Molecular characterization of a novel nuclear transglutaminase that is expressed during starfish embryogenesis Hiroyuki Sugino*, Yudai Terakawa, Akiko Yamasaki, Kazuhiro Nakamura, Yoshiaki Higuchi, Juro Matsubara, Hisato Kuniyoshi and Susumu Ikegami Department of Applied Biochemistry, Hiroshima University, Japan We report the constitution and molecular characterization of a novel tran sglutaminase (EC 2.3.2.13) that starts to accumulate specifically in the nucleus in the starfish (Asterina pectinifera) embryo after progression through the early blastula stage. The cDNA for the nuclear transglutaminase was cloned and the cDNA-deduced sequence defines a single open reading frame encoding a protein with 737 amino acids and a predicted molecular mass of 83 kDa. A comparison of this transglutaminase with other members of the gene family revealed an overall sequence identity of 33–41%. A special sequence feature of this transglutaminase, which is not found in other transglutaminases, is t he presence of nuclear local- ization signal-like sequences in the N-terminal region. Microinjection of hybrid constructs that encode the N-ter- minal segment fused to reporter proteins into the germinal vesicle of an oocyte produced chimeric proteins by transcription-coupled translation. It was foun d that the N-terminal segmen t alone was sufficient t o effect nuclear accumulation of an otherwise cytoplasmic protein. These results suggest that the nuclear accumulation of the trans- glutaminase may play an important role in nuclear remod- eling during early starfish embryogenesis. Keywords: transglutaminase; nucleus; starfish; e mbryo; cloning. The class of enzymes that are commonly referred to as transglutaminases (TG) (EC 2.3.2.13) are known mostly for their role in the post-translational remodeling of proteins (reviewed in [1]). These enzymes catalyze protein cross- linking reactions via the formation of e-(c-glutamyl)lysine bonds between the c-carboxyl group of a Gln residue in one polypeptide chain and the e-amino group of a Lys residue in a second polypeptide chain. Well-documented examples of TG are p lasma factor X IIIa [2], keratinocyte TG [ 3], epidermalTG[4],tissueTG[5],andprostaticTG[6]. Recent findings have shown that, apart f rom their protein modifying capabilities, tissue TG is also able to function as a component of the signal-transducing G protein complex [7]. The cDNA of G ha , involved in the transmission of adrenergic stimuli, is identical to that of tissue TG of human endothelial cells [7]. Tissue TG is localized mainly in the cytosol, but detectable tissue TG expression has been reported in the nucleus [8–10]. However, TG a ctivity in the nucleus and the mechanisms of its translocation is not well understood, and nucleus-specific TG has not been reported. It is accepted that many proteins are able to cross nuclear membranes and accumulate against gradients to c oncen- trate in the nucleus [11,12]. The nuclear translocation of proteins via the nuclear pore complex is dependent on a nuclear localization signal in the protein, which is rich in basic amino acids and may be bipartite [13–1 5]. T he functional assays of such nuclear localization signals are usually based on the ability of a signal to confer nuclear localization to an otherwise non-nuclear protein. The present paper describes the occurrence of a novel TG that is localized exclusively in the nucleus of starfish (Asterina pectinifera) embryonic cells and is designated nuclear TG (nTG). The amino-acid sequence derived from the cDNA sequence contains putative nuclear localization signals [15] in the N -terminal region. We demonstrate here that the N-terminal region promotes the nuclear accumu- lation of an otherwise cytoplasmic protein, namely pyruvate kinase (PK), in t he A. pectinifera oocyte system. This finding suggests that nuclear localization signals in the N-terminal region of nTG are functional in the starfish embryonic cells. Northern b lot analyses carried out in this study demonstrate that nTG mRNA appears at the early blastula stage a nd increases thereafter. The nTG protein level inc reases in parallel w ith m RNA levels. These results suggest that nTG is, directly or indirectly, involved in the modification of the nuclear structure or intranuclear signaling pathways during starfish embryogenesis [16–18]. MATERIALS AND METHODS Cultivation of embryos Specimens of the starfish, A. pec tinifera, were collected from coastal waters off Japan during their breeding season and maintained in artificial sea water in laboratory aquaria at Correspondence to S. Ikegami, Department of Applied Biochemistry, Hiroshima University, 1-4-4 Kagamiyama, Higashi-hiroshima, Hiroshima 739-8528, Japan. Fax: + 81 824 22 7059, Tel.: + 81 824 24 7948, E-mail: sssike@hiroshima-u.ac.jp Abbreviations: nTG, nuclear transglutaminase; GFP, g reen fluorescent protein; PK, pyruvate kinase; TG, transglutaminase. *Present address: Department of Applied Life Science, Faculty of Engineering, Sojo University, Japan. Note: the nucleotide sequence reported in this paper has been sub- mitted to the DDBJ Data Bank with accession number AB036064. (Received 26 October 2001, revised 8 February 2002, accepted 20 February 2002) Eur. J. Biochem. 269, 1957–1967 (2002) Ó FEBS 2002 doi:10.1046/j.1432-1033.2002.02847.x 15 °C. Eggs and sperm were obtained as described previ- ously [18–20]. Eggs were fertilized and embryos were cultured in artificial s ea water that contained 5 mgÆmL )1 streptomycin sulfate and 50 lgÆmL )1 penicillin G. Cultures were maintained in jars at a density of < 5000 embryos per mL with gentle stirring. Only cultures w ith a fertilization rate in excess of 95% and normal morphological develop- ment were used for experimentation. RT-PCR Poly(A) + RNA was prepared from blastulae (packed vol- ume, 300 lL) using a QuickPrep micro mRNA purification kit (Amersham Pharmacia B iotech). RNA (0.1 lg) was reverse-transcribed into cDNA in a total volume of 20 lL using the RNA LA PCR kit (Takara, Tokyo, Japan) with oligo(dT) primer. cDNA coding for tissue TG of bovine endothelial cells [21] was used as a n internal c ontrol for PCR. PC R w as carried out with 1.25 U of KOD DNA polymerase (Toyobo, Osaka, Japan) in the reaction mixture (50 lL) that contained 120 m M Tris/HCl (pH 8.0), 10 m M KCl, 6 m M (NH 4 ) 2 SO 4 , 0.1% Triton X-100, 0.001% BSA, 1m M MgCl 2 ,0.2m M each of four deoxyribonucleoside 5¢-triphosphates, and 4 l M each of the TG-specific degen- erate oligonucleotide primers, TG5 (5¢-TAYGGNCARTG YTGGGT-3¢;N¼ A, C, G or T; Y ¼ CorT;R¼ Aor G) and TG 3V (5¢-CCANACRTGRAARTTCCA-3¢). The PCR cycles were 15 s at 98 °C, 2 s at 55 °C, and 10 s at 74 °C. A total of 25 cycles were run, with the first cycle containing