9), pEGFP-OlSUMO-3gg

521(lane 10), pEGFP-OlSUMO-3g (lane 11), pEGFP-OlSUMO-4gg (lane 12) or

522

pEGFP-OlSUMO-4g (lane 13). After 24 hours, total cell lysates were analyzed by523

immunoblotting using anti-EGFP antibody. The position of the EGFP-OlSUMO

524

monomer (~45 kDa) is indicated by an arrowhead. The high molecular mass bands are525

indicated by a bracket with an asterisk. Molecular mass standards are expressed in526

kilodaltons (kDa).

5),

pEGFP-OlSUMO-1gg

(lane

527
18

6),

pEGFP-OlSUMO-1g

(lane

7),

528

Figure 3. Distinct biochemical properties among OlSUMO-1/2/3/4 proteins.

529

A, subcellular localization of OlSUMOs. Hela cells were transfected with

530

pEGFP-HsSUMO-1gg,

531

pEGFP-OlSUMO-2gg, pEGFP-OlSUMO-3gg and pEGFP-OlSUMO-4gg. After 24

532

hours, the cells were fixed and EGFP signals were detected under the fluorescent533

microscope. DAPI-stained images are also indicated (inset).

534

B, interaction among OlSUMOs and SIM-containing proteins. A bacterial lysate

535

expressing recombinant (His)6-RanBP2-IR (upper panel) or (His)6-MCAF1 (bottom

536

panel) was incubated with beads of GST-OlSUMO-1gg (lane 3), GST-OlSUMO-2gg537(lane 4), GST-OlSUMO-3gg (lane 5) and GST-OlSUMO-4gg (lane 6). Following

538

incubation, a GST-pulldown assay was carried out and the proteins associated with the539

beads were subjected to immunoblot analysis using anti-(His)6 antibody. For positive

540

controls, GST-HsSUMO-1 (lane 1) and GST-HsSUMO-3 (lane 2) were used. Amounts

541

of GST-SUMOs used in the pull-down assay were visualized by immunoblot analysis

542

with anti-GST antibody (bottom).

pEGFP-HsSUMO-3gg,

pEGFP-OlSUMO-1gg,

543
544

Figure 4. Expression of OlSUMO paralogs embryos at different developmental

545

stages.

546

Quantitative detection of the transcripts of OlSUMO paralogs in 0-, 3-, 6- and 9-dpf

547

embryos. Quantitative RT-PCR was used to compare the amounts of OlSUMO-1 (black),

548

OlSUMO-2 (gray), OlSUMO-3 (white) and OlSUMO-4 (dark gray) transcripts using

549

total RNA prepared from at least three individual embryos at the different

550

developmental stages. In this experiment we used β-actin for the internal control; it551

showed a 2-fold reduction during medaka embryogenesis.

552

19

553

554
555

20

556
557

21

558
559

22

560
561

23

562

24