Kazuto Nunomura*, Satoru Kawakami†, Takahiko Shimizu†, Tomohiro Hara, Kazuhiro Nakamura,Yudai Terakawa, Akiko Yamasaki and Susumu Ikegami‡ Department of Applied Biochemistry, Hiroshima U
Trang 1Kazuto 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 Ne(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 Ca2+, histone H2B was modified only in the
presence of Ca2+ 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
a Ca2+-dependent nuclear transglutaminase Treatment of sperm with the egg jelly, which activates Ca2+influx to induce the acrosome reaction, resulted in a significant elevation of the p28 content in the nucleus This is the first demonstration of an in vivo activation of transglutaminase leading to the formation of a cross-link in intracellular proteins
Keywords: starfish; sperm; transglutaminase; chromatin; acrosome reaction
The nucleosome, which contains four core histones (H2A,
H2B, H3, and H4), is the primary unit of chromatin The
higher-order chromatin structure is stabilized by
inter-nucleosomal interactions involving the N-terminal region of
core histones and by linker histone H1 Histones undergo a
variety of post-translational modifications, including
acety-lation, methyacety-lation, phosphoryacety-lation, ubiqutination, and
ADP-ribosylation, which lead to alterations in the charge
and conformation of the molecules [1] The majority of these
modifications occur at the N-terminal region of the core
histones and possibly these reactions modulate the affinity
of the histones for DNA [2,3]
We previously reported on a new post-translational modification of the N-terminal region of core histones, namely transamidation, which led to the formation of an
Ne(c-glutamyl)lysine bridge between histones H2B and H4
in starfish (Asterina pectinifera) sperm [4] The structure of the cross-linked histone dimer, p28, includes a cross-link 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
Ne(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,
Nagahama, Shiga 562-0829, Japan.
Fax: + 81 749 64 8140, Tel.: + 81 749 64 8103,
E-mail: s_ikegami@nagahama-i-bio.ac.jp
Abbreviations: CBB, Coomassie Brilliant Blue; DCA,
monodansyl-cadaverine.
Enzymes: transglutaminase (EC 2.3.2.13).
Present addresses: *Division of Proteomics Research (ABJ &
Millipore), Institute of Medical Science, University of Tokyo,
Minato-ku, Tokyo 108-8639, Japan;
Department of Molecular Gerontology, Tokyo Metropolitan
Insti-tute of Gerontology, Itabashi-ku, Tokyo 173-0015, Japan; and
Laboratory of Environmental Biology, Nagahama Institute of
Bioscience and Technology, Tamura-cho, Nagahama,
Shiga 562-0829, Japan.
(Received 14 April 2003, revised 25 June 2003,
accepted 21 July 2003)
Trang 2undertaken to investigate whether histone H2B is modified
by the incubation of sperm nuclei with
monodansylcada-verine (DCA) as an amine donor As expected,
DCA-conjugated histone H2B was produced in the presence of
Ca2+, but not in its absence Gln9, at the N-terminal region,
was specifically modified, but other Gln residues in the
internal region were not These results suggest that p28 is
produced by a nuclear transglutaminase which is able to
utilize nucleosomal histone H2B as an amine acceptor
In the starfish, the jelly coat of the eggs cause sperm to
undergo the acrosome reaction [15] The reaction is an
essential event in fertilization and requires a Ca2+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 Ca2+ 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
15C 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 [0.25Msucrose, 50 mMTris/
HCl (pH 7.5), 5 mMMgCl2, 40 mM NaCl, 1 mM
phenyl-methanesulfonyl fluoride, and 1% (v/v) Triton-X-100]
Nuclei were precipitated from the homogenate by
centri-fugation at 2000 g for 6 min, followed by washing three
times with buffer A without Triton-X-100 Nuclei were then
resuspended in 25% (v/v) glycerol, 65 mM KCl, 15 mM
NaCl, 15 mM Tris/HCl (pH 7.0), 0.5 mM spermine,
0.15 mM spermidine, 0.2 mM EDTA, 0.2 mM EGTA,
10 mM 2-mercaptoethanol and 0.1 mM
phenylmethane-sulfonyl fluoride, and stored at 4C
Chromatin fractionation
Chromatin was released from sperm nuclei by digestion
with micrococcal nuclease [16] Nuclear suspensions were
washed three times with digestion buffer [0.32Msucrose,
50 mM Tris/HCl (pH 7.5), 4 mM MgCl2, 1 mM CaCl2,
1 mM phenylmethanesulfonyl fluoride], and digested at