an extended denaturation p eriod (2 min). The 195-bp PCR product was gel-purified and sequenced by means of the dideoxy chain termination method using the Thermo sequenase II dye terminator cycle sequencing kit (Amersham Pharmacia Biotech) with TG5 and TG3V primers. Isolation of cDNA clones and DNA sequencing Adaptor-ligated double stranded cDNA was prepared from poly(A) + RNA of A. pectinifera blastulae using the Mara- thon cDNA amplification kit (Clontech) in conjunction with the oligo(dT) primer and Marathon cDNA adaptor. TG sequences were amp lified by PCR in both directions using TG-specific oligonucleotide primers VG5-3 (5¢-ACCC TCCTCCAGATCGGG-3¢)andTG3-1(5¢-GGACTGTG CAGAAGTCT-3¢), and the adaptor-specific primer AP1 (5¢-CCATCCTAATACGACTCACTATAGGGC-3¢). The PCR cycles were 15 s at 98 °C, 2 s at 55 °C, and 30 s at 74 °C. A total of 40 cycles were run, w ith the fi rst cycle containing an extended denaturation period (2 min). Nested PCR reactions were performed using the product of the first PCR under the conditions described above with adaptor specific primer AP2 (5¢-ACTCACTATAGGGCTCGAGC GGC-3¢), and internal TG-specific primers TG5-4 (5¢ CCA TCCAGCAGTCATTCC-3¢)andTG3-2(5¢-AATTTTGC CTCGGCTCA-3¢). The PCR products were gel-purified using an Ultraclean DNA purification kit (Mo Bio Labo- ratories), cloned, and both strands were sequenced from both d irections under the conditions described a bove. The deduced cDNA sequence was devoid of a termination codon. To isolate an oligonucleotide that codes for the C-terminal region of nTG, the 3¢-RACE approach was carried out using TG3-3 (5¢-ATCGTGTCGCTGACCAA C-3¢)andTG3-4(5¢-CC ATTGCCGTACCCGCTG-3¢), the sequences of which were d erived from the determined internal region, and the adaptor-specific primers AP1 and AP2. TG-specific primers designed from the 5¢ and 3 ¢ ends of the obtained products, 5¢GSP1 (5¢-CGATTACAGTCG TGGTCAGAGCTG-3¢), 5¢GSP2 (5¢-TCGTGGTCAGAG CTGTTGTTTGTG-3¢), 3¢GSP1 (5¢-CAAGGACTGACC TTCACTGAGATG-3¢)and3¢GSP2 (5¢-GTGGCGTTGG GATGCAACATTGTG-3¢), were used to amplify the full- length cDNA, and the BamHI (5¢-GCGGATCCATGGTT CGTCGATCCACTCGC-3¢)andNotI primers (5¢-CT GCGGCCGCTTAAGCACTCTTGACATTGAG-3¢)to amplify the coding sequence (Fig. 1). RNA isolation and Northern blot hybridization Samples of poly(A) + RNA (0.5 lg) were prepared from staged embryos as described previously [22]. They were denatured and separated by formaldehyde gel electropho- resis and transferred to nylon filters (Amersham Pharmacia Biotech). The blots were hybridized overnight at 42 °Cin hybridization buffer with a probe and washed according to the manufacture’s recommended protocol. Digoxygenin- labeled antisense RNA p robes were p repared from a linearized plasmid DNA template, which contained a 0.27-kbp StuI–NotI restriction fragment of nTG cDNA or 0.15-kbp BamHI–EcoRI restrictio n fragmen t of A. pecti- nifera ubiquitin cDNA (H. Sugino, unpublished data) using the digoxygenin-RNA labeling kit (Roche Molecular Bio- chemicals). D igoxygenin-labeled RNA probes were immu- nodetected with an Fab fragment of anti-digoxygenin Ig conjugated to alkaline phosphatase. The bound Ig conju- gate was then visualized with the chemiluminescent sub- strate CDP-Star (Roche Molecular Biochemicals). Expression and purification of glutathione S -transferase-conjugated nTG To generate a recombinant protein of nTG with N-terminally placed glutathinone S-transferase, the 2214-bp BamHI–NotI fragment, which contained the entire coding region of nTG (nTG fragment), was inserted between the BamHI and NotI sites of p GEX-4T-1(Amersham Pharma- cia Biotech). Escherichia coli [strain BL21 (DE3)] were transformed and transcription was induced with 0.5 m M 10 2 3 (kb) 5' 3' TG3-3 + AP1 / TG3-4 + AP2 BamHI primer + NotI primer TG5 + TG3V 5'GSP1 + 3'GSP1 / 5'GSP2 + 3'GSP2 TG3-1 + AP1 / TG3-2 + AP2 TG5-3 + AP1 / TG5-4 + AP2 Fig. 1. PCR strategy for amplification of nTG cDNA. The horizontal bar i ndicates the n TG cDNA. Thic k horizontal b ars indicate the sequences of PCR-amplified clones. T he p rimers used for PCR a re given on the right. The sequences of the oligonucleotide primers are given in Materials and methods. 1958 H. Sugino et al. (Eur. J. Biochem. 269) Ó FEBS 2002 isopropyl thio-b- D -galactoside. Bacteria were lysed in 1% Triton X-100 in NaCl/P i , sonicated with six bursts of 10 s, and incubated at 4 °C for 1 h. Insoluble materials were removed by centrifugation at 13 000 g for 10 min. Gluta- thione S-transferase-conjugated nTG w as purified from th e supernatant using glutathione–Sepharose 4B beads (Amer- sham Pharmacia Biotech) essentially following the protocol provided by the manufacturer. Biochemical fractionation of embryos Embryos were washed with ice-cold solution 1 [0.25 M sucrose, 10 m M Tris/HCl (pH 8.0), and 0 .1 m M EDTA]. They were then resuspended in the same volume of solution 1, to which had been added 0.15 m M spermine and 0.5 m M spermidine. The suspension was homogenized by 10 strokes with a Dounce homogenizer. To the homogenate was added 1.3 v ol. of 2.0 M sucrose, 65 m M KCl, 15 m M NaCl, 15 m M Tris/HCl (pH 8.0), 0.15 m M spermine, 0.5 m M spermidine, 10 m M 2-mercaptoethanol, and 0.1 m M phenylmethane- sulfonyl fluoride. The mixture was centrifuged for 50 min at 50 000 g, to give the nuclear fraction in the form of a pellet. Subnuclear fractionation was carried out according to the method described by Singh et al. [8]. In brief, the nuclear suspension was suspended in 10% sucrose, 10 m M trieth- anolamine/HCl (pH 7.5), and 0.1 m M MgCl 2 . The suspen- sion was treated with 5 lgÆmL )1 of deoxyribonuclease I (Worthington Biochemical) and 2 lgÆmL )1 of ribonuc- lease A (Sigma Chemicals) for 15 min at 22 °C, followed by centrifugation for 1 0 m in at 4 °C (20 000 g). The superna- tant was collected and designated as Sup1. The pellet obtained after this step was treated with 1% Triton X-100 and recentrifuged. T he supernatant was separated and designated as Sup2. The pellet was resuspended in 25 m M Tris/HCl (pH 7.5), 1% Triton X-100, and 0.5 M NaCl. This suspension was incubated for 30 min at 4 °Candthen centrifuged f or 10 min a t 20 000 g. T he supernatant was separated and designated as Sup3. The pellet was resus- pended in 10S buffer [50 m M Hepes/HCl (pH 7.2), 10 m M sodium phosphate, 250 m M NaCl, 0.3% Nonidet P-40, 0.1% Triton X-100, 0.005% SDS, 1 m M NaF, 0.5 m M dithiothreitol, a nd 0.1 m M phenylmethanesulfonyl fluoride] and the suspension incubated for 30 min, followed by centrifugation for 10 m in at 17 000 g. The supernatant, designated as Sup4, was separated from the pellet. For the immunoprecipitation experiment, Sup4 was concentrated to 1 : 26 of the original volume using Centricon-10 (Amicon). Proteins were determined by the modified method of alkaline copper (Lowry) protein assay [23] using BSA as the standard. Transglutaminase activity assays TG activity was assayed by fluorometric measurement of monodansylcadaverine conjugation to N,N-dimethylcasein [24]. S tandard reaction mixtures contained 2.5 mgÆmL )1 N,N-dimethylcasein,0.5 m M monodansylcadaverine,10 m M Tris/HCl (pH 7.5), 5 m M CaCl 2 ,and5m M dithiothreitol in 400 lL. Incubation was c arried out at 37 °C for 30 min. Reactions were quenched by the addition of 400 lLof10% (w/v) trichloroacetic acid and the suspension was chilled on ice for 20 min. Precipitated protein was collected by centrifugation for 20 min at 16 000 g,andwashedthree times with cold ethanol/diethyl ether (1 : 1, v/v), before solubilization in 4 mL of 50 m M Tris/HCl (pH 7.5), 8 M urea, and 0.5% (w/v) SDS. The amount of incorporated monodansylcadaverine was determined by measuring the fluorescence o f the solubilized protein using a Shimazu RF-540 fluorescence spectrophotometer with an excitation wavelength of 340 nm, emission wavelength of 525 nm, and a 5-nm slit. The instrument was calibrated with m ono- dansylcadaverine in 50 m M Tris/HCl (pH 7.5), 8 M urea, and 0.5% (w/v) SDS prior to each run. One unit of enzyme activity defined as AIU (amine incorporation unit per min) was calculated as described previously [24]. Preparation of nTG-specific antibodies Two portions of the putative amino acid sequence of nTG, Leu-Asp-Tyr-His-Tyr-Asp-Glu-Asn-Ser-Glu-Pro-Leu-Asp- Asp and Arg-Arg-Ser-Thr-Arg-Thr-Arg-Ser-Thr-Pro-Thr- Arg-Phe-Gly-Tyr-Thr-Asp-Arg, were used to produce nTG-specific polyclonal antibodies, anti-(nTG-M) Ig and anti-(nTG-N) Ig, respectively. The peptides were synthe- sized such that each of them contained an artificial Cys residue at the N- or C-terminus, respectively, for coupling purposes. E ach s ynthesized peptide was conjugated to maleimide-activated keyhole limpet hemocyanin (Amer- sham Pharmacia B iotech) a ccording to manufacturer’s instructions. New Zealand White rabbits were then immu- nized with a keyhole limpet hemocyanin-conjugated peptide (0.5 mg for each injection). Anti-nTG Ig in the antisera were affinity purified on the antigenic peptide cross-linked to 2-fluoro-1-methylpyridinium-toluene-4-sulfonate-activated cellulose (Seikagaku Kogyo, Tokyo, Japan). The bound nTG-specific Ig were e luted with 100 m M glycine-HCl (pH 2.5). The eluates were neutralized with 1 M Tris, and stored at )80 °C. Polyacrylamide gel electrophoresis and immunoblotting SDS/PAGE was c arried out accor ding to the method described by Laemmli [25]. Immunoblotting was performed on poly(vinylidene difluoride) membranes using anti- (nTG-M) I g (1.1 lgÆmL )1 ), and horseradish peroxidase- coupled goat a nti-(rabbit I gG) I g (Bio-Rad). D etection o f the peroxidase was carried out with 3,3¢-diaminobenzidine and H 2 O 2 . A control experiment w as performed using the anti- (nTG-M) Ig t hat h ad been preincubated for 1 h at37 °Cwith the antigenic peptide (0.65 lgÆmL )1 of affinity-purified Ig). Immunoprecipitation Concentrated Sup4 (10 lL) was incubated with the affinity- purified anti-(nTG-N) Ig (3 lg) for 3 h at 4 °C in 400 lLof IP buffer [50 m M Tris/HCl (pH 7.5), 150 m M NaCl, 0 .5% Triton X-100, and 0.1% SDS]. After the incubation, 100 lL of protein A–Sepharo se that had be en equilibrated in IP buffer was added, and t he mixture w as then rotated moderately for 1 h at 4 °C. Following centrifugation and removal of the supernatant, the pellets were washed twice with IP buffer, and resuspended with 4 00 lL of IP buffer (total volume, 500 lL). C ontrol experiments were per- formed using the affinity-purified anti-ANOC Ig, which was raised against the C-terminal portion of ANO 39, a starfish protein unrelated to nTG [22]. Ó FEBS 2002 Nuclear transglutaminase in starfish embryos (Eur. J. Biochem. 269) 1959 CGATTACAGTCGTGGTCAGAGCTGTTGTTTGTGTTCCTTGTAAATCGTAATCATCCAAA 59 ATGGTTCGTCGATCCACTCGCACCCGCAGCACCCCTACCCGCTTCGGCTACACCGACCGG 119 M V R R S T R T R S T P T R F G Y T D R TTTGAGCCGTATGCCCGCAAGCCTAAACGGGAAACGACGCGCACAGAGGGGCGACGCTAC 179 F E P Y A R K P K R E T T R T E G R R Y GTACCCGCCACACCACTGACTCTGCCTACGCTGAAAGAAAAAAAGACGCAACTCAAGGTG 239 V P A T P L T L P T L K E K K T Q L K V GTGTCAGTTGATCTATGTGTGGAGCGAAACCAGCAGGAGCATAAGACCAGCAAGTACAAG 299 V S V D L C V E R N Q Q E H K T S K Y K GTTGACAATCTGGTCCTGCGTCGTGGTCAACCGTTCCACCTCAATGTCAAGTTTGACCGA 359 V D N L V L R R G Q P F H L N V K F D R GACTTCAAGCCGAGTACCGATGAACTTGTATTGGAATTACGAATGGGCAGCCGTGCCAAC 419 D F K P S T D E L V L E L R M G S R A N GTGACCAAGGGCACACGCTGTGTGGCCCCCGTGGTAACGTCAGCCCCCGACCACGACGAT 479 V T K G T R C V A P V V T S A P D H D D TGGGGCATTAAGGTGGAGAGTGCCAAAGGCGCCAACGTGACGCTGAAGGTCTTCTGTAGT 539 W G I K V E S A K G A N V T L K V F C S TCGGAGGCTCTTATTGGCTACTACAATCTGTACATCTTGACGATGAGCGGTGGGGATGAA 599 S E A L I G Y Y N L Y I L T M S G G D E TACGAGTATGAATCTCCTAAGGAGCTCATCATGCTGTTCAACGCCTGGTGCAAAGATGAT 659 Y E Y E S P K E L I M L F N A W C K D D GATGTGTATATGGCTGATGAGGTGAAACGGCAGGAGTACGTCATGGGCGAAGTCAGCCTG 719 D V Y M A D E V K R Q E Y V M G E V S L TACTTCTATGGTTCCAAGTATCGCATCGGCTCATCCCCATGGAACTACGGGCAGTTTGAG 779 Y F Y G S K Y R I G S S P W N Y G Q F E AAAATGTCGTTGGACTGTGCCCTGTATTTGCTGCAGAAGTCCGGCATGCCCGACTCTAGC 839 K M S L D C A L Y L L Q K S G M P D S S CGCAAGAGCCCCATCCAGGTTTCCAGGGTTTTATCTGCCTTGGTCAATGCCCAAGATGAT 899 R K S P I Q V S R V L S A L V N A Q D D GACGGAGTTCTCGTGGGAAGATGGGATGGGGAGTATGACGACGGCATTTCCCCTACCACC 959 D G V L V G R W D G E Y D D G I S P T T TGGACTGGGAGCATCGCCATCTTGTCCCAGTACATGAAGACTCGGGAATCGGTCAAATAC 1019 W T G S I A I L S Q Y M K T R E S V K Y GGCCAGTGTTGGGTGTTCGGGAGTCTGCTCACTGGACTGTGCAGAAGTCTGGGTCTACCC 1079 G Q C W V F G S L L T G L C R S L G L P ACCCGGACCATCACCAATTTTGCCTCGGCTCACGACACCGATGGCAACCTGACTCTTGAC 1139 T R T I T N F A S A H D T D G N L T L D TACCACTACGATGAGAACTCGGAACCGTTGGATGACTATGACGAAGATAGTATCTGGAAT 1199 Y H Y D E N S E P L D D Y D E D S I W N TTCCACGTATGGAATGACTGCTGGATGGCTAGACCCGATCTGGAGGAGGGTTACGGGGGC 1259 F H V W N D C W M A R P D L E E G Y G G TGGCAGGCCGTGGACGCAACCCCTCAGGAAACAAGCAACGGTGTGTACTGCATGGGACCT 1319 W Q A V D A T P Q E T S N G V Y C M G P ACCTCTCTGCGCGCCATCAAGCAGGGTCACGTGTACATGCAGTATGACACCAAGTTTGCC 1379 T S L R A I K Q G H V Y M Q Y D T K F A TTTGCTGAGGTCAACGCTGAAAAGGTCTACTGGAAGGTCTTCACGAAATCTAGAAAGGCC 1439 F A E V N A E K V Y W K V F T K S R K A CCGGAGGTCATAGACATTGACTCCGATGATGTCGGATGCAAGATCAGCACCAAAGCCGTC 1499 P E V I D I D S D D V G C K I S T K A V GGCAAATTTGAGCGTGAGGACATCACTGAGCAGTACAAGTACAAGGAAGGAACGGAGTTG 1559 G K F E R E D I T E Q Y K Y K E G T E L GAGCGCATCGCCGTCAGAGAAGCCAGCCGTCATGTACGCAAAGCAAAGAGAATTCTCAAG 1619 E R I A V R E A S R H V R K A K R I L K AACCTTGTCCGCGACGTGGACTTTGACGTGGACATGGCGGAGGAGTTCCCCATTGGGAAA 