Mechanism of salmon sperm decondensation by nucleoplasmin

International Journal of Biological Macromolecules 26 (1999) 95 – 101 www.elsevier.com/locate/ijbiomac

Mechanism of salmon sperm decondensation by nucleoplasmin Kazumichi Iwata, Kentaro Hozumi, Akiko Iihara, Motoyoshi Nomizu, Nobuo Sakairi, Norio Nishi * Di6ision of Bioscience, Graduate School of En6ironmental Earth Science, Hokkaido Uni6ersity, Kita-ku, Sapporo 060 -0810, Japan Received 8 October 1998; received in revised form 5 March 1999; accepted 10 March 1999

Abstract Removal of protamine from DNA–protamine (salmine, protamine from salmon sperm) complexes by nucleoplasmin was examined and compared with that of poly-L-glutamic acid (PLGA) using turbidity and ethidium bromide (EB) treatment methods. When nucleoplasmin or PLGA was added to a DNA– protamine complex solution, turbidity was decreased and the amount of EB intercalated into DNA was increased. These results suggest that nucleoplasmin and PLGA can remove protamine from DNA –protamine complexes. The effect of nucleoplasmin was more potent than that of PLGA. Direct interaction of nucleoplasmin with protamine was confirmed by mixing experiments using circular dichroism (CD) and fluorescence spectroscopies. Results suggest that nucleoplasmin is bound to protamine in a 1:1 ratio and that Trp126 is located near a hydrophilic region containing a polyglutamic acid tract of nucleoplasmin which was obviously influenced by its binding with protamine. It would appear that the polyglutamic acid tract in nucleoplasmin plays a critical role for binding with protamine. © 1999 Elsevier Science B.V. All rights reserved. Keywords: Nucleoplasmin; Salmon sperm; Protamine; Fluorescence spectra; Circular dichroism

1. Introduction Spermatogenesis in higher animal species has been characterized by dramatic morphological and biological changes in nuclear organization. In spermatozoa, somatic-type histones in spermatid chromatin are generally replaced by protamines or sperm-specific histone variants [1]. As spermatogenesis proceeds, various changes occur in protamines associated with DNA which cause a progressive condensation of chromatin. It is assumed that protamines completely repress gene expression and condense DNA to a minimum volume [2]. Decondensation of the sperm nucleus occurs dramatically on entry into the egg cytoplasm. This decondensation process is indispensable for successful fertilization. Once fertilization is complete, protamines are replaced by histones and DNA is packaged into a nucleosome structure. The nucleosome structure is constituted with 146 DNA base pairs coiled around a * Corresponding author. Tel.: +81-11-706-2200; fax: + 81-11-7479780. E-mail address: [email protected] (N. Nishi)

histone octamer complex, which consists of histones H2A, H2B, H3 and H4 [3–5]. Although decondensation of nucleoprotamines has been thought to be caused by transient phosphorylation [6,7] or by enzymatic degradation of protamines, the exact mechanism(s) is not yet known. Nucleoplasmin is first named protein named for a molecular chaperone that was isolated from the egg of Xenopus lae6is [8] and is the most abundant protein in the oocyte and egg nucleus of this species [9–11]. Nucleoplasmin transfers histones to DNA and promotes nucleosome assembly [8,12,13]. It has been reported that nucleoplasmin removes a protamine-like protein from the Xenopus sperm nuclei and converts DNA into a nucleosome structure [14–16]. Nucleoplasmin was also found to decondense human and Mytilus sperm nuclei [16,17]. These findings suggest that the disassembling activity of nucleoplasmin in sperm nuclei is a universal role for this process. In fact, p22 protein, a nucleoplasmin- like protein isolated from Drosophila embryos, can also promote the decondensation of Xenopus sperm nuclei [18].

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Nucleoplasmin is acidic, phosphorylated and thermostable and it forms a stable homo-pentamer [8,12]. The amino acid sequence of nucleoplasmin has been deduced from its cDNA and a polyglutamic acid tract has been located in the carboxyl terminal half [19,20]. It is presumed that this unique acidic region is important for the main function of nucleoplasmin [21]. Other acidic macromolecules have been shown to mediate nucleosome reconstitution under physiological conditions [22,23]. Poly-L-glutamic acid (PLGA) was also found to decondense mouse sperm and to mediate reassembly into a nucleosome structure in in vitro experiments [24]. Recently, it was observed that nucleoplasmin promotes disassembly of salmon sperm nuclei [25]. In the present study, elimination of protamine by nucleoplasmin from DNA–protamine (salmine, protamine from salmon sperm) complexes was determined and compared with that of PLGA. The DNA – protamine complex is a simple model of fish sperm nuclei which is useful to analyze mechanisms of nucleoplasmin- mediated reactions at a molecular level. The interaction of nucleoplasmin with protamine was also investigated using circular dichrosim (CD) and fluorescence spectroscopies.