37C for 5 min in digestion buffer containing 80 U of micrococcal nucleaseÆmg)1 of DNA [estimated by the absorbance at 260 nm (A260)] The reaction was stopped
by the addition of EDTA to a final concentration of 5 mM
and then cooled on ice The S1 fraction was obtained as the supernatant by centrifugation of the reaction mixture at
11 600 g for 10 min at 4C The pellet was resuspended in lysis buffer containing 1 mM Tris/HCl (pH 7.4), 0.2 mM
EDTA and 0.2 mM phenylmethanesulfonyl fluoride, and the mixture was dialyzed against the same buffer for 12 h at
4C Centrifugation of the suspension at 600 g for 10 min
at 4C yielded the supernatant, designated the S2 fraction, and the pellet, designated the P fraction [17]
Sucrose density-gradient centrifugation The S2 fraction was layered onto 40 mL of a 5–20% (w/v) linear sucrose-density gradient containing 1 mM Tris/HCl (pH7.4), 20 mM EDTA, 0.4M NaCl, and a mixture of protease inhibitors at appropriate concentrations, followed
by centrifugation at 140 000 g for 13 h at 4C [17] Gradients were fractionated into 9 or 18 fractions from the bottom and their A260value was measured A 220-ll aliquot
of each 2.2-mL fraction was digested with proteinase K and DNA was extracted by using the conventional phenol/ chloroform extraction method To the aqueous solution was added a one-tenth volume of 3Msodium acetate (pH5.2) 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 mMTris/HCl (pH 7.5), 5 mMCaCl2, and 5 mM dithio-threitol] by centrifugation at 2000 g for 6 min at 4C, followed by resuspension in labeling buffer containing 0.5 mM DCA in the presence or absence of different concentrations of alutacenoic acid B methyl ester [19] (kindly provided by Dr K Ogita, Sankyo Co., Tokyo, Japan) Incubations were carried out at 20C for 8 h and the reactions were quenched by the addition of EDTA to give a final concentration of 20 mM The reaction mixture was centrifuged at 2000 g for 6 min at 4C to sediment Fig 1 Histone dimers in starfish sperm Primary structure of p28 Open bars below the sequence (single-letter code) of histones H2B and H4 indicate fragments produced by treatment with Achromobacter lyticus protease I.
Trang 3times in 0.14MNaCl and 50 mMNaHCO3by
centrifuga-tion at 1000 g for 5 min at 4C The histones were
extracted with cold (4C) 0.2M H2SO4 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%, 5 min; 25%, 10 min; 32%, 20 min; 40%, 55 min; and
55%, 65 min) in 0.1% trifluoroacetic acid at a flow rate of
1.4 mLÆmin)1 The A229of 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
37C 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 mM Tris/HCl
(pH9.0) Proteolytic digests were loaded onto an
RP-HPLC column (25· 0.46 cm; Inertsil ODS; GL Science,
Tokyo, Japan) Fragments were eluted at a flow rate of
1.0 mLÆmin)1with a linear gradient of 5–60% acetonitrile
(1%Æmin)1) in 0.1% trifluoroacetic acid, after a 10-min hold
The A214and fluorescence (excitation wavelength, 330 nm;
and emission wavelength, 510 nm) were monitored The
molecular mass of the separated peptides was determined by
MALDI-TOF MS using a Voyager-DE PRO mass
spectrometer (Applied Biosystems)
Production of antibodies
Spleen cells were collected from BALB/c mice, which had
been immunized with p28, and these spleen cells were fused
with SP2/0-Ag14 cells Supernatants from the hybridomas
grown in selective media were first screened in an ELISA for
immunoglobulins that reacted with p28 Then, positive
supernatants were subjected to immunoblot analysis on
histones separated by SDS/PAGE and transferred from the
gel to a poly(vinylidene difluoride) membrane One
hybri-doma, designated as 5C7, was subcloned until clonal A
polyclonal antibody specific for nuclear transglutaminase
was prepared as described previously [14]
Preparation of egg jelly and the acrosome reaction
Soluble egg jelly was prepared as described previously [21]
The quantity of egg jelly fraction was expressed as neutral
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
of histone H2A, H2B or H3 (T Shimizu, K Hozumi,
K Nunomura & S Ikegami, unpublished results)
Fig 2 Total histones (8 lg of BSA-equivalent per lane) separated on an SDS/15% polyacrylamide gel and stained with Coomassie Brilliant Blue (lane 2) Histone dimers were detected by Western blotting and immunostaining with mAb 5C7 (lane 1) Sizes of molecular-mass-marker proteins are shown at the left.