1679 N L V R D V D F D V D M A E E F P I G K GATATCAAGTTCACTATCACTATGGTGAATAAGTCACAACAGACACGTAATGTCTTTCTG 1739 D I K F T I T M V N K S Q Q T R N V F L GGTGTGACAGGAAGCACCGTGTACTACACAGGTGTTAAGAAGGCCAAGGTGTCATCCTAC 1799 G V T G S T V Y Y T G V K K A K V S S Y AATGGCACCCTGCCACTGAAGGCAAAGGAAACGCGAGTGATTCCTGTGACTGTACCTGCG 1859 N G T L P L K A K E T R V I P V T V P A TCTGACTACCTGCCGCAGCTCACTGACTATGCTGGCGTAACGTTCTTCATCATGGCTTCC 1919 S D Y L P Q L T D Y A G V T F F I M A S GTCAAGGAGACCAAGCAACCATTCAGCAGGCAGTATGACGCCGTGCTTGATAAGCCTGAC 1979 V K E T K Q P F S R Q Y D A V L D K P D CTGGAGGTCAAGACGGAGGGGCCCATTGTGCGTGGCAAGCCGTTCACAGCTATCGTGTCG 2039 L E V K T E G P I V R G K P F T A I V S CTGACCAACCCATTGCCGTACCCGCTGACTGACTGCAGCCTACTTATGGAGGGGTCCATC 2099 L T N P L P Y P L T D C S L L M E G S I ATTGAGGGCGCCAAACGGGTCAAAGCTCCACATGTTCCAGTGAACGGTAAGATGGCCCAG 2159 I E G A K R V K A P H V P V N G K M A Q CGAGTGCAGCTGACACCCAAGACTGCTGGATCGTGCGACCTCATCGTCAGCTTCAGTTCC 2219 R V Q L T P K T A G S C D L I V S F S S CCGCAGCTCAGTGGTGTCAAGGCCCATGTCACACTCAATGTCAAGAGTGCTTAATTTGCT 2279 P Q L S G V K A H V T L N V K S A * ATGCGAGGTCAGCATTTATCCAACCAGAAGCTTCACGGAGCTAGCTGGGCAAGGAAATTT 2339 GATAATCGCAAGAAATAATTTCCCCCCAAAAACAAAAGGTTGTTGGCTGAAAATACTTCT 2399 ACATGTACATGTATATCACTTTGAACTGGTTTTCATTAAAAAAAAAAAACCATCAATTTG 2459 AGAAGAAACAATTACTTCTTAAGTCAATTAATTTTTCTAGAAATGCAAAAGATATTCCCC 2519 TTAACAGCTGTTTGAAATGAGGCCTCGGTCTCAAGTTTAAGAGTGCCCCCATATGTAAGC 2579 TAAAAAGCTCCAGGAAGTTGACCCAGAAGAAATTTGTTAAGAGTTCACGGATAAGCAAGG 2639 TATTTGGATAAGGTGCATTTGTACATTTTGTGTGTACTGGTTTAGTGTAGAATTTAATTT 2699 TTTTTGGTTAATTCTGTCACAAGAACATAATTCTATGGTTACTACACAATGTTGCATCCC 2759 AACGCCACCTTTTTATTTTTAATCATATATCATCTCAGTGAAGGTCAGTCCTTG 2813 A 1960 H. Sugino et al. (Eur. J. Biochem. 269) Ó FEBS 2002 To measure TG activity r ecovered in e ach fraction, aliquots (200 lL) of the supernatants or the resuspend ed pellets were incubated in the same condition as described above, except that incubation was carried out for 1 h. Immunofluorescence microscopy Embryos were processed for immunofluorescence as whole mounts. In some experiments, embryos were dissociated by the method described by Kaneko & Dan-Sohkawa [26]. The whole embryos or dissociated cells were fixed with 3.5% formaldehyde for 30 min at room temperature. After washing in NaCl/P i without divalent cations, the cells were incubated in 1% T riton X-100 in NaCl/P i ,theninNaCl/P i alone, then in acetone ()20 °C), and finally in NaCl/P i again. The samples were blocked with 3% BSA in NaCl/P i for 30 min at 37 °C. Incubations wi th primary and secondary antibodies were carried out for 2 h at 37 °C. Monospecific anti-(nTG-M) Ig (1.9 lgÆmL )1 ), which had been preincubated with the antigenic peptide (1.1 lgÆmL )1 of affinity-purified Ig), was used as the negative control. The secondary antibody was cEry MGGP 4 lHem MYGFGRGNMFRNRSTRYRRRPRYRAENYHSYMLDLLENMNEEFGRNWWGTPESHQPDS 58 nTG MVRRSTRTRSTPTRFGYTDRFEPYARKPKRETTRTEGRRYVPATPLTL 48 hKer MMDGPRSDVGRWGGNPLQPPTTPSPEPEPEPDGRSRRGGGRSFWARCCGCCSCRNAADDDWGPEPSDSRGRGSSSGTRRPGSRGSDSRRPVSRGSGVNAA 100 gpLiv MAEDLILERCDLQLEV NGRDHRTADLCRERLVLRRGQPFWLTLHFEGRGYEAGVDTLTFNAVTGPDPSEEAGTMARFSLSSAV EGGTW 88 cEry GPDGTMAEELVLETCDLQCER NGREHRTEEMGSQQLVVRRGQPFTITLNFAGRGYEEGVDKLAFDVETGPCPVETSGTRSHFTLTDCP EEGTW 97 lHem GPSSLQVESVELYTRDNAREH NTFMYDLVDGTKPVLILRRGQPFSIAIRFK-RNYNPQQDRLKLEIGFGQQPLITKGTLIMLPVSGSDTFTKDKTQW 154 nTG PTLKEKKTQLKVVSVDLCVER NQQEHKTSKYKVDNLVLRRGQPFHLNVKFD-RDFKPSTDELVLELRMGSRANVTKGTRCVAPVVTSAP DHDDW 141 hKer GDGTIREGMLVVNGVDLLSSRSDQNRREHHTDEYEYDELIVRRGQPFHMLLLLS RTYESSDRITLELLIGNNPEVGKGTHVIIPVGKGG SGGW 193 hPro MMDASKELQVLHIDFLNQD NAVSHHTWEFQTSSPVFRRGQVFHLRLVLN QPLQSYHQLKLEFSTGPNPSIAKHTLVVLDPRTPS DHYNW 89 . . * . * . . **** * . . . . . . * . * * gpLiv SASAVDQQDSTVSLLLSTPADAPIGLYRLSLEASTGYQG SSFVLGHFILLYNPRCPADAVYMDSDQERQEYVLTQQGFIYQGSAKFINGIPWN 181 cEry SAVLQQQDGATLCVSLCSPSIARVGRYRLTLEASTGYQG SSFHLGDFVLLFNAWHPEDAVYLKEEDERREYVLSQQGLIYMGSRDYITSTPWN 190 lHem DVRLRQHDGAVITLEIQIPAAVAVGVWKMKIVSQLTSEEQPNVSAVTHECKNKTYILFNPWCKQDSVYMEDEQWRKEYVLSDVGKIFTGSFKQPVGRRWI 254 nTG GIKVESAKGANVTLKVFCSSEALIGYYNLYILTMSGGDE YEYESPKELIMLFNAWCKDDDVYMADEVKRQEYVMGEVSLYFYGSKYRIGSSPWN 235 hKer KAQVVKASGQNLNLRVHTSPNAIIGKFQFTVRTQSDAGEFQLP FDPRNEIYILFNPWCPEDIVYVDHEDWRQEYVLNESGRIYYGTEAQIGERTWN 289 hPro QATLQNESGKEVTVAVTSSPNAILGKYQLNVKTGNHILK SEENILYLLFNPWCKEDMVFMPDEDERKEYILNDTGCHYVGAARSIKCKPWN 180 . . . . . .* . . .*.*. * * . *.** . . . *. * gpLiv FGQFEDGILDICLMLLDTNPKFLKNAGQDCSRRSRPVYVGRVVSAMVNCND-DQGVLQGRWDNNYSDGVSPMSWIGSVDILRRWKDYGCQRVKYGQCWVF 280 cEry FGQFEDEILAICLEMLDINPKFLRDQNLDCSRRNDPVYIGRVVSAMVNCNDEDHGVLLGRWDNHYEDGMSPMAWIGSVDILKRWRRLGCQPVKYGQCWVF 290 lHem FGQFTDSVLPACMLILER S-GLDYTARSNPIKVVRAISAMVNNID-DEGVLEGRWQEPYDDGVAPWMWTGSSAILEKYLKTRGVPVKYGQCWVF 346 nTG YGQFEKMSLDCALYLLQKS G MPDSSRKSPIQVSRVLSALVNAQD-DDGVLVGRWDGEYDDGISPTTWTGSIAILSQYMKTRES-VKYGQCWVF 326 hKer YGQFDHGVLDACLYILDRR G MPYGGRGDPVNVSRVISAMVNSLD-DNGVLIGNWSGDYSRGTNPSAWVGSVEILLSYLRTGYS-VPYGQCWVF 380 hPro FGQFEKNVLDCCISLLTES SLKPTDRRDPVLVCRAMCAMMSFEK-GQGVLIGNWTGDYEGGTAPYKWTGSAPILQQYYNTKQA-VCFGQCWVF 271 .*** . * .* * *. . *. . . *** *.* * * * * ** ** . * .****** gpLiv AAVACTVLRCLGIPTRVVTNFNSAHDQNSNLLIEYFRNESGE-IEGNKSEMIWNFHCWVESWMTRPDLEPGYEGWQALDPTPQEKSEGTYCCGPVPVRAI 379 cEry AAVACTVMRCLGVPSRVVTNYNSAHDTNGNLVIDRYLSETGM-EERRSTDMIWNFHCWVECWMTRPDLAPGYDGWQALDPTPQEKSEGVYCCGPAPVKAI 389 lHem AGVANTVSRALGIPSRTVTNYDSAHDTDDTLTIDKWFDKNGDKIEDATSDSIWNFHVWNDCWMARPDLPTGYGGWQAYDSTPQETSEGVYQTGPASVLAV 446 nTG GSLLTGLCRSLGLPTRTITNFASAHDTDGNLTLDYHYDENSEPLDDYDEDSIWNFHVWNDCWMARPDLEEGYGGWQAVDATPQETSNGVYCMGPTSLRAI 426 hKer AGVTTTVLRCLGLATRTVTNFNSAHDTDTSLTMDIYFDENMKPLEHLNHDSVWNFHVWNDCWMKRPDLPSGFDGWQVVDATPQETSSGIFCCGPCSVESI 480 hPro AGILTTVLRALGIPARSVTGFDSAHDTERNLTVDTYVNENGKKITSMTHDSVWNFHVWTDAWMKRPDLPKGYDGWQAVDATPQERSQGVFCCGPSPLTAI 371 . *.** * .* **** . .* . .**** * ** **** *. ***. *.**** *.* . ** gpLiv KEGHLNVKYDAPFVFAEVNADVVNWIRQK DGSLRKSIN-HLVVGLKISTKSVGRDE REDITHTYKYPEGSEEEREAFVRANHLNKLATKE- 468 cEry KEGDLQVQYDIPFVFAEVNADVVYWIVQS DGEKKKSTH-SSVVGKNISTKSVGRDS REDITHTYKYPEGSEKEREVFSKAEHEKSSLG 476 lHem QRGEIGYMFDSPFVFSEVNADVVHWQEDDSS-ETGYKKLKIDSYRVGRLLLTKKIGVDDDFGDADAEDITDQYKNKEGTDEERMSVLNAARSSGFNYAFN 545 nTG KQGHVYMQYDTKFAFAEVNAEKVYWKVFTKS-RKAPEVIDIDSDDVGCKISTKAVGKFE REDITEQYKYKEGTELERIAVREASRHVRKAKR 517 hKer KNGLVYMKYDTPFIFAEVNSDKVYWQRQD DGSFKIVYVEEKAIGTLIVTKAISSNMR EDITYLYKHPEGSDAERKAVETAAAHGSKPNVYA 571 hPro RKGDIFIVYDTRFVFSEVNGDRLIWLVKMVNGQEELHVISMETTSIGKNISTKAVGQDR RRDITYEYKYPEGSSEERQVMDHAFLLLSSERE 463 * . .* * *.*** . * .* . ** .