MgCl2. The [Arg]/[nucleotide] ratio in DNA–protamine complexes was fixed to 1/1. Average molecular weights of protein–DNA complexes were determined by turbidities using the equation of Klevan and Schumaker [28].

2.4. Fluorescence spectrophotometry study Measurements of fluorescence spectra were carried out on a Hitachi F4500 fluorescence spectrophotometer (Hitachi). Sample solutions of 1 mM nucleoplasmin with various concentrations of protamine were excited at 285 nm and fluorescence emissions from 300 to 500 nm were measured. Nuleoplasmin concentrations were determined by absorbance at 276 nm and were fixed at 1 mM in 10 mM Tris–HCl buffer (pH 7.4) in the presence of 100 mM KCl and 2 mM MgCl2.

2.5. CD study All measurements were carried out on a JASCO J-720 CD spectropolarimeter (Japan Spectroscopic, Tokyo, Japan). Nucleoplasmin concentration was fixed at 0.25 mg/ml in 10 mM Tris–HCl buffer (pH 7.4) in the presence of 100 mM KCl and 2 mM MgCl2. CD spectra were recorded at 20°C from 200 to 300 nm in a quartz cell with a 1.0 mm path length.

2. Materials and methods

2.1. Materials

3. Results and discussion

DNA (Na salt from salmon testis, MW= 5 000 000) and protamine (salmine sulfate from salmon sperm) were purchased from Sigma (St. Louis, MO). PLGA was synthesized using a diphenylphosphoryl azide (DPPA) method as previously described [26]. Other reagents were purchased from Wako Pure Chemical (Osaka, Japan).

3.1. Effect of nucleoplasmin and PLGA on turbidity of DNA–protamine complex solutions

2.2. Isolation and purification of nucleoplasmin Nucleoplasmin was prepared from Xenopus lae6is oocyte as described by Laskey et al. [13], Sealy et al. [27] and Iwata et al. [25].

2.3. Turbidity measurements Turbidity was determined by UV scattering of DNA–protamine (salmine) complexes. Nucleoplasmin and DNA–protamine complexes were mixed in various ratios and UV absorbances of the resulting solutions was measured at 320 nm on a Hitachi U-2000A spectrophotometer (Hitachi, Tokyo, Japan). Nucleotide concentration was determined from absorbance at 260 nm and was fixed at 50 mM in 10 mM Tris – HCl buffer (pH 7.4) in the presence of 100 mM KCl and 2 mM

In order to identify effects of nucleoplasmin on DNA–protamine complexes, a turbidity of mixing solutions was examined. DNA and protamine solutions were mixed in diverse ratios and turbidities were determined. When protamine was added to a DNA solution, turbidity increased and reached equilibrium within 30 min. When protamine was added continuously, the average molecular weight of DNA–protamine complexes was further increased. When [Arg]/[nucleotide] in the DNA–protamine complex was 1/1, the physiological ratio of [Arg]/[nucleotide] in sperm [29], the average molecular weight of DNA–protamine complexes was 2.7× 108 as determined by the equation of Klevan and Schumaker [28]. When nucleoplasmin was added to DNA–protamine complex solutions, turbidity rapidly decreased (Fig. 1(a)). Upon further addition of nucleoplasmin, turbidity decreased continuously and when nucleoplasmin was added to the extent of more than 0.13 mM (mean residue concentration of nucleoplasmin; 24.7 mM), turbidity reached a constant value. However, absorbance did not reach zero, owing to formation of different complexes, such as protamine–nucleoplasmin.

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Fig. 1. (a) Time course of OD320 in the absorption spectra of DNA – protamine complexes by the addition of nucleoplasmin. [nucleotide] = 50 mM, [Arg]/[nucleotide] in the DNA–protamine complex = 1/1 in 10 mM Tris – HCl buffer (pH 7.4) in the presence of 100 mM KCl and 2 mM MgCl2. Arrows show points at which nucleoplasmin was added. (b) Average molecular weights of complexes in mixing solutions of 50 mM DNA– protamine complexes in the presence of various amounts of nucleoplasmin.

Fig. 2. (a) Time course of OD320 in the absorption spectra of DNA – protamine complexes by the addition of PLGA. [nucleotide] = 50 mM, [Arg]/[nucleotide] in DNA–protamine complex = 1/1 in 10 mM Tris – HCl buffer (pH 7.4) in the presence of 100 mM KCl and 2 mM MgCl2. Arrows show points at which PLGA was added. (b) Average molecular weights of complexes in mixing solution of 50 mM DNA – protamine complex in the presence of poly-L-glutamic acid (PLGA).

