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 m
Trang 1Molecular 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 transglutaminase (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 the 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 gerN-ter-minal vesicle of an oocyte produced chimeric proteins by transcription-coupled translation It was found that the N-terminal segment alone was sufficient to 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; embryo; 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 plasma factor XIIIa [2], keratinocyte TG [3],
epidermal TG [4], tissue TG [5], and prostatic TG [6]
Recent findings have shown that, apart from 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 Gha, 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 activity 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 concen-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 [13–15] The 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 the 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 blot analyses carried out in this study demonstrate that nTG mRNA appears at the early blastula stage and increases thereafter The nTG protein level increases in parallel with mRNA 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]
M A T E R I A L S A N D M E T H O D S Cultivation of embryos
Specimens of the starfish, A pectinifera, were collected from coastal waters off Japan during their breeding season and maintained in artificial sea water in laboratory aquaria at
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, green 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)
Trang 215°C Eggs and sperm were obtained as described
previ-ously [18–20] Eggs were fertilized and embryos were
cultured in artificial sea water that contained 5 mgÆmL)1
streptomycin sulfate and 50 lgÆmL)1penicillin G Cultures
were maintained in jars at a density of < 5000 embryos per
mL with gentle stirring Only cultures with a fertilization
rate in excess of 95% and normal morphological
develop-ment were used for experidevelop-mentation
RT-PCR
Poly(A)+RNA was prepared from blastulae (packed
vol-ume, 300 lL) using a QuickPrep micro mRNA purification
kit (Amersham Pharmacia Biotech) 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 an internal control for
PCR PCR was carried out with 1.25 U of KOD DNA
polymerase (Toyobo, Osaka, Japan) in the reaction mixture
(50 lL) that contained 120 mMTris/HCl (pH 8.0), 10 mM
KCl, 6 mM(NH4)2SO4, 0.1% Triton X-100, 0.001% BSA,
1 mM MgCl2, 0.2 mM each of four deoxyribonucleoside
5¢-triphosphates, and 4 lMeach of the TG-specific
degen-erate oligonucleotide primers, TG5 (5¢-TAYGGNCARTG
YTGGGT-3¢; N ¼ A, C, G or T; Y ¼ C or T; R ¼ A or
G) and TG3V (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 period (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 amplified by PCR in both directions
using TG-specific oligonucleotide primers VG5-3 (5¢-ACCC
TCCTCCAGATCGGG-3¢) and TG3-1 (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, with the first 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¢) and TG3-2 (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 directions under the conditions described above 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¢) and TG3-4 (5¢-CCATTGCCGTACCCGCTG-3¢), the sequences of which were derived 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¢) and 3¢GSP2 (5¢-GTGGCGTTGG GATGCAACATTGTG-3¢), were used to amplify the full-length cDNA, and the BamHI (5¢-GCGGATCCATGGTT CGTCGATCCACTCGC-3¢) and NotI 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°C in hybridization buffer with a probe and washed according to the manufacture’s recommended protocol Digoxygenin-labeled antisense RNA probes were prepared 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 restriction fragment of A pecti-niferaubiquitin cDNA (H Sugino, unpublished data) using the digoxygenin-RNA labeling kit (Roche Molecular Bio-chemicals) Digoxygenin-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 pGEX-4T-1(Amersham Pharma-cia Biotech) Escherichia coli [strain BL21 (DE3)] were transformed and transcription was induced with 0.5 m
1
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 indicates the nTG cDNA Thick horizontal bars indicate the sequences of PCR-amplified clones The primers used for PCR are given on the right The sequences of the oligonucleotide primers are given in Materials and methods.
