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DNA-interacting Proteins in the Spermiogenesis of the MolluscMurex brandaris

1999; Elsevier BV; Volume: 274; Issue: 2 Linguagem: Inglês

10.1074/jbc.274.2.649

1083-351X

Carme Càceres, Pepita Gimenez‐Bonafé, Enric Ribes, D. Wouters‐Tyrou, Arlette Martinage, Mostafa Kouach, Pierre Sáutière, Sylviane Muller, Jaume Palau, Juan A. Subirana, Luís Cornudella, Manel Chiva,

Epigenetics and DNA Methylation

Sperm chromatin of Murex brandaris (a neogastropod mollusc) undergoes a series of structural transitions during spermiogenesis. The DNA-interacting proteins responsible for these changes as well as the mature protamines present in the ripe sperm nucleus have been characterized. The results reveal that spermiogenic nuclear proteins are protamine precursors that are subjected to a substantial number of small N-terminal deletions that gradually modify their overall charge. The composition of mature protamines is remarkably simple in turn, promoting an efficient and extremely tight packaging of DNA. The pattern of spermiogenic chromatin condensation in M. brandaris clearly departs from that corresponding to vertebrate chromatin. Sperm chromatin of Murex brandaris (a neogastropod mollusc) undergoes a series of structural transitions during spermiogenesis. The DNA-interacting proteins responsible for these changes as well as the mature protamines present in the ripe sperm nucleus have been characterized. The results reveal that spermiogenic nuclear proteins are protamine precursors that are subjected to a substantial number of small N-terminal deletions that gradually modify their overall charge. The composition of mature protamines is remarkably simple in turn, promoting an efficient and extremely tight packaging of DNA. The pattern of spermiogenic chromatin condensation in M. brandaris clearly departs from that corresponding to vertebrate chromatin. The current model for the nuclear changes occurring in spermiogenesis has been essentially drawn from studies on bony fishes and other vertebrates (1Louie A.J. Dixon G.H. J. Biol. Chem. 1972; 247: 7962-7968Abstract Full Text PDF PubMed Google Scholar, 2Christensen M.E. Dixon G.H. Dev. Biol. 1982; 93: 404-415Crossref PubMed Scopus (74) Google Scholar, 3Oliva R. Mezquita C. Nucleic Acids Res. 1982; 10: 8049-8059Crossref PubMed Scopus (75) Google Scholar, 4Oliva R. Bazzet-Jones D.P. Locklear L. Dixon G.H. Nucleic Acids Res. 1990; 188: 2739-2747Crossref Scopus (98) Google Scholar, 5Oliva R. Dixon G.H. Prog. Nucleic Acids Res. and Mol. Biol. 1991; 40: 25-94Crossref PubMed Scopus (363) Google Scholar). The model considers that in early spermatids, histones become acetylated preceding their replacement by a highly basic protein (protamine). During histone displacement, the protamine is found polyphosphorylated, although it undergoes progressive dephosphorylation in the subsequent spermiogenic development. The processes of protamine phosphorylation and dephosphorylation imply a mechanism regulating the interaction with DNA. This mechanism allows for the orderly substitution of protamine for the nucleohistones and for the ensuing binding to DNA. The protamine is a small molecule displaying a very high positive charge density (6Saperas N. Ausió J. Lloris D. Chiva M. J. Mol. Evol. 1994; 39: 282-295Crossref PubMed Scopus (50) Google Scholar, 7Oliva R. Jamieson B.G.M. Ausió J. Justine J.L. Advances in Spermatozoal Taxonomy and Phylogeny. 166. Memoires du Museum National d'Histoire Naturelle, Paris1995: 537-548Google Scholar). Consequently, the nuclear replacement of histones by protamine induces profound transitions in chromatin structure directly leading to the compactness of the sperm nucleus. Although these events are well established for some bony fishes and related vertebrates, there is not a universal pattern, or even a “more frequent” model, to account for spermiogenic processes. Evolution has generated an enormous diversity of both DNA-condensing proteins (8Kasinsky H.E. Hnilica L.S. Stein G.S. Stein J.L. Histones and Other Basic Nuclear Proteins. CRC Press, Inc., Boca Raton, FL1989: 73-163Google Scholar, 9Chiva M. Saperas N. Càceres C. Ausió J. Jamieson B.G.M. Ausió J. Justine J.L. Advances in Spermatozoal Taxonomy and Phylogeny. 166. Memoires du Museum National d'Histoire Naturelle, Paris1995: 501-514Google Scholar, 10Ausió J. Jamieson B.G.M. Ausió J. Justine J.L. Advances in Spermatozoal Taxonomy and Phylogeny. 166. Memoires du Museum National d'Histoire Naturelle, Paris1995: 447-462Google Scholar) and structural conformations for spermiogenic and/or sperm chromatin (11Subirana J.A. Andrè J. The Sperm Cell. Martine Nijhoff, The Hague, 1993: 197-213Google Scholar, 12Suzuki M. Wakabayashi T. J. Mol. Biol. 1988; 204: 653-661Crossref PubMed Scopus (18) Google Scholar). Traditionally, electron microscopy studies have focused on cenogastropod molluscs due to the complexity of their chromatin condensation patterns (13Buckland-Nicks J. Chia F.S. Cell Tissue Res. 1976; 170: 455-475Crossref PubMed Scopus (63) Google Scholar, 14Buckland-Nicks J. Williams D. Chia F.S. Fontaine A. Cell Tissue Res. 1982; 227: 235-255Crossref PubMed Scopus (32) Google Scholar, 15Jaramillo R. Garrido O. Jorquera B. Veliger. 