Trang 4Native soluble chromatin (S2 fraction) was prepared by
the digestion of chromatin in situ in nuclei, by using
micrococcal nuclease The DNA fragments, when resolved
on an agarose gel, appeared as typical oligonucleosome
ladders in the S2 fraction (Fig 3A) The S2 fraction was
subjected to SDS/PAGE and immunoblotted using 5C7 as a
probe Nearly all of the p28 in sperm nuclei was recovered in
the S2 fraction (Fig 3B,C) This fraction was then
size-fractionated by sucrose density-gradient centrifugation to
obtain 18 fractions (Fig 4A) Although histone H1 can be
dissociated easily from chromatin of almost all eukaryotes
examined, by incubation in sucrose density-gradient buffer
containing 0.4MNaCl, starfish sperm histone H1 could not
be removed from nucleosomes under these conditions
(Fig 4C) Almost all the mononucleosomes were recovered
in fractions 14 and 15, which were devoid of p28 (Fig 4B,D)
However, p28 was recovered in the di-, oligo- and
poly-nucleosome fractions (Fig 4B–D) This suggests that histone
cross-linking occurs between histone H2B of a nucleosome
core and histone H4 of an adjacent nucleosome core
Incorporation of DCA into histones
We next addressed the issue of whether transglutaminase is involved in the formation of an Ne(c-glutamyl)lysine bridge
of p28 Sperm nuclei prepared from testes were incubated with DCA (as an amine donor) in buffer containing 5 mM
CaCl2, 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 mMEDTA (a divalent ion chelator) was included in the reaction mixture which deprives transglu-taminase of necessary Ca2+[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
Fig 3 Histone dimers in native soluble chromatin Chromatin was released from sperm nuclei (SN) by digestion with micrococcal nuclease After centrifugation at 11 600 g for 10 min at 4 C, the supernatant (S1) was separated from the pellet, which was resuspended in lysis buffer and dialyzed against the same buffer Centrifugation of the suspension at 600 g for 10 min at 4 C yielded the supernatant (S2) and the pellet (P) (A) Size distribution of DNA purified from undigested nuclei (SN) and that from S1, S2, and P fractions DNA preparations were resolved on a 2% agarose gel then stained with ethidium bromide Lane M, a 123-bp DNA ladder marker Sizes of the marker DNAs are shown at the left (B) Proteins of SN, S1, S2, and P fractions were resolved on an SDS/15% polyacrylamide gel and stained with CBB Sizes of molecular-mass-marker proteins are shown at the left (C) Proteins of SN, S1, S2, and P fractions were resolved on an SDS/15% polyacrylamide gel, Western blotted and immuno-stained with mAb 5C7.
Trang 5H3 in sperm nuclei serve as glutaminyl substrates for
endogenous nuclear transglutaminase
Ballestar et al [23] reported that chicken erythrocyte
nucleosome cores were labeled with DCA by incubation
with guinea-pig liver transglutaminase as an exogenous
enzyme, and that dansyl fluorescence was observed only
in histones H2B and H3 Consistent with these results,
nucleosomal histones H2B and H3, but not histones H2A
and H4, are substrates of an endogenous nuclear
trans-glutaminase in starfish sperm nuclei In this study, we also
showed that linker histone of starfish sperm was a substrate
of the nuclear transglutaminase
Determination of the DCA-binding sites of histone H2B
Because the carboxamide group of Gln9 of histone H2B is
selectively transamidated with the e-amino group of Lys5 or
Lys12 of histone H4 in p28 (Fig 1), we studied whether
DCA modification takes place only at Gln9 of histone H2B
DCA-modified H2B was digested with A lyticus protease I,
and the digests were then separated by RP-HPLC (Fig 6)
A single fluorescent peak was obtained, and the
corres-ponding peak in the UV-absorbance profile was well
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 (Mr) of 650.32 This
Mrvalue is 17 units less than the sum of the Mrof 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 Mrunits is consistent with the prediction that DCA and the tripeptide are cross-linked
by transamidation with the loss of NH3 Digestion of 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 Mr values (Fig 1) [5] Therefore, substance X was determined to be Gly–Gln–Lys, the carboxamide group of which is transamidated with DCA This result clearly demonstrates that Gln9 is the sole glutaminyl substrate for endogenous nuclear transglutami-nase in histone H2B
To investigate the issue of whether histone H2B in the nucleosome was modified by endogenous nuclear trans-glutaminase, DCA-labeled nuclei were digested with micro-coccal nuclease to yield the S2 fraction, which was separated
