In vivo
cross-linking of nucleosomal histones catalyzed by nuclear
transglutaminase in starfish sperm and its induction by egg jelly
triggering the acrosome reaction
Kazuto Nunomura*, Satoru Kawakami†, Takahiko Shimizu†, Tomohiro Hara, Kazuhiro Nakamura,
Yudai Terakawa, Akiko Yamasaki and Susumu Ikegami‡
Department of Applied Biochemistry, Hiroshima University, Higashi-hiroshima, Hiroshima, Japan
A histone heterodimer, designated as p28, which contains
an N
e
(c-glutamyl)lysine cross-link between Gln9 of histone
H2B and Lys5 or Lys12 of histone H4, is present in starfish
(Asterina pectinifera) sperm. Treatment of sperm nuclei with
micrococcal nuclease produced soluble chromatin, which
was size-fractionated by sucrose-gradient centrifugation to
give p28-containing oligonucleosome and p28-free mono-
nucleosome fractions, indicating that the cross-link is inter-
nucleosomal. When sperm nuclei were incubated with
monodansylcadaverine, a fluorescent amine, in the presence
or absence of Ca
2+
, histone H2B was modified only in the
presence of Ca
2+
. Gln9, in the N-terminal region, was
modified, but the other Gln residues located in the internal
region were not, suggesting that the modification takes place
on the surface of the nucleosome core by the in situ action of
aCa
2+
-dependent nuclear transglutaminase. Treatment of

between Gln9 of histone H2B, and Lys5 or Lys12 of histone
H4 (Fig. 1) [5]. In addition to p28, histone dimers cross-
linked between Gln9 of histone H2B and a Lys residue of
histone H2A, H2B or H3, are also present [5]. These histone
dimers are referred to as histones d.
An isopeptide is formed by a transglutaminase (EC
2.3.2.13), which is largely known for its role in catalyzing
protein cross-linking reactions via the formation of an
N
e
(c-glutamyl)lysine bond 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 [6].
Well-documented examples of transglutaminases include
plasma factor XIIIa [7], keratinocyte transglutaminase [8],
epidermal transglutaminase [9], tissue transglutaminase [10],
and prostatic transglutaminase [11]. Tissue transglutami-
nase is localized mainly in the cytosol, but detectable tissue
transglutaminase expression has been reported in the rabbit
liver nucleus [12] and Huntington’s disease brain nucleus
[13]. However, transglutaminase activity in the nucleus, and
the mechanisms of its translocation, are not well under-
stood. We recently reported that a novel type of transglu-
taminase is present specifically in the nuclei of starfish
embryo and that it contains functional nuclear localization
signals in the N-terminal region [14].
In order to establish the involvement of nuclear trans-
glutaminase in the formation of p28, the present study was
Correspondence to S. Ikegami, Laboratory of Environmental Biology,
Nagahama Institute of Bioscience and Technology, Tamura-cho,

essential event in fertilization and requires a Ca
2+
influx. In
this study, we observed a significant increase in the content
of p28 in the sperm nucleus, possibly through the acrosome
reaction-induced activation of nuclear transglutaminase by
an increase in the intracellular concentration of Ca
2+
.To
our knowledge, this is the first demonstration of intracellular
transglutaminase-catalyzed protein cross-linking in vivo.
Experimental procedures
Collection of sperm
Specimens of A. pectinifera were collected, during their
breeding season, from the coastal waters off Japan and were
maintained in artificial sea water in laboratory aquaria at
15 °C. Sperm were obtained using a previously described
procedure [5].
Preparation of nuclei
Sperm were homogenized by means of a Dounce homo-
genizer (A-pestlle) in buffer A [0.25
M
sucrose, 50 m
M
Tris/
HCl (pH 7.5), 5 m
M
MgCl
2
,40m

Chromatin fractionation
Chromatin was released from sperm nuclei by digestion
with micrococcal nuclease [16]. Nuclear suspensions were
washed three times with digestion buffer [0.32
M
sucrose,
50 m
M
Tris/HCl (pH 7.5), 4 m
M
MgCl
2
,1m
M
CaCl
2
,
1m
M
phenylmethanesulfonyl fluoride], and digested at
37 °C for 5 min in digestion buffer containing 80 U of
micrococcal nucleaseÆmg
)1
of DNA [estimated by the
absorbance at 260 nm (A
260
)]. The reaction was stopped
by the addition of EDTA to a final concentration of 5 m
M
and then cooled on ice. The S1 fraction was obtained as the