Trang 3isopropyl thio-b-D-galactoside Bacteria were lysed in 1%
Triton X-100 in NaCl/Pi, 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 was purified from the
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 [0.25M
sucrose, 10 mM Tris/HCl (pH 8.0), and 0.1 mM EDTA]
They were then resuspended in the same volume of solution
1, to which had been added 0.15 mMspermine and 0.5 mM
spermidine The suspension was homogenized by 10 strokes
with a Dounce homogenizer To the homogenate was added
1.3 vol of 2.0Msucrose, 65 mMKCl, 15 mMNaCl, 15 mM
Tris/HCl (pH 8.0), 0.15 mMspermine, 0.5 mMspermidine,
10 mM 2-mercaptoethanol, and 0.1 mM
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 mM
trieth-anolamine/HCl (pH 7.5), and 0.1 mMMgCl2 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 10 min 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 The supernatant was separated and
designated as Sup2 The pellet was resuspended in 25 mM
Tris/HCl (pH 7.5), 1% Triton X-100, and 0.5MNaCl This
suspension was incubated for 30 min at 4°C and then
centrifuged for 10 min at 20 000 g The supernatant was
separated and designated as Sup3 The pellet was
resus-pended in 10S buffer [50 mMHepes/HCl (pH 7.2), 10 mM
sodium phosphate, 250 mM NaCl, 0.3% Nonidet P-40,
0.1% Triton X-100, 0.005% SDS, 1 mM NaF, 0.5 mM
dithiothreitol, and 0.1 mMphenylmethanesulfonyl fluoride]
and the suspension incubated for 30 min, followed by
centrifugation for 10 min 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] Standard reaction mixtures contained 2.5 mgÆmL)1
N,N-dimethylcasein,0.5 mMmonodansylcadaverine,10 mM
Tris/HCl (pH 7.5), 5 mMCaCl2, and 5 mMdithiothreitol in
400 lL Incubation was carried out at 37°C for 30 min
Reactions were quenched by the addition of 400 lL of 10%
(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, and washed three
times with cold ethanol/diethyl ether (1 : 1, v/v), before solubilization in 4 mL of 50 mM Tris/HCl (pH 7.5), 8M
urea, and 0.5% (w/v) SDS The amount of incorporated monodansylcadaverine was determined by measuring the fluorescence of 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 mono-dansylcadaverine in 50 mM Tris/HCl (pH 7.5), 8M 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 Each synthesized peptide was conjugated to maleimide-activated keyhole limpet hemocyanin (Amer-sham Pharmacia Biotech) according 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 eluted with 100 mM glycine-HCl (pH 2.5) The eluates were neutralized with 1MTris, and stored at)80 °C
Polyacrylamide gel electrophoresis and immunoblotting SDS/PAGE was carried out according to the method described by Laemmli [25] Immunoblotting was performed
on poly(vinylidene difluoride) membranes using anti-(nTG-M) Ig (1.1 lgÆmL)1), and horseradish peroxidase-coupled goat anti-(rabbit IgG) Ig (Bio-Rad) Detection of the peroxidase was carried out with 3,3¢-diaminobenzidine and
H2O2 A control experiment was performed using the anti-(nTG-M) Ig that had been preincubated for 1 h at 37°C with the antigenic peptide (0.65 lgÆmL)1of 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 lL of
IP buffer [50 mMTris/HCl (pH 7.5), 150 mMNaCl, 0.5% Triton X-100, and 0.1% SDS] After the incubation, 100 lL
of protein A–Sepharose that had been equilibrated in IP buffer was added, and the mixture was 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 400 lL of IP buffer (total volume, 500 lL) Control 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]
Trang 4CGATTACAGTCGTGGTCAGAGCTGTTGTTTGTGTTCCTTGTAAATCGTAATCATCCAAA 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 ACATGTACATGTATATCACTTTGAACTGGTTTTCATTAAAAAAAAAAAACCATCAATTTG 2459 TTAACAGCTGTTTGAAATGAGGCCTCGGTCTCAAGTTTAAGAGTGCCCCCATATGTAAGC 2579 TATTTGGATAAGGTGCATTTGTACATTTTGTGTGTACTGGTTTAGTGTAGAATTTAATTT 2699
A
Trang 5To measure TG activity recovered in each fraction,
aliquots (200 lL) of the supernatants or the resuspended
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/Pi without divalent cations, the cells were incubated in 1% Triton X-100 in NaCl/Pi, then in NaCl/Pialone, then in acetone ()20 °C), and finally in NaCl/Piagain The samples were blocked with 3% BSA in NaCl/Pi for 30 min at 37°C Incubations with 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)1of 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 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 .* *.* * * *.** * *
gpLiv FGQFEDGILDICLMLLDTNPKFLKNAGQDCSRRSRPVYVGRVVSAMVNCND-DQGVLQGRWDNNYSDGVSPMSWIGSVDILRRWKDYGCQRVKYGQCWVF 280 cEry FGQFEDEILAICLEMLDINPKFLRDQNLDCSRRNDPVYIGRVVSAMVNCNDEDHGVLLGRWDNHYEDGMSPMAWIGSVDILKRWRRLGCQPVKYGQCWVF 290 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 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 * .* * *.*** * * ** .*** ** ** ** *
gpLiv -EAQEETGVAMRIRVGQNMTMGSDFDIFAYITNGTAESHECQLLLCARIVSYNGVLGPVCSTNDLLNLTLDPFSENSIPLH-ILYEKYGDYLTESNLIKV 566 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 hKer NVSGHVKESGQVLAKQHTFRLRTPDLSLTLLGAAVVGQECEVQIVFKNPLPVTLTNVVFRLEGSGLQRPKI LNVGDIGGNETVTLRQSFVPVRPGPRQ 765 hPro FIIAEIVESKEIMASEVFTSFQYPEFSIELPNTGRIGQLLVCNCIFKNTLAIPLTDVKFSLESLGISSLQT SDHGTVQPGETIQSQIKCTPIKTGPKK 661 * *.* * * * *
gpLiv LVVNFECDKLKAVKGYRNVIIGPA 690
cEry LMVDFESDKLTGVKGYRNVIIAPLPK 698
lHem IVATFSSRQLIQVVGSKQVEVLD 764
nTG LIVSFSSPQLSGVKAHVTLNVKSA 737
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) Deduced amino-acid sequences for guinea pig liver TG (gpLiv) [5], chicken erythrocyte TG (cEry) [30],
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 His and Asp residues of the catalytic triad [35] Putative nuclear localization signals [11] are underlined.