Fig 4 Sucrose density-gradient analysis of chromatin solubilized by treatment with micrococcal nuclease The S2 fraction was prepared from the digest by using the method described in the legend to Fig 3 S2 was then subjected to size fractionation on sucrose gradients (A) Sedimentation pattern S2 was layered onto 40 mL of a 5–20% (w/v) linear sucrose-density gradient containing 1 m M Tris/HCl (pH 7.4), 20 m M EDTA, 0.4 M
NaCl, and a mixture of protease inhibitors at appropriate concentrations, followed by centrifugation at 140 000 g for 13 h at 4 C Gradients were fractionated to 18 fractions from the bottom The absorbance at 260 nm (A 260 ) is shown as a bold line and the sucrose density as a dotted line (B) The sizes of DNA fragments obtained from the fractions were analyzed by electrophoresis on a 2% agarose gel, followed by staining with ethidium bromide The number below each lane represents the fraction number Lane M, a 123-bp DNA ladder marker Sizes of the marker DNAs are shown at the left (C) Fractions of the sucrose density gradient and unfractionated S2 resolved on an SDS/15% polyacrylamide gel and stained with CBB Sizes of molecular-mass-marker proteins are shown at the left (D) Proteins separated on a gel, as described in (C), were Western blotted and immunostained with mAb 5C7.
Trang 6by sucrose density-gradient centrifugation Each fraction
was subjected to RP-HPLC in an attempt to purify the
DCA-labeled histone H2B The relative height of the
fluorescent peaks corresponding to DCA-labeled histone
H2B in the chromatogram (Fig 5B), was calculated As
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 Ca2+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 Ca2+ flux takes place in the egg jelly-induced acrosome reaction, we postulated that high levels of intracellular Ca2+might activate the nuclear transglutami-nase and induce histone cross-linking As expected, treat-ment of a sperm suspension (1.0· 107spermÆmL)1) with egg jelly (25 lg of L-fucose-equivalentÆmL)1) induced a significant elevation in p28 and p32 levels, and a concomi-tant reduction in the level of monomeric histones H2B and H3 (Fig 9A) Notably, the intensity of the histone H1 band was significantly reduced According to Amano et al [21], treatment of sperm with the egg jelly induces proteolytic cleavage of sperm histone H1 Treatment of sperm with
Fig 5 In situ monodansylcadaverine (DCA) labeling of histones in sperm nuclei A sperm nuclear suspension was incubated for 8 h at 20 C in
10 m M Tris/HCl (pH 7.5), 5 m M CaCl 2 , 5 m M dithiothreitol, and 0.5 m M DCA, in the presence or absence of 20 m M EDTA or 80 ngÆmL)1methyl 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.
Trang 71.25 lMof 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 Ca2+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
protamines, which are present in mammals, birds, and
various species of fish During this replacement, the
nucleosomal structure [24], which is common to somatic
cells, is abolished [25] A less radical transition in the
chromatin structure occurs during spermatogenesis in sea
urchins In mature sea urchin sperm, no protamines are
present and the nucleosomal structure is retained [26]
Chromatin of sea urchin spermatozoa contains two
sperm-specific histones, SpH1 and SpH2B, which differ most
strikingly from their embryonic counterparts by the fact
that they contain extended N-terminal regions [26] The
phosphorylation/dephosphorylation events of sea urchin
sperm-specific histones might involve a modulation in
charge shielding to achieve the high packaging density that
is characteristic of sperm chromatin [26] In mature sperm
of starfish, no protamines are present and linker and core histones are retained [21,27] Sperm chromatin is essentially
Fig 8 Occurrence of nuclear transglutaminase in sperm chromatin Immunoblot analysis of nuclear transglutaminase in the sucrose den-sity-gradient fractions Chromatin was released from sperm nuclei (1.0 · 10 8 ) by digestion with micrococcal nuclease The S2 fraction was prepared from the digest by the method described for Fig 3 The S2 fraction was then subjected to size fractionation, on 5–20% sucrose gradients, to 18 fractions, as described for Fig 4A Aliquots of the fractions were dissolved in SDS sample buffer and proteins were sep-arated on an SDS/7.5% polyacrylamide gel, followed by immuno-staining using anti-(nuclear transglutaminase) Ig SN denotes undigested sperm nuclei Sizes of molecular-mass-marker proteins are 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-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 Reacreac-tions 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 circles), were determined.