DNA was extracted by using the conventional phenol/
chloroform extraction method. To the aqueous solution was
added a one-tenth volume of 3
M
sodium acetate (pH 5.2)
and 3 lL of ethachinmate (Nippon Gene, Tokyo); ethanol
was then added to precipitate the DNA. The sizes of the
DNA fragments obtained from the fractions were analyzed,
by electrophoresis, on a 2% agarose gel, followed by staining
with ethidium bromide. The proteins precipitated from each
2.2-mL fraction, by adding 2.8 mL of 10% (w/w) trichloro-
acetic acid and 20 lg of BSA, were separated by SDS/
PAGE, which was carried out on SDS/15% polyacrylamide
gels, as described previously [18], followed by staining with
Coomassie Brilliant Blue (CBB) or immunostaining.
DCA-labeling
Sperm nuclei were washed three times in the labeling buffer
[10 m
M
Tris/HCl (pH 7.5), 5 m
M
CaCl
2
,and5m
M
dithio-
threitol] by centrifugation at 2000 g for 6 min at 4 °C,
followed by resuspension in labeling buffer containing
0.5 m
M

and collected by
adding ethanol, as described previously [5]. The histone
fraction was separated by RP-HPLC using a Zorbax
300SB-CN (Hewlett Packard) column (25 · 0.46 cm). Elu-
tion was carried out using a 0–55% acetonitrile gradient
(0%,5min;25%,10min;32%,20min;40%,55min;and
55%, 65 min) in 0.1% trifluoroacetic acid at a flow rate of
1.4 mLÆmin
)1
.TheA
229
of each fraction was monitored and
relevant fractions were collected. The purity of each histone
in the fractions was confirmed by SDS/PAGE. The protein
concentration was determined by the method of Lowry
et al. [20] using BSA as the standard. In the case of histones
prepared from DCA-labeled nuclei, fluorescence was
monitored (excitation wavelength, 330 nm; and emission
wavelength, 510 nm) by using an F-1050 fluorescence
spectrophotometer (Hitachi), and fractions that contained
DCA-labeled histones were recovered. Specimens of p28
were purified as described previously [5].
Proteolytic digestion of histones
DCA-labeled or -unlabeled histone H2B was digested at
37 °C for 16 h using Achromobactor lyticus protease I
(Wako Pure Chemical Inc., Osaka, Japan), at an enzyme/
substrate molar ratio of 1 : 100, in 20 m
M
Tris/HCl
(pH 9.0). Proteolytic digests were loaded onto an RP-

sugar equivalents, determined by the phenol/sulfuric acid
method [22] using
L
-fucose as a standard. A sperm
suspension was incubated with the egg jelly fraction at
20 °C for 60 min. After centrifugation of the suspension at
5000 g for 20 min, the pelleted sperm were suspended in
SDS-sample buffer and the lysate was subjected to SDS/
PAGE on a 15% gel, followed by staining with CBB or
immunoblotting.
Results
Presence of p28 in chromatin
The purpose of this study was to develop a better
understanding of the molecular mechanisms of p28 forma-
tion in mature starfish sperm. In order to detect p28 in a
Western blot analysis of sperm chromatin, we prepared a
mAb that reacts with p28, but not with monomeric core
histones, by immunizing mice with p28 as antigen. One such
antibody, designated 5C7, was obtained. This mAb was of
the IgG2bj isotype. The total histone fraction prepared
from starfish sperm was separated by SDS/PAGE to
produce several bands of histone d subspecies [5], which
migrated more slowly than those of histones H2B, H2A, H3
and H4 that have ordinary molecular masses (Fig. 2). An
immunoblot analysis, using 5C7, revealed that p28 and
histones d with a molecular mass of 32 kDa were reactive to
5C7, whereas histones H2B, H2A, H3 and H4 were not
(Fig. 2). Preliminary studies revealed that this histone d
subspecies, designated p32, was a mixture of histone dimers
cross-linked between Gln9 of histone H2B and a Lys residue

Incorporation of DCA into histones
We next addressed the issue of whether transglutaminase is
involved in the formation of an N
e
(c-glutamyl)lysine bridge
of p28. Sperm nuclei prepared from testes were incubated
with DCA (as an amine donor) in buffer containing 5 m
M
CaCl
2
, and the reaction products were resolved by SDS/
PAGE. Three fluorescent bands, the positions of which
were identical to those of histones H1, H2B and H3, were
detected (Fig. 5A). Furthermore, RP-HPLC of the DCA-
labeled histone fraction produced three fluorescent peaks
(Fig. 5B), corresponding to modified histones H1, H2B,
and H3. When 20 m
M
EDTA (a divalent ion chelator) was
included in the reaction mixture which deprives transglu-
taminase of necessary Ca
2+
[14], the fluorescent bands were
not observed (Fig. 5A). When the reaction was carried out
in the presence of the methyl ester of alutacenoic acid B, a
specific and potent inhibitor of transglutaminase [19], at a
concentration of 80 ngÆmL
)1
, the bands were not detected
(Fig. 5A). These results suggest that histones H1, H2B and