Molecular weights were calculated from differences of absorbance at OD320 between DNA – protamine and protamine–nucleoplasmin complexes and were plotted at each mean residue concentration of nucleoplasmin (Fig. 1(b)). As shown in Fig. 1, average molecular weights were drastically decreased by addition of nucleoplasmin and with equilibrium being achieved at approximately 20 mM mean residue concentration of nucleoplasmin. Turbidity of DNA – protamine complexes was also decreased by addition of PLGA (Fig. 2(a)). PLGA required higher residual molar concentrations relation to nucleoplasmin in order to reach constant absorbance. Average molecular weights were determined from differences of absorbances at OD320 between DNA–protamine and protamine – PLGA complexes

(Fig. 2(b)). Average molecular weights decreased by addition of PLGA and reached constant values at 40–50 mM of mean residue concentration of PLGA. These results show that PLGA also disintegrates DNA–protamine complexes, but its ability is less than that of nucleoplasmin. The mechanism of decondensation of salmon sperm at the molecular level as an in vitro model of the fertilization process was studied. The molecular ratio of phosphoric acid residues of DNA to arginine residues of protamine is approximately 1: 1 in nucleoprotamine. As a result, DNA–protamine complex form giant molecules through electrostatic interactions. PLGA was shown previously to affect the nucleoprotamines of other species, however, this activity was less potent than that of nucleoplasmin [30]. PLGA was required at

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Fig. 3. UV spectra (a) and absorption maxima (b) of ethidium bromide (EB) in the presence of 50 mM DNA – protamine complex with various amounts of nucleoplasmin. Mean residue concentrations of nucleoplasmin are (1) 0 mM; (2) 277 mM; (3) 499 mM. [nucleotide] = 50 mM, [Arg]/[nucleotide] in DNA–protamine complex = 1.0 in 10 mM Tris – HCl buffer (pH 7.4) in the presence of 100 mM KCl and 2 mM MgCl2.

Fig. 4. UV spectra (a) and absorption maxima (b) of ethidium bromide (EB) in the presence of 50 mM DNA – protamine complex with various amounts of poly-L-glutamic acid (PLGA). Mean residue concentrations of PLGA are (1) 0 mM; (2) 122 mM; (3) 690 mM. [nucleotide]=50 mM, [Arg]/[nucleotide] in the DNA–protamine complex = 1/1 in 10 mM Tris – HCl buffer (pH 7.4) in the presence of 100 mM KCl and 2 mM MgCl2.

levels five times greater than nucleoplasmin for decondensation of salmon sperm (data not shown). These results suggest that the effective removal of protamine from DNA–protamine complexes by nucleoplasmin is caused not only by its negative charges but also by other factors such as conformation and hydrophobicity.

3.2. Effect of nucleoplasmin on absorption spectra of ethidium bromide (EB) containing DNA– protamine complex solutions EB, a reagent which intercalates into DNA base pairs, was added to DNA – protamine complex solutions in the presence or absence of nucleoplasmin, with amounts of free DNA then being evaluated. EB can intercalate into free DNA, but EB can not affect DNA which has formed complexes with protamines [31]. If nucleoplasmin removes protamines from DNA–protamine complexes and produces free DNA, EB can then intercalate into base pairs of free DNA. Amounts

of the EB intercalated into DNA were determined by measuring UV absorbance from 400 to 600 nm. When EB was added to DNA–protamine complex solutions, absorbance peak maxima at 480 nm in UV spectrum were observed (Fig. 3(a)). When nucleoplasmin was added to the solutions, maxima shifted to longer wavelength and intensity decreased (Fig. 3(a)). This result suggests that free DNA was released from DNA–protamine complexes by addition of nucleoplasmin. Maximum wavelengths at different concentrations of nucleoplasmin were plotted (Fig. 3(b)). When all DNA was liberated from DNA–protamine complexes, the concentration of nucleoplasmin was approximately 2.5 mM, which is comparable to that of protamine (salmine) (2.3 mM). These results suggest that nucleoplasmin binds protamine in a 1:1 ratio and removes protamine from DNA–protamine complexes. The effect of PLGA on UV absorption spectra of EB in DNA–protamine complexes was also examined (Fig. 4). When PLGA was added to the solutions, EB ab-

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Fig. 5. Fluorescence spectra (a) and fluorescence intensity maxima (b) of nucleoplasmin in the presence of various amounts of protamine. [nucleoplasmin] =1 mM in 10 mM Tris–HCl buffer (pH 7.4) in the presence of 100 mM KCl and 2 mM MgCl2. Samples were exited at 285 nm.

sorption maxima shifted to longer wavelengths and intensity decreased (Fig. 4(a)). This result suggests that PLGA also interacts with the DNA – protamine complexes and produces free DNA. The effect of PLGA on the DNA–protamine complexes was weaker than that of nucleoplasmin, suggesting that negative charges are critical but not sufficient for the effective removal of protamine from the DNA – protamine complexes.