Trang 8organized into typical core nucleosome [28] In this and
preceding papers [5], we showed that p28 is present in
starfish sperm chromatin and that the structure of p28
involves an Ne(c-glutamyl)lysine cross-link between Gln9
of histone H2B and a Lys residue of histone H4 The fact
that histones H2B and H4 are cross-linked
internucleo-somally (Fig 4) implies that in the chromatin of starfish
sperm, Gln9 of histone H2B and Lys5 or Lys12 of histone
H4 must be in close proximity This may have important
implications for the chromatin organization in the sperm of
starfish Such cross-linking might be involved in chromatin
compaction
Starfish sperm histone H2B contains Gln43 and Gln91
in addition to Gln9 [5] Our finding, that Gln9 in the N-terminal region is the only DCA-labeled residue, suggests that Gln residues in the internal region are not accessible to nuclear transglutaminase Furthermore, when DCA-labeled nuclei were treated with micrococcal nuclease followed by separation of the digested chromatin by sucrose density-gradient centrifugation, DCA-labeled histones were detec-ted in the mononucleosome fraction (Fig 7) This therefore suggests that histone H2B in the nucleosome structure is a substrate of nuclear transglutaminase Ballestar et al [23] reported that when chicken erythrocyte nucleosome cores
Fig 9 Elevation of the p28 content in sperm by Ca2++influx Sperm (1.0 · 10 7 ) were incubated at 20 C for 60 min in 1 mL of artificial sea water, with or without egg jelly (25 lg of 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.
Trang 9reaction mixture with guinea-pig liver transglutaminase,
Gln95 was labeled, whereas Gln22 and Gln47 were not
Their observations strongly support our view that Gln9 of
starfish histone H2B, constituting the nucleosomal
struc-ture, is modified by transglutaminase in situ
The fact that histone H3 was also labeled with DCA
(Fig 5A,B) is in agreement with our finding that a histone d
molecule contains an Ne(c-glutamyl)lysine cross-link
between histones H3 and H4 (T Shimizu, K Hozumi,
K Nunomura & S Ikegami, unpublished results) Our
preliminary experiments demonstrate that Gln5 of histone
H3 was labeled with DCA in sperm nuclei (data not shown)
Moreover, Ballestar et al [23] showed that histone H3 was
also labeled with DCA when chicken erythrocyte
nucleo-some cores were used as substrates for exogenously supplied
guinea-pig liver transglutaminase In this case, Gln5 or
Gln19 of histone H3 was DCA-labeled They also showed
that histones H2A and H4 were not modified in the
nucleosome, whereas free histones H2A and H4 were In
agreement with their results, histones H2A and H4 were not
labeled with DCA in situ, in starfish sperm nuclei (Fig 5)
Free histone H1 has been shown to be a good amine
acceptor substrate for guinea-pig liver transglutaminase and
bacterial transglutaminase [29] Our results demonstrate
that histone H1, in the chromatin of starfish sperm, is a
substrate of endogenous nuclear transglutaminase (Fig 5)
The cDNA which encodes nuclear transglutaminase was
cloned from starfish embryo and the cDNA-deduced
sequence defines a single open-reading frame encoding a
protein with a predicted molecular mass of 83 kDa [14]
The amino acid sequence of nuclear transglutaminase
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
Ca2+-binding site [31], were also conserved In agreement
with their sequence features, Ca2+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
completed the acrosome reaction and from those prepared from acrosome-unreacted sperm, and found that the acrosome reaction is accompanied by a reduction in the amount of histone monomers and appearance of new bands located between histone H1 and the core histones, which can be attributed to histones d The present study shows a significant increase in the content of p28 and p32
in the nucleus as the result of treatment of sperm with egg jelly or calcium ionophore A 23187 (Fig 9B) These data strongly suggest that the elevation in intracellular Ca2+ concentrations induced by egg jelly activates nuclear transglutaminase, thus leading to histone cross-linking This may have important implications for the role of histone cross-linking Such histone cross-linking may induce chromatin compaction, which allows passage of the sperm head through the narrow acrosomal tube to the egg cytoplasm Studies are currently underway to deter-mine the fate of histones d in the sperm nucleus in the egg cytoplasm
Acknowledgements
We are thankful to Dr H Fukuda (Institute of Medical Science, University of Tokyo) for MALDI-TOF MS measurements, and Dr K 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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