resolved from the neighboring peptide peaks (Fig. 6). This
fluorescent, UV-absorbing peak was not present in RP-
HPLC of fragments produced by the digestion of unlabeled
histone H2B with A. lyticus protease I (data not shown).
The fluorescent substance, designated as substance X, was
recovered and analyzed by MALDI-TOF-MS, and was
found to have a relative molecular mass (M
r
) of 650.32. This
M
r
value is 17 units less than the sum of the M
r
of DCA
(335.50) and that of a tripeptide, Gly–Gln–Lys (331.37), the
sequence of which corresponds to Gly8–Gln9–Lys10 of
histone H2B. The difference of 17 M
r
units is consistent with
the prediction that DCA and the tripeptide are cross-linked
by transamidation with the loss of NH
3
.Digestionof
unlabeled histone H2B with A. lyticus protease I produced
Gly8–Gln9–Lys10 and the two other Gln-containing pep-
tides (K11 and K18) with higher M
r
values (Fig. 1) [5].
Therefore, substance X was determined to be Gly–Gln–Lys,
the carboxamide group of which is transamidated with

shown in Fig. 7, histone H2B in the mononucleosome
fractions was extensively modified. These results strongly
suggest that the modification of nucleosomal histone H2B is
catalyzed by an endogenous nuclear transglutaminase.
Occurrence of transglutaminase in sperm chromatin
We next examined whether nuclear transglutaminase is
present in the chromatin fraction by carrying out an
immunoblot analysis using anti-(nuclear transglutaminase)
Ig [14]. These experiments verified that nuclear transgluta-
minase is a constituent of native soluble chromatin (Fig. 8).
The immunoreactive band of a sperm nuclear specimen
migrated at the same position as that obtained from
embryonic nuclei (date not shown). Size fractionation of the
S2 fraction by sucrose density-gradient centrifugation
showed that the nuclear transglutaminase sediments at
positions corresponding to mono-, di-, and oligonucleo-
some fractions (Fig. 8). These results suggest that nuclear
transglutaminase exists in the vicinity of the nucleosome and
is responsible for the nucleosomal histone modification.
Elevation of p28 content in sperm by Ca
2+
influx
When sperm move towards the egg surface, they come into
contact with the jelly layer that surrounds an egg and which
has the ability to induce the acrosome reaction in sperm [15].
Because Ca
2+
flux takes place in the egg jelly-induced
acrosome reaction, we postulated that high levels of
intracellular Ca

DCA, in the presence or absence of 20 m
M
EDTA or 80 ngÆmL
)1
methyl
alutacenoate B (MAB). Reactions were terminated and the reaction mixtures centrifuged to isolate the nuclear pellets from which histones were
acid-extracted and then precipitated with ethanol. (A) Histones separated on an SDS/15% polyacrylamide gel. The gel was stained with CBB (left)
after being illuminated with light (365 nm wavelength; right). Lanes 1 and 4, DCA only; lanes 2 and 5, DCA and EDTA; lanes 3 and 6, DCA and
MAB. Sizes of molecular-mass-marker proteins are shown at the left. (B) RP-HPLC profile of DCA-labeled histones using a Zorbax 300SB-CN
column (4.6 · 250 mm) and an acetonitrile gradient (0–55%) in 0.1% trifluoroacetic acid. The concentration of acetonitrile is shown by a dotted
line. The relative absorbance at 229 nm (upper panel) and fluorescence (excitation, 330 nm, emission, 510 nm; lower panel) are shown by bold lines.
Ó FEBS 2003 In vivo cross-linking of histones (Eur. J. Biochem. 270) 3755
1.25 l
M
of the calcium ionophore A 23187, which induces
the acrosome reaction, also resulted in the formation of p28
and p32 (Fig. 9B). The addition of methyl alutacenoate B
(final concentration 320 ngÆmL
)1
), to the sperm suspension,
suppressed the A 23187-induced formation of p28 and p32
(Fig. 9B). These results strongly suggest that nuclear
transglutaminase-induced histone cross-linking is activated
by an increase in intracellular Ca
2+
concentrations.
Discussion
Chromatin condensation in sperm is usually associated
with changes in basic nuclear proteins. The most radical
change involves the complete replacement of histones by