*** ** **. ** . * gpLiv -EAQEETGVAMRIRVGQNMTMGSDFDIFAYITNGTAESHECQLLLCARIVSYNGVLGPVCSTNDLLNLTLDPFSENSIPLH-ILYEKYGDYLTESNLIKV 566 cEry EQEEGLHMRIKLSEGANNGSDFDVFAFISNDTDKERECRLRLCARTASYNGEVGPQCGFKDLLNLSLQPHMEQSVPLR-ILYEQYGPNLTQDNMIKV 572 lHem LPSPEKEDVYFNLLDIEKIKIGQPFHVTVNIENQSSETRRVSAVLSASSIYYTGITGRKIKRENGN-FSLQPHQKEVLSIE-VTPDEYLEKLVDYAMIKL 643 nTG ILKNLVRDVDFDVDMAEEFPIGKDIKFTITMVNKSQQTRNVFLGVTGSTVYYTGVKKAKVSSYNGT-LPLKAKETRVIPVT-VPASDYLPQLTDYAGVTF 615 hKer N-RGSAEDVAMQVEAQDAVMG-QDLMVSVMLINHSSSRRTVKLHLYLSVTFYTGVSG-TIFKETKKEVELAPGASDRVTMP-VAYKEYRPHLVDQGAMLL 667 hPro HRRPVKENFLHMSVQSDDVLLGNSVNFTVILKRKTAALQNVNILGSFELQLYTGKKMAKLCDLNKTSQIQGQVSEVTLTLDSKTYINSLAILDDEPVIRG 563 . . . . . . . *.* . * . . gpLiv RGLLIEPAANSYVLAERDIYLENPEIKIRVLGEPKQNRKLIAEVSLKNPLPVPLLGCIFTVEGAGLTKDQKSVEVPDPVEAGEQAKVRVDLLPTEVGLHK 666 cEry VALLTEYETGDSVVAIRDVYIQNPEIKIRILGEPMQERKLVAEIRLVNPLAEPLNNCIFVVEGAGLTEGQRIEELEDPVEPQAEAKFRMEFVPRQAGLHK 672 lHem YAIATVKETQQTWSEEDDFMVEKPNLELEIRGNLQVGTAFVLAISLTNPLKRVLDNCFFTIEAPGVTGAFR VTNRDIQPEEVAVHTVRLIPQKPGPRK 741 nTG FIMASVKETKQPFSRQYDAVLDKPDLEVKTEGPIVRGKPFTAIVSLTNPLPYPLTDCSLLMEGSIIEGAKR VKAPHVPVNGKMAQRVQLTPKTAGSCD 713 hKer NVSGHVKESGQVLAKQHTFRLRTPDLSLTLLGAAVVGQECEVQIVFKNPLPVTLTNVVFRLEGSGLQRPKI LNVGDIGGNETVTLRQSFVPVRPGPRQ 765 hPro FIIAEIVESKEIMASEVFTSFQYPEFSIELPNTGRIGQLLVCNCIFKNTLAIPLTDVKFSLESLGISSLQT SDHGTVQPGETIQSQIKCTPIKTGPKK 661 . . * . . *.* * . . .*. . . * * . gpLiv LVVNFECDKLKAVKGYRNVIIGPA 690 cEry LMVDFESDKLTGVKGYRNVIIAPLPK 698 lHem IVATFSSRQLIQVVGSKQVEVLD 764 nTG LIVSFSSPQLSGVKAHVTLNVKSA 737 hKer LIASLDSPQLSQVHGVIQVDVAPAPGDGGFFSDAGGDSHLGETIPMASRGGA 817 hPro FIVKLSSKQVKEINAQKIVLITK 684 . . . . B Fig. 2. Nucleotide and deduced amino acid sequences of nTG. (A) The nucleotide sequence of the cDNA clone which encodes nTG and the amino acid sequence deduced therefrom. (B) D educed amino-acid sequences for guinea pig liver TG (gpLiv) [ 5], chicken erythrocyte TG (cEry) [30], Limulus hemocyte TG (lHem) [31], human keratinocyte TG (hKer) [3], human prostate TG (hPro) [6], and nTG are shown using the single letter amino acid codes. Gaps have been inserted to achieve maximum similarity. Asterisks and dots at the bottom of the aligned sequences indicate positions that are occupied by identical or chemically similar amino acids in all TG. The arrowhead indicates the active site Cys residue [31]. The arrows indicate the positions of the H is and Asp residues of the c atalytic triad [35]. Putative nuclear localization signals [11] are underlined. Ó FEBS 2002 Nuclear transglutaminase in starfish embryos (Eur. J. Biochem. 269) 1961 donkey anti-(rabbit IgG) Ig labeled with fluorescein (Amersham Pharmacia Biotech). Specimens were observed with a Nikon Eclipse E600 equipped with d ifferential interphase and epifluorescence optics. Generation of green fluorescent protein–fusion protein constructs The 500 -bp BamHI–HindIII fragment, which contained the Drosophila heat shock protein promoter, was inserted into the BglII–HindIII site of pEGFP-1 (Clontech) t o generate pHEG. To generate the fusion protein between the green fluorescent protein (GFP) and nTG, the 800-bp KpnI– BamHI fragment that contained the GFP gene (GFP fragment) was first generated by subclo ning the 800-bp Eco47III–PstI fragment of pEGFP-C1 ( Clontech) into the HincII–PstI sites of pBluescriptII KS(+) (Toyobo), and then digested with KpnIandBamHI. The 2214-bp BamHI – NotI fragment, which contains the entire coding region of nTG (nTG f ragment), was generated via PCR with BamHI and NotI primers. The 2043-bp BamHI–NotIfragment, which contains the coding region of nTG, bu t without the N-terminal 57 amino-acids residue (nTGDN57 fragment), was generated via PCR with BamHI-2 (5¢-AGGGATCCCT CAAGGTGGTGTCAGTTGATC-3¢)andNotI primers. The 171-bp BamHI–XhoI fragment, which contains the N-terminal 57 amino acid residues of nTG (N57 fragment), was generated via PCR with BamHI and XhoI(5¢-GACTC GAGTTGCGTCTTTTTTTCTTTCAGC-3¢) primers. The 1596-bp BamHI–NotI fragment, which contains the en tire coding region of rat muscle P K [27] ( PK fragment), was generatedviaPCRwithPK-N(5¢-GCCGGATCCGGC CTCGAGATGCCCAAGCCAGACAGC-3¢)andPK-C (5¢-GAGCGGCCGCTCATCAGCCGAGCTCTGGTAC AGGCACTAC-3¢) primers. The PK fragment was digested with XhoI and ligated with N57 fragment to give the N57PK fragment. The nTG, nTGDN57, PK, and N57PK fragments were separately ligated with the GFP fragment and the vector fragment derived from KpnI/NotI-digestion of pHEG to produce pHE-TG, pHE-TGDN57, pHE-PK, and pHE-N57PK, respectively. Expression of cloned cDNAs in vivo The constructs were separately dissolved in 10 m M Tris/HCl (pH 8.5) to g ive a final concentration o f 200 ngÆlL )1 . Twenty picoliters of the solution, along with a small amount of KF96 silicone oil (Shin-Etsu Chemical, Tokyo, Japan), were then microinjected into the germinal vesicle of an oocyte as described previously [22]. T hree to four hours later, the injected oocytes we re examined for localization of fluorescent proteins under a fluorescence microscope equipped with differential interphase and epifluorescence optics. RESULTS Molecular cloning of A. pectinifera transglutaminase Comparison of the amino-acid sequences among already- known TGs shows highly conserved regions, including the TG active site [28], in t he middle portions of the polypep- tides. Based on the sequence of the con served regions, a single set of degenerate oligonucleotide primers, TG5 and TG3V, were designed and used for an RT-PCR experiment. Using poly(A) + RNA f rom A. pectinifera embryos a t the early b lastula stage as a template, a single PCR product which encodes a 65-amino-acid sequence similar to that of the catalytic site-containing region of the other TGs was amplified. To obtain further sequences upstream of primer TG5 and downstream of primer TG3V, we used the r apid amplification of cDNA ends (RACE) approach with the strategy summarized in Fig. 1. Finally, a 2813-bp cDNA was amplified utilizing the primers 5¢GSP1,2 and 3¢GSP1,2, which correspond to the 5¢ or 3¢ edges, respectively, of B 200 - 97.2 - 66.2 - 45.0 - 31.0 - 21.5 - 14.4 - 6.5 - 321 112.0 - 81.0 - 645 A Fig. 3. Western blot analysis of nTG during embryogenesis. (A) Cyto- solic fractions (lanes 1–3 and 7–9) and nuclear fractions (lanes 4–6 and 10–12) we re prepared from 8-h-old early b lastulae (lanes 1, 4, 7, and 10), 12-h-old mid-blastulae (lanes 2, 5, 8, and 11), and 24-h-old mid- gastrulae (lanes 3, 6, 9, and 12). An a liquot of each fraction (60 lg bovine serum albumin-equivalent per lane) was separated by SDS/ PAGE, and the gel was stained with Coomasssie blue (lanes 1–6) or transferred to poly(vinylidene difluoride) membrane, followed by staining using anti-(nTG-M) Ig as a probe (lanes 7–12). Sizes of the molecular mass marker proteins in kDa are shown to the left. (B) Nuclear fractions were prepared from 29- h-old midgastrulae (lan es 1 and 4), 40-h-old late gastrulae (lanes 2 and 5), and 51-h-old bipinnariae (lanes 3 and 6). An aliquot of each fraction (3000 embryos-equivalent per lane) was separated by SDS/PAGE, and the gel was stained with Coomasssie blue (lanes 1–3) or transferred to poly(vinylidene difluo- ride) membrane, followed b y s taining using an ti-(nTG-N) Ig a s a p robe (lanes 4–6). S izes of the molecular mass marker prote ins in kD a are shown to the left. 1962 H. Sugino et al. (Eur. J. Biochem. 