Trang 6donkey anti-(rabbit IgG) Ig labeled with fluorescein
(Amersham Pharmacia Biotech) Specimens were observed
with a Nikon Eclipse E600 equipped with differential
interphase and epifluorescence optics
Generation of green fluorescent protein–fusion protein
constructs
The 500-bp BamHI–HindIII fragment, which contained the
Drosophilaheat shock protein promoter, was inserted into
the BglII–HindIII site of pEGFP-1 (Clontech) to 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 subcloning the 800-bp
Eco47III–PstI fragment of pEGFP-C1 (Clontech) into the
HincII–PstI sites of pBluescriptII KS(+) (Toyobo), and
then digested with KpnI and BamHI The 2214-bp BamHI–
NotI fragment, which contains the entire coding region of
nTG (nTG fragment), was generated via PCR with BamHI
and NotI primers The 2043-bp BamHI–NotI fragment,
which contains the coding region of nTG, but without the
N-terminal 57 amino-acids residue (nTGDN57 fragment),
was generated via PCR with BamHI-2 (5¢-AGGGATCCCT
CAAGGTGGTGTCAGTTGATC-3¢) and NotI 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 entire
coding region of rat muscle PK [27] (PK fragment), was
generated via PCR with PK-N (5¢-GCCGGATCCGGC
CTCGAGATGCCCAAGCCAGACAGC-3¢) and PK-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 cDNAsin vivo
The constructs were separately dissolved in 10 mMTris/HCl
(pH 8.5) to give a final concentration of 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] Three to four hours
later, the injected oocytes were examined for localization
of fluorescent proteins under a fluorescence microscope
equipped with differential interphase and epifluorescence
optics
R E S U L T S
Molecular cloning ofA pectinifera transglutaminase
Comparison of the amino-acid sequences among
already-known TGs shows highly conserved regions, including the
TG active site [28], in the middle portions of the
polypep-tides Based on the sequence of the conserved regions, a
single set of degenerate oligonucleotide primers, TG5 and TG3V, were designed and used for an RT-PCR experiment Using poly(A)+RNA from A pectinifera embryos at the early blastula 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 rapid 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
-3 2 1
112.0 81.0
-6
4 5
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) were prepared from 8-h-old early blastulae (lanes 1, 4, 7, and 10), 12-h-old blastulae (lanes 2, 5, 8, and 11), and 24-h-old mid-gastrulae (lanes 3, 6, 9, and 12) An aliquot 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 (lanes 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 by staining using anti-(nTG-N) Ig as a probe (lanes 4–6) Sizes of the molecular mass marker proteins in kDa are shown to the left.
Trang 7the sequence obtained by the RACE experiments The
cDNA contained a single open reading frame (ORF)
beginning with an ATG codon in an adequate context for
the initiation of translation (Fig 2A); the sequence
CCAAAATGG surrounding the ATG fits the consensus
sequence CC(G/A)CCATGG for 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 the deduced
amino-acid sequence with those of the 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 Ca2+
-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-dimethylcasein with Km and Vmax values of 0.35 mM and 13.3 nmolÆmin)1Æmg)1, respectively, indicating that the predicted protein is a transglutaminase
Subcellular localization ofA pectinifera transglutaminase
A major characteristic feature of the A pectinifera TG is the presence of two putative nuclear 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
C
a
b
A
Fig 4 Subcellular localization of nTG in embryos (A) The distribution of nTG in a midgastrula, as detected by indirect immunofluorescence 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 fluorescein-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 above Immunofluorescence micrographs (a,c,e,g), and the corresponding Nomarski differential interference-contrast micrographs (b,d,f,h) Bar, 5 lm.