shown at the left.
Fig. 6. Separation of the monodansylcadaverine (DCA)-labeled frag-
ments obtained by Achromobacter lyticus protease I digestion of DCA-
labeled histone H2B. DCA-labeled histone H2B, prepared by the
method described in the legend to Fig. 5, was digested with A. lyticus
protease I, and the peptides were resolved using an Inertsil ODS col-
umn (4.6 · 250 mm) and an acetonitrile gradient (10–70%) in 0.1%
trifluoroacetic acid. The relative absorbance at 214 nm (upper panel)
and fluorescence (excitation, 330 nm, emission, 510 nm; lower panel)
are shown by bold lines. The concentration of acetonitrile is shown by
a dotted line. Arrows indicate the position of substance X.
Fig. 7. Sucrose density-gradient analysis of nucleosome containing
monodansylcadaverine (DCA)-labeled histone H2B. A suspension of
sperm nuclei (8.0 · 10
8
) was incubated in the DCA-containing reac-
tion mixture by the method described in the legend to Fig. 5. Reactions
were terminated, nuclei were collected by brief centrifugation, and then
treated with micrococcal nuclease to afford soluble chromatin (S2)
from which nucleosomal core particles were purified on 5–20% sucrose
gradients, as described for Fig. 4A. Gradients were fractionated to
nine fractions from the bottom. After fractionation, DNA fragments
were prepared by phenol/chloroform extraction and analyzed by
agarose-gel electrophoresis followed by staining with ethidium bro-
mide, as described for Fig. 4B. Only fraction 7 contained mono-
nucleosomes (data not shown). The histones were acid-extracted from
each fraction and resolved by HPLC using a Zorbax 300SB-CN col-
umn (4.6 · 250 mm). For each chromatographic run, the relative
height of the fluorescent peak corresponding to labeled histone H2B in
the chromatogram (box), and the amount of histone H2B (closed

+
influx. Sperm (1.0 · 10
7
) were incubated at 20 °Cfor60minin1mLofartificialseawater,
with or without egg jelly (25 lgof
L
-fucose equivalentÆmL
)1
). In a separate run, sperm were incubated in artificial sea water containing 1.25 l
M
calcium ionophore A 23187 in place of egg jelly, with or without methyl alutacenoate B (MAB) (320 ngÆmL
)1
), or in their absence. Sperm were
collected by centrifugation at 5000 g for 20 min and dissolved in SDS-sample buffer. (A) Detection of p28 and p32 in egg-jelly treated and
-untreated sperm. Lysates were subjected to SDS/15% PAGE, followed by staining of the gel with Coomassie Brilliant Blue (CBB) (upper panel) or
immunoblot analysis of p28 and p32 (lower panel). Lane 1, sperm incubated without egg jelly; lane 2, sperm incubated with egg jelly. Actin bands
were used as an internal control. Sizes of molecular-mass-marker proteins are shown at the left. (B) Sperm treated with calcium ionophore A 23187.
Proteins separated on an SDS/15% polyacrylamide gel were Western blotted and immunostained using 5C7 (upper panel). Lane 1, sperm incubated
with calcium ionophore A 23187; lane 2, sperm incubated with calcium ionophore A 23187 and MAB; lane 3, sperm incubated without calcium
ionophore A 23187. The lower panel shows CBB-stained actin on the gel.
Ó FEBS 2003 In vivo cross-linking of histones (Eur. J. Biochem. 270) 3757
were incubated with DCA and exogenously supplied
guinea-pig liver transglutaminase, only Gln22 in the
N-terminal region of histone H2B was labeled, although
two other Gln residues – Gln47 and Gln95 – are present in
chicken erythrocyte histone H2B. On the other hand, when
free histone H2B was incubated in a DCA-containing
reaction mixture with guinea-pig liver transglutaminase,
Gln95 was labeled, whereas Gln22 and Gln47 were not.
Their observations strongly support our view that Gln9 of

showed a 33–41% overall similarity with other transglu-
taminases [11,30]. The residues comprising the catalytic
triad are conserved in the nuclear transglutaminase
(Cys323, His382, Asp405). Three acidic residues –
Glu447, Glu496, and Glu501 – which could act as a
Ca
2+
-binding site [31], were also conserved. In agreement
with their sequence features, Ca
2+
is essential for nuclear
transglutaminase activity in sperm (Fig. 5A,B) [14]. A
special sequence feature of this nuclear transglutaminase,
which is not found in other transglutaminases identified
thus far, is the presence of an extension of 57 amino acid
residues in the N-terminal region, which contained nuclear
localization signal-like sequences [32,33]. An antibody is
produced by immunizing the peptide which contains a
region of the N-terminal sequence of the nuclear transgl-
utaminase (residues 3–20) [14]. Immunoblot analysis of
starfish sperm using this antibody, which specifically
recognizes nuclear transglutaminase, showed that only
one polypeptide was recognized and that it was localized
in the sperm nuclear fraction (Fig. 8). This study showed
that nuclear transglutaminase with the same molecular
mass as embryonic nuclear transglutaminase (date not
shown) is a constituent of chromatin and suggests that this
enzyme is involved in the formation of p28 and p32 in
sperm nuclei.
The acrosome reaction in sperm is necessary for gamete

Ogita (Sankyo Co., Japan) for the supply of alutacenoic acid B methyl
ester. This work was supported, in part, by grants-in-aid for scientific
research from the Ministry of Education, Science, Sports and Culture,
Japan.
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