269) Ó FEBS 2002 the sequence obtained by t he RACE experiments. The cDNA contained a single open reading fram e ( ORF) beginning with an ATG codon in an adequate context for the initiation of translation (Fig. 2A); the sequence CCAAAATGG s urrounding the A TG fits t he consensus sequence CC(G/A)CCATGG f or the eukaryotic initiator site [29]. The predicted protein consists of 737 amino acids, with a molecular mass of 83 105 Da and an isoelectric point of 7.9. Neither a polyadenylation signal (AATAAA) nor a poly(A) + tail was found in the 540-bp untranslated region following the termination codon (TAA), suggesting that the cDNA might not be full-length. Figure 2B shows the alignment of t he deduced amino- acid sequence w ith those o f t he other known TGs from various species [2,3,5,6,30,31]. The predicted protein exhi- bited 33–41% identity with other TGs. The residues comprising the catalytic triad are perfectly conserved (Cys323, His382, Asp405; Fig. 2B). Three acidic residues, Glu447, Glu496, and Glu501, which could act as a Ca 2+ - binding site [32], were also conserved. The sequence surrounding His351, i.e. Ser349-Ala-His-Asp352, was con- served, suggesting its interaction with Glu443 by analogy with the crystallography data on factor XIIIa [32] (Fig. 2B). On the other hand, residues for the putative GTP-binding region [33] found in tissue TGs were not well conserved. To confirm whether the predicted protein carries TG activity, a bacterially expressed protein in which the putative ORF was fused in-frame at its N-terminal end to glutathi- one–S-transferase was prepared, and assayed for TG acti- vity. The recombinant protein catalyzed the incorporation of monodansylcadaverine into N,N-dimethylc asein with K m and V max values of 0.35 m M and 13.3 n molÆmin )1 Æmg )1 , respectively, indicating that the predicted protein is a transglutaminase. Subcellular localization of A. pectinifera transglutaminase A major characteristic feature of the A. pectinifera TG is the presence of two putative nucle ar localization signals in the N-terminal region, a monopartite (residues 26–30) and a bipartite (residues 38–39 and 52–55) ones [12–14], suggest- ing nuclear localization of this protein. To examine if the A. pectinifera TG is a nuclear protein, we raised an B ab cd ef gh C ab cd ef gh a b A Fig. 4. Subcellular localization of nTG in embryos. (A) The distribution of nTG in a mid gastrula, as detected by indirect immuno fluorescen ce microscopy using a rabbit anti-(nTG-M) Ig and a fluorescein-conjugated secondary antibody. Immunofluorescence micrographs (a) and corre- sponding Normaski differential interference-contrast images (b). Bar, 50 lm (B) The distribution of nTG in cells dissociated from 24-h-old midgastrulae, as detected by indirect immunofluorescence microscopy using a rabbit anti-(nTG-M) Ig and a fluoresce in-conjugated secondary Ig (a,b). As a negative control, parallel immunofluorescence was performed using the anti-(nTG -M) Ig preincubated with the antigenic peptide (c,d) or preimmune sera (e,f), or omitting the primary antibody (g,h). Immunofluorescence micrographs (a,c,e,g) and the corresponding Nomarski differential interference-contrast micrographs (b,d,f,h). Bar, 5 lm (C) Subcellular localization of nTG during embryogenesis. The distribution of nTG in cells dissociated from 8-h- (a,b), 12-h- (c,d), 15-h- (e,f), and 24-h-old embryos (g,h) as detected by indirect immunofluorescence microscopy as described abo ve. Immu nofluorescenc e micrographs (a,c,e ,g), and th e corresponding Nomarski differential interference-contrast micrographs (b,d,f,h). Bar, 5 lm. Ó FEBS 2002 Nuclear transglutaminase in starfish embryos (Eur. J. Biochem. 269) 1963 antibody, d esignated anti-(nTG-M) Ig, against the peptide whose sequence (Leu359–Asp372) was deduced from the nucleotide sequence of cloned cDNA, and used it for Western blot analysis (Fig. 3A) and immunocytochemistry (Fig. 4B). On blots shown in Fig. 3A, this antibody specifically reacted with a single 90-kDa protein of the nuclear fraction which was prepared from mid-blastulae (12 h after fertilization: lane 11) or midgastrulae (24 h after fertilization: lane 12) whereas n o band was detected in the cytosol fraction (lane 8, 9). When formalin-fixed prepara- tions of single cel ls, which had been dissociated from the midgastrulae, were stained with the same antibody, the signal was limited to the nucleus (Fig. 4 B, a and b). The staining was fully blocked by preincubation of the antibody with the antigenic peptide (Figs 4B, c and d), demonstrating that the observed fluorescence was not derived from nonspecific labeling of the nucleus. These results collectively indicate that the protein encoded by the cloned cDNA is localized to the nucleus. Hence, we designated the protein Ônuclear transglutaminase (nTG) Õ. Expression pattern of nTG during embryogenesis Early starfish development may be directed by two sources of mRNA: (a) a pool stored in the immature oocyte transcribed from the maternal genome during oogenesis such as ANO39 mRNA [22], and (b) newly synthesized mRNA transcribed from the embryonic genome. Northern blot hybridization on poly(A) + RNA from blastulae and gastrulae showed a gradual increase in the signal at 5.0-kb during the progression of embryonic development (Fig. 5), whereas hybridization on poly(A) + RNA from f ertilized eggs showed little or no signals, s uggesting that nTG mRNA belongs to the latter. The developmental Western blot analysis revealed that the 90-kDa band corresponding to the nTG protein was first detected in the mid-blastula embryo and that the level of the band increased by t he bipinnaria stage (Fig. 3A, lanes 10–11, Fig. 3B, lanes 4–6). Therefore, the nTG protein s ynthesis starts at mid-blastula stage and continues thereafter. Immunostaining o f t he dissociated cells of embryos at different developmental stages revealed a specific pattern of accumulation. At the 8- to 12-h-old early blastula stages, nTG was undetectable in the nucleus (Fig. 4C, a–d). The nucleus of the early blastula is larger and looser than that of embryos collected at later developmental stages (Figs 4C, b,d,f,h). nTG starts to accumulate in the compact nucleus of the 15-h-old mid-blastula (Figs 4C, e,f) and its amount increases over the next 9 h (Figs 4C, g,h). To identify the cells expressing nTG, formalin-fixed whole-mount embryos at the midgastrula stage were stained