3.3. Effect of protamine on a nucleoplasmin fluorescence spectrum Next, interactions between nucleoplasmin and protamine using fluorescence spectrophotometry are examined (Fig. 5). Nucleoplasmin contains two tryptophan residues [19,20] and the fluorescence spectrum exhibits a maximum peak at 340 nm and a shoulder peak at 315 nm. When protamine was added to a nucleoplasmin solution, the fluorescence intensity at 340 nm was drastically decreased while that at 315 nm was minimally decreased. Two tryptophan residues are located in different surroundings as suggested by the hydrophilicity plot (Fig. 6). It is known that fluorescence emission spectra of tryptophan exhibit a maximum at 348 nm with excitation at 285 nm. When tryptophan residues are examined within hydrophobic surroundings, the fluorescence emission maximum shifts toward a shorter wavelength. The maximum at 340 nm could therefore be attributed to Trp126 located within a hydrophilic region which was strongly affected by protamine binding. On the contrary, the maximum at 315 nm (Trp19) is hardly influenced, since it exists within the nucleoplasmin molecule. The relative fluorescence intensity at 340 nm was decreased by addition of protamine (Fig. 5(b)), and reached a constant value at [nucleoplasmin]/[protamine]= 1/1. According to studies on both fluorescence analysis and EB intercalation to DNA–protamine complexes (Fig. 3), nucleoplasmin

was found to bind to protamine in a 1:1 molar ratio. As a result of protamine binding to nucleoplasmin by a 1:1 ratio, the Trp126 residue which is situated in close proximity to the polyglutamic acid tract, could be influenced. However, detailed structural analysis of binding mechanisms is required using other techniques such as NMR and X-ray.

3.4. Effect of protamine on a CD spectrum of nucleoplasmin Fig. 7 shows CD spectra of nucleoplasmin –protamine mixtures in various molecular ratios. As reported previously, the CD spectrum of nucleoplasmin shows two negative maxima at 208 and 222 nm, suggesting the existence of an a-helical conformation. The a-helix content was calculated to be 30–40% [25]. When the ratio of protamine increased, the ellipticity decreased compared with the spectrum of nucleoplasmin only. For example, at a ratio of 5:1 (nucleoplasmin/protamine molar ratio), about 20% of the apparent a-helix content decreased compared with that of nucleoplasmin. At a ratio of 1:1, the spectrum changed completely. This pattern was considered not to be a genuine CD spectrum of the nucleoplasmin –protamine

Fig. 6. Hydrophilicity plot of amino acid sequence of nucleoplasmin according to Hopp and Woods method [32]. Trp19, Trp126 and the polyglutamic acid tract are indicated.

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Fig. 8. Schematic drawing describing of a proposed function of nucleoplasmin.

complex, but to be a spectrum induced by light-scattering due to aggregate formation, although the mixture was still transparent. These results suggest that nucleoplasmin strongly interacts with protamine. As suggested by the fluorescence study, protamine appears to interact with nucleoplasmin in a 1: 1 ratio. Protamine could bind mainly to the polyglutamic acid tract within nucleoplasmin. Protamine has 21–22 arginine residues in its amino acid sequence, whereas polyglutamic acid tract in nucleoplasmin has only 16– 17 acidic amino acid residues. Remaining arginine residues may bind to glutamic acids in the core region of nucleoplasmin. In the present study, we have described how nucleoplasmin causes a removal of protamine from DNA–

protamine (salmine, protamine from salmon sperm) complexes. Mixing experiments described here are a useful models to evaluate molecular interactions and identify further functions of nucleoplasmin. A schematic drawing of the proposed function of nucleoplasmin is shown in Fig. 8. Nucleoplasmin is suggested to play a critical role in sperm nucleus decondensation in fertilization process.

Acknowledgements We thank Dr Toru Itoh and Professor Chiaki Katagiri (Division of Biological Science, Graduate School of Science, Hokkaido University) for their valuable suggestions and discussions. We also thank Dr Terrence Burke (National Cancer Institute, National Institutes of Health, Bethesda, Maryland, USA) for critical reading of the manuscript.

References

Fig. 7. Circular dichroism (CD) spectra of nucleoplasmin–protamine (salmine) mixtures at various molecular ratios; (a) nucleoplasmin, (b) nucleoplasmin (monomer):protamine = 10:1, (c) 5:1, (d) 1:1, (e) protamine in 10 mM Tris – HCl buffer (pH 7.4) in the presence of 100 mM KCl and 2 mM MgCl2. [u] was calculated based on the sequence of nucleoplasmin (a – d) and protamine (e).

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