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 sperm with the egg jelly, which activates Ca 2+ 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, methylation, phosphorylation, 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 N e (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 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, 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 àLaboratory 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) Eur. J. Biochem. 270, 3750–3759 (2003) Ó FEBS 2003 doi:10.1046/j.1432-1033.2003.03761.x undertaken 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 Ca 2+ , 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 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 M NaCl, 1 m M 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 m M KCl, 15 m M NaCl, 15 m M Tris/HCl (pH 7.0), 0.5 m M spermine, 0.15 m M spermidine, 0.2 m M EDTA, 0.2 m M EGTA, 10 m M 2-mercaptoethanol and 0.1 m M phenylmethane- sulfonyl fluoride, and stored at 4 °C. 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 supernatant by centrifugation of the reaction mixture at 11 600 g for 10 min at 4 °C. The pellet was resuspended in lysis buffer containing 1 m M Tris/HCl (pH 7.4), 0.2 m M EDTA and 0.2 m M phenylmethanesulfonyl fluoride, and the mixture was dialyzed against the same buffer for 12 h at 4 °C. Centrifugation of the suspension at 600 g for 10 min at 4 °C 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 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 [17]. Gradients were fractionated into 9 or 18 fractions from the bottom and their A 260 value 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 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 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 20 °Cfor8hand the reactions were quenched by the addition of EDTA to give a final concentration of 20 m M . The reaction mixture was centrifuged at 2000 g for 6 min at 4 °Ctosediment 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. Ó FEBS 2003 In vivo cross-linking of histones (Eur. J. Biochem. 270) 3751 nuclear pellets, which were washed three times by centri- fugation and resuspension in labeling buffer. Purification of histones Nuclei, before or after DCA-labeling, were washed three times in 0.14 M NaCl and 50 m M NaHCO 3 by centrifuga- tion at 1000 g for 5 min at 4 °C. The histones were extractedwithcold(4°C) 0.2 M H 2 SO 4 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- HPLC column (25 · 0.46 cm; Inertsil ODS; GL Science, Tokyo, Japan). Fragments were eluted at a flow rate of 1.0 mLÆmin )1 with a linear gradient of 5–60% acetonitrile (1%Æmin )1 ) in 0.1% trifluoroacetic acid, after a 10-min hold. The A 214 and 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 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 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. 3752 K. Nunomura et al. (Eur. J. Biochem. 270) Ó FEBS 2003 Native 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 0.4 M NaCl, starfish sperm histone H1 could not be removed from nucleosomes under these conditions (Fig. 4C). Almost all the mononucleosomes were recovered infractions14and15,whichweredevoidofp28(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 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 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. Ó FEBS 2003 In vivo cross-linking of histones (Eur. J. Biochem. 270) 3753 H3 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 (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 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. 3754 K. Nunomura et al. (Eur. J. Biochem. 270) Ó FEBS 2003 by 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 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 2+ might activate the nuclear transglutami- nase and induce histone cross-linking. As expected, treat- ment of a sperm suspension (1.0 · 10 7 spermÆmL )1 )with egg jelly (25 lgof 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 °Cin 10 m M Tris/HCl (pH 7.5), 5 m M CaCl 2 ,5m M dithiothreitol, and 0.5 m M 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 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-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 circles), were determined. 3756 K. Nunomura et al. (Eur. J. Biochem. 270) Ó FEBS 2003 organized 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 N e (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 Ca 2+ + 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 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 N e (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 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 fusion. The initiation of the acrosome reaction is brought about by an abrupt increase in Ca 2+ uptake, which is induced by molecules contained in the jelly coat of the egg [15]. Amano et al. [21] performed SDS/PAGE of total histones prepared from A. pectinifera sperm which had 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 Ca 2+ concentrations induced by egg jelly activates nuclear transglutaminase, thus leading to histone cross-linking. 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Dingwall, C., Robbins, J., Dilworth, S.M., Roberts, B. & Richardson, W.D. (1988) The nucleoplasmin nuclear location sequence is larger and more complex than that of SV-40 large T antigen. J. Cell Biol. 107, 841–849. Ó FEBS 2003 In vivo cross-linking of histones (Eur. J. Biochem. 270) 3759 . 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*,. 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. 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