with the anti-(nTG-M) Ig. Staining was not limited to specific areas but was observed in cells of all the germ layers (Fig. 4 A). Extraction of nTG from midgastrulae We measured the TG activity in nuclear preparations obtained from 8-h-old early blastulae, 12-h-old mid-blast- ulae, and 24-h-old midgastrulae. As the total TG activity (Fig. 6A) as well as the amount of nTG protein (Fig. 3A) was the highest in the nuclear fraction prepared from midgastrulae, we e xtracted nTG f rom this preparation according to the methods of Singh et al. [8]. After treatment with deoxyribonuclease I and ribonuclease A (Sup1), the nuclear preparation was subjected to extraction with 1% Triton X-100 t o afford Sup2 (nuclear membrane fraction), andthenwithacombinationof1%TritonX-100and0.5 M NaCl to afford Sup3 (nuclear pore–lamina complex fraction). Substantial TG activity remained insoluble after extraction of the nuclear pore–lamina complex. Extraction of the pellet with 10S buffer, which contained 0.005% SDS along with a nonionic detergent, successfully solubilized the enzyme; the total activity recovered in Sup4 was nearly twice as large as that in the nuclear fraction (Fig. 6B). The apparent activation of the enzymatic activity recovered in Sup4 could be due to the r emoval of putative i nhibitors during subnuclear fractionation. SDS/PAGE of Sup4 resulted in a prominent band with an apparent molecular mass of 90 kDa (Fig. 6C, lane 1), which was re cognized by the anti-(nTG-M) Ig in Western blot analysis (Fig. 6C, lane 3). To determine if the TG activity in Sup4 results from t he nTG protein, Sup4 was subjected to immunoprecipitation with the antibody raised against the N-terminal portion (Arg3–Arg20) of nTG [anti- (nTG-N) Ig]. As a result, the TG activity was mainly recovered in the immunoprecipitate (Fig. 7), showing t hat the molecule, which predominantly generates the TG activity in Sup4, is the nTG. Identification of the segment containing nuclear localization signals in nTG To identify the elements(s) in nTG that determine nucleus- specific topogenesis, we examined the localization of the 12345 10.0 - 4.0 - 3.0 - 6.0 - Fig. 5. Expression of nTG gene. Northern blots of poly(A) + RNA from fertilized eggs (lane 1), 8-h-old early blastulae (lane 2), 12-h-old mid-blastulae (lane 3), 15-h-old late b lastulae (lane 4), and 24-h-old midgastrulae (lane 5). The filter was hybridized with a digoxygenin- labeled RNA probe obtained from the c DNA of nTG (upper pane l) and of A. pectinifera ubiquitin (lower panel). Each lane was loaded with 0.5 lgofpoly(A) + RNA. Sizes of the transcripts expressed in kb were determined by comparison to the relative migration of RNA markers. 1964 H. Sugino et al. (Eur. J. Biochem. 269) Ó FEBS 2002 GFP–nTG fusion protein produced in an oocyte by microinjection of pHE-TG, which c ontains the Drosophila heat shock protein promoter and a gene that encodes the fusion protein (Fig. 8A), into the germinal vesicle, a nucleus that is arrested in prophase of division I of meiosis [22]. Transcription-coupled translation produced the fluorescent fusion protein and the major fraction was accumulated in the germinal vesicle as shown by fluorescence microscopy (Fig. 8B, a,e). On the other hand, microinjection of pHE- TGDN57, which contains the Drosophila heat shock protein promoter and the gene encoding nTG, in which the N-terminal 57 amino-acid residues had been deleted and fused with GFP, led to the formation of a fluorescent protein which was not located in the nucleus but, rather, Fig. 6. Extraction of nTG from the nuclear fraction. (A) TG activity in the nuclear fraction during emb ryogenesis. Enzyme activity was measured on the nuclear fractions prepared from 8-h-old early blast- ulae, 12-h-old mid-blastulae, and 24-h-old midgastrulae. The activity is expressed as the percentage of maximum activity observed in 24-h-old midgastrulae. (B) TG activity extracted from the n uclear fraction. Sup1, Sup2, Sup3, and Sup4 ( S1, S2, S3, and S4, respective ly) were prepared from the nuclear fraction of 24-h-old midgastrulae as described in Materials and methods, and assayed for TG activity. Results are shown as t he percent age activity relative to th e total activity in the nuclear fraction. (C) Western blot analysis of nTG r ecovered in Sup4. A n a liquot of Sup4 prepared from the nuclear fraction of 24-h-old midgastrulae (400 embryos-equivalent per lane) was sepa- rated by SDS/PAGE, and the gel was stained with Coomasssie blue (lane 1) or transferred to poly(vinylidene difluoride) membrane, fol- lowed by staining using anti-(nTG-M) Ig as a p robe (lanes 3). As a negative control, parallel immunoblotting was performed using anti- (nTG-M) Ig preincubate d with the peptid e antigen (lane 2) . Sizes of the molecular mass marker proteins in kDa are shown to the left. 100 50 0 Input TG activity (% control) SIPSIP anti-nTG-N Control Ig Fig. 7. Immunoprecipitation of nTG recovered in Sup4. Ten microliters of concentrated Sup4 were subjected to immunoprecipitation wit h anti-(nTG-N) Ig or control Ig (anti-ANOC Ig [22]). Total TG activity recovered in the supernatants (S) or the immunoprecipitates (IP) was measured, and is expressed as the percentage of the total activity in the 10 lL of concentrated Sup4 (Input). The results shown are the aver- ages of three experiments. Error bars indicate plus one SEM. A nTGGFPhsp nTG∆N57 hsp GFP PKhsp GFP N57hsp GFP PK pHE-TG pHE-TG∆N57 pHE-PK pHE-N57PK B a b c d e f g h Fig. 8. Subcellular localization of the green fluorescent protein-nTG fusion protein after express ion in oocytes. (A) Schematic drawings of constructs pHE-TG, pHE-TGDN, pHE-PK, and pHE-N57PK, which encode fusion proteins, GFP–nTG, GFP–N57-delete d nTG, GFP– PK, and GF P–N5 7PK, respectively. hsp, the Drosophila heat sho ck protein promoter; GFP, green fluorescent protein; N57, N-terminal region (residues 1–57); nTGDN57, N57-deleted nTG; PK, rat pyruvate kinase. (B) Subcellular localization of hybrid proteins after expression in oocytes. Four picograms each of pHE-TG (a,e), pHE-TGDN57 (b,f), pHE-PK (c,g), and pHE-N57PK (d,h) were separately microin- jected into the germinal vesicle of an Asterina pectinifera oocyte. Three to four hours later, the injected oocytes were examined for localization of fluorescent proteins with a fluorescence microscope (a–d) and with Nomarski differential interphase-contrast optics (e–h). Bar, 50 lm. Ó FEBS 2002 Nuclear transglutaminase in starfish embryos (Eur. J. Biochem. 