Trang 8antibody, designated 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 no band was detected in the
cytosol fraction (lane 8, 9) When formalin-fixed
prepara-tions of single cells, which had been dissociated from
the midgastrulae, were stained with the same antibody, the
signal was limited to the nucleus (Fig 4B, 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 fertilized
eggs showed little or no signals, suggesting 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 the
bipinnaria stage (Fig 3A, lanes 10–11, Fig 3B, lanes 4–6)
Therefore, the nTG protein synthesis starts at mid-blastula
stage and continues thereafter
Immunostaining of the 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 4A)
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 extracted nTG from 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 to afford Sup2 (nuclear membrane fraction), and then with a combination of 1% Triton X-100 and 0.5M
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 removal of putative inhibitors 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 recognized by the anti-(nTG-M) Ig in Western blot analysis (Fig 6C, lane 3) To determine if the TG activity in Sup4 results from the 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 that 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
10.0
4.0 3.0 6.0
from fertilized eggs (lane 1), 8-h-old early blastulae (lane 2), 12-h-old mid-blastulae (lane 3), 15-h-old late blastulae (lane 4), and 24-h-old midgastrulae (lane 5) The filter was hybridized with a digoxygenin-labeled RNA probe obtained from the cDNA of nTG (upper panel) and of A pectinifera ubiquitin (lower panel) Each lane was loaded
were determined by comparison to the relative migration of RNA markers.
Trang 9GFP–nTG fusion protein produced in an oocyte by
microinjection of pHE-TG, which contains 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 embryogenesis Enzyme activity was
measured on the nuclear fractions prepared from 8-h-old early
blast-ulae, 12-h-old mid-blastblast-ulae, 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 nuclear fraction.
Sup1, Sup2, Sup3, and Sup4 (S1, S2, S3, and S4, respectively) 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 the percentage activity relative to the total activity
in the nuclear fraction (C) Western blot analysis of nTG recovered
in Sup4 An aliquot 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 probe (lanes 3) As a
negative control, parallel immunoblotting was performed using
anti-(nTG-M) Ig preincubated with the peptide antigen (lane 2) Sizes of the
molecular mass marker proteins in kDa are shown to the left.
100
50
0
Input
S IP S IP anti-nTG-N Control Ig
Fig 7 Immunoprecipitation of nTG recovered in Sup4 Ten microliters
of concentrated Sup4 were subjected to immunoprecipitation with 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
nTG GFP
hsp
nTG∆N57 hsp GFP
PK hsp GFP
N57
pHE-TG
pHE-TG∆N57 pHE-PK
pHE-N57PK
B
Fig 8 Subcellular localization of the green fluorescent protein-nTG fusion protein after expression in oocytes (A) Schematic drawings of constructs pHE-TG, pHE-TGDN, pHE-PK, and pHE-N57PK, which encode fusion proteins, GFP–nTG, GFP–N57-deleted nTG, GFP–
PK, and GFP–N57PK, respectively hsp, the Drosophila heat shock 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.
Trang 10almost exclusively in the cytoplasm (Fig 8B, b,f) The
possibility that the N-terminal 57 amino-acid residues (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 and the sequence 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 8B, c,g)
D I S C U S S I O N
During the early development of A pectinifera, the embryo
undergoes extremely 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 transcriptional 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 the
mid-blastula transition and that the level of the enzyme protein
increases gradually thereafter (Figs 3 and 4)
nTG is similar to the 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 been
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 by 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 pectinifera sperm, which involves an
e-(c-glutamyl)lysine cross-link between a Gln residue of
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 the present paper indicate that a significant amount of p28 is produced in
A pectiniferaembryos 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 nTG 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 and selective inhibitor of histone deacetylase [39], induces hyperacetyla-tion of histone H4 and causes developmental arrest at the early gastrula 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 an acetylated lysine residue 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 be obtained and produce similar developmental arrest as observed by trichostatin A-treated embryos which are devoid of p28 but whose histone H4 is in the normal acetylation-deacetylation cycle Such investigations are currently in progress in our laboratory
A C K N O W L E D G E M E N T S
We thank Drs S Hirose (Tokyo Institute of Technology), K Okano (Akita Prefectural University), and T Noguchi (Nagoya University) for the plasmids harboring bovine endotherial TGase, the Drosophila heat shock protein promoter, 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 the Science and Technology Agency of the Japanese Government.
R E F E R E N C E S
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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., Jetten, A.M & Rice, R.H (1990) Primary structure of keratinocyte transglutaminase Proc Natl Acad Sci USA 87, 9333–9337.