269) 1965 almost exclusively in the cytoplasm (Fig. 8B, b,f). The possibility that the N-terminal 57 amino-acid resid ues (N57) act as an autonomous signal that is capable of specifying nuclear translocation was tested by directly transferring it to the N-terminus of PK, a cytoplasmic protein. A cDNA encoding rat muscle PK [27] was engineered to include the GFP sequence a nd the s equence of N57 that precedes the fusion junction with PK. The construct, pHE-N57PK, was microinjected into the germinal vesicle of an oocyte and the subcellular localization of the expressed protein was moni- tored. The results, as shown in Fig. 8B, clearly demonstrate the ability of N57 to promote the nuclear accumulation of PK (Figs 8B, d,h). Without N57, the expressed GFP–PK fusion protein is located exclusively in the cytoplasm (Figs 8 B, c,g). DISCUSSION During the early development of A. pectinifera, the embryo undergoes e xtremely rapid cellular replication [16,18]. Slower rates of cell division characterize the embryo from the early to mid-blastula stages. Concomitant with this rate reduction, an increase in embryonic t ranscriptional activity is also observed. The large swollen nuclei become smaller and more compact, and dispersed chromatin becomes more condensed. The present study demonstrates that nTG initially appears in A . pectinifera embryos at t he mid- blastula transition and that the level of the enzyme protein increases gradually thereafter (Figs 3 and 4). nTG is similar to t he TG of vertebrates and arthropods [34]. Its molecular mass is within the 75–90-kDa range known for the TG of these organisms [34]. The most unique property of nTG, not found in other TGs, is that its distribution is confined to the nucleus. Nuclear localization of TG has been reported in the studies on tissue TG [8,9]. A nuclear transport protein, importin-a3, has been shown to be involved in the active transport of tissue TG into the nucleus in NCI-H596 cells [10]. Recently, it has b een demonstrated that tissue TG interacts with histone H2B in lysates of neural cells which had been committed to apoptosis and that this interaction might takes place in vivo, as indicated b y the subcellular localization of the enzyme in the nuclear matrix [35]. Furthermore, retinoblas- toma protein has been identified as a nuclear substrate of the TG activity of tissue TG in promonocytic cells undergoing apoptosis [36]. These studies have revealed that tissue TG translocates to the nucleus of mammalian cells and catalyzes transamidation under certain circumstances. However, the amount of tissue TG in the nucleus is lower than that in the cytosol of normally growing cells [9]. On the other hand, nTG is located exclusively in the nucleus of starfish embryos (Figs 3 and 4). This could be due to the presence of functional nuclear localization signals in the N-terminal region, which are not found in other TGs (Fig. 2B). The results of in vivo transcription-coupled translation experi- ments using a series of mutants within the nTG coding region in-frame with the GFP established that the N-terminal region is strictly necessary for nuclear targeting (Fig. 8), implying that nTG has to be transported as other nuclear proteins across the nuclear membrane. We recently reported the occurrence of a novel histone modification in A. pe ctinifera sperm, wh ich invo lves an e-(c-glutamyl)lysine cross-link between a Gln residue o f histone H2B and a Lys residue of histone H4 to form a histone dimer, which has been designated p28 [37,38]. Experimental data not described in t he present paper indicate that a significant a mount of p28 is p roduced in A. pectinifera embryos at the mid-blastula stage but not at earlier stages (T. Shimizu & S. Ikemagi, unpublished results). Although the formation of an e-(c-glutamyl) lysine cross-link could be accounted for by several mech- anisms such as the activation of a c-carbonyl of histone H2B by esterification, followed by a nucleophilic attack by an e-amino group of Lys residue of histone H4 [38], the fact that the cross-link is formed between Gln9 of H2B and Lys5 of H4 strongly suggests that p28 is produced by a transamidation reaction catalyzed by TG. Although the possibility that p28 is produced in the cytoplasm and then translocated into the nucleus cannot be excluded, our data show the simultaneous appearance of both n TG and p28 in the nucleus of embryonic cells during the progression of embryogenesis. This finding is consistent with nTG being involved in the histone dimerization reaction. We have shown that the treatment of A. pectinifera embryos with trichostatin A, a potent a nd selective inhibitor of histone d eacetylase [39], induces hyperacetyla- tion of histone H4 and causes developmental arrest at the early gast rula stage [18]. Trichostatin A treatment causes suppression of the appearance of p28 in A. pectinifera embryos (T. Shimizu & S. Ikemagi, unpublished results). The acetylation of Lys5 of histone H4 competes with the TG-catalyzed histone dimerization reaction because a n acetylated lysine re sidue is not a functional amine donor substrate for TG. Deprivation of the amine donor for the TG reaction to produce p28 could be the cause of developmental arrest. However, this issue will only be settled if a very specific inhibitor of the TG activity can b e obtained and p roduce similar d evelopmental a rrest as observed b y t richostatin A -treated embryos which are devoid of p28 but whose histone H4 is in the normal acetylation-deacetylation c ycle. Such investigations are currently in progress in our laboratory. ACKNOWLEDGEMENTS We thank D rs S. Hirose (Tokyo Institute of Technology), K. Okano (Akita Prefectural University), and T . Noguchi (Nagoya U niversity) for the plasmids harboring bovine endotherial TGase, the Drosophila heat shock protein p romoter, and rat muscle pyruvate kinase, respectively. This work was supported, in part, by a Grant-in-Aid for Scientific Research from the Ministry of Education, Science, Sports and Culture, Japan, and by Special Coordination Funds for Promoting Science and Technology of t he Science and Technology Agency of the Japanese Government. REFERENCES 1. Folk, J.F. (1980) Transglutaminases. Annu. Rev. B iochem . 49, 517–531. 2. Ichinose, A., Henderickson, L.E., Fujiwara, K. & Davie, E.W. (1986) Amino acid sequence the a subunit of human factor XIII. Biochemistry 25, 6900–6906. 3. Phillips, M.A., Stewart, B.I., Qin, Q., Charkravarty, R., Floyd, E.E., J etten, A.M. & Rice, R.H. 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ATGCGAGGTCAGCATTTATCCAACCAGAAGCTTCACGGAGCTAGCTGGGCAAGGAAATTT 2339 GATAATCGCAAGAAATAATTTCCCCCCAAAAACAAAAGGTTGTTGGCTGAAAATACTTCT 2399 ACATGTACATGTATATCACTTTGAACTGGTTTTCATTAAAAAAAAAAAACCATCAATTTG 2459 AGAAGAAACAATTACTTCTTAAGTCAATTAATTTTTCTAGAAATGCAAAAGATATTCCCC. AGAAGAAACAATTACTTCTTAAGTCAATTAATTTTTCTAGAAATGCAAAAGATATTCCCC 2519 TTAACAGCTGTTTGAAATGAGGCCTCGGTCTCAAGTTTAAGAGTGCCCCCATATGTAAGC 2579 TAAAAAGCTCCAGGAAGTTGACCCAGAAGAAATTTGTTAAGAGTTCACGGATAAGCAAGG