1986; 29: 217-225Google Scholar, 16Healy J.M. Harasewych M.G. Nautilus. 1992; 106: 1-14Google Scholar). In this regard, Murex brandaris is a paradigmatic species (17Amor M.J. Durfort M. Mol. Reprod. Dev. 1990; 25: 348-356Crossref PubMed Scopus (23) Google Scholar). Chromatin in the early haploid spermatid displays a somatic-like appearance, which immediately undergoes a peripheral migration, giving rise gradually to granular and fibrilar structures and finally to 18-nm lamellae (18Càceres C. Ribes E. Muller S. Cornudella L. Chiva M. Mol. Reprod. Dev. 1994; 38: 440-452Crossref PubMed Scopus (17) Google Scholar). The nascent lamellar structures are first seen in a disordered arrangement but later become progressively ordered in a regular concentric pattern surrounding the nuclear axis. The progression of structural changes in chromatin should be paralleled by simultaneous modifications of DNA-protein interactions, concurrently with thermodynamic restrictions on the size of the DNA-protein complexes (19Subirana J.A. FEBS Lett. 1992; 302: 105-107Crossref PubMed Scopus (19) Google Scholar, 20Casas M.T. Ausió J. Subirana J.A. Exp. Cell Res. 1993; 204: 192-197Crossref PubMed Scopus (22) Google Scholar). It was found in a previous study (18Càceres C. Ribes E. Muller S. Cornudella L. Chiva M. Mol. Reprod. Dev. 1994; 38: 440-452Crossref PubMed Scopus (17) Google Scholar) that the histone complement of the immature gonads of M. brandarisbecomes replaced by a large set of intermediate proteins that lead to the final appearance of three small and simple protamine molecules in the sperm nucleus. It was also observed that these intermediate proteins reacted positively to antibodies elicited against a sperm protamine. In this paper, we report the primary structure of theM. brandaris mature protamines and show that one of them (protamine P1) is synthesized as a precursor molecule that undergoes a complex series of partial deletions in its N terminus in conjunction with a single step of dephosphorylation. The final forms of these mature protamines generate an almost extreme packing of sperm DNA . Male specimens of the mollusc M. brandaris were collected periodically on the Mediterranean coast of Spain and moved live to the laboratory in cold sea water. Either free-flowing sperm cells or gonadal tissue was homogenized separately in ice-cold buffer (0.25m sucrose, 10 mm MgCl2, 3 mm CaCl2, 10 mm Tris-HCl, pH 7.0, 0.1% Triton X-100, 50 mm benzamidine chloride as protease inhibitor) and centrifuged at 3000 × g for 5 min. Crude nuclear pellets were then rehomogenized and resedimented three times in the same buffer. The purified nuclear pellets were next homogenized in 10 mm Tris-HCl, pH 7.0, 20 mmNa2EDTA, followed by centrifugation and then one more time in the same buffer, omitting the chelating agent. The nuclear sediments thus obtained were used to extract proteins as well as for x-ray diffraction analyses. Proteins were extracted from purified sperm or gonadal nuclei with 0.4 n HCl, precipitated with 6 volumes of cold acetone, and finally rinsed with acidified acetone (21Chiva M. Kasinsky H.E. Subirana J.A. FEBS Lett. 1987; 2: 237-240Crossref Scopus (30) Google Scholar). On one occasion, nuclear sediments were reduced with 50 mm Tris-HCl, pH 8.8, 2 mm Na2EDTA, 10 mm dithiothreitol for 1 h at 37 °C under N2 atmosphere and then alkylated with 12.5 mmiodoacetamide in the same buffer prior to HCl extraction (22Gusse M. Sautière P. Chauvière M. Chevaillier P. Biochim. Biophys. Acta. 1983; 748: 93-98Crossref PubMed Scopus (26) Google Scholar, 23Saperas N. Chiva M. Bols N. Kulak D. Kasinsky H.E. Biol. Bull. 1993; 185: 186-196Crossref PubMed Google Scholar). Polyclonal antibodies against protamine P1 were prepared as described previously (18Càceres C. Ribes E. Muller S. Cornudella L. Chiva M. Mol. Reprod. Dev. 1994; 38: 440-452Crossref PubMed Scopus (17) Google Scholar) with some modifications. Briefly, outbred New Zealand White female rabbits were multi-injected intradermally with 750 μg of protamine/animal. The antigen was previously emulsified with Freund's complete adjuvant. The anti-protamine P1 antisera was tested by an enzyme-linked immunosorbent assay with purified protamines. Western blotting of nuclear proteins from both sperm and gonads was performed according to Harlow and Lane (24Harlow E. Lane D. Antibodies: A Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1988Google Scholar). Following acetic acid-urea polyacrylamide gel electrophoresis, proteins were electrotransferred onto nitrocellulose membranes at 500 mA for 1 h at 4 °C in a solution of 0.7% acetic acid, 10% methanol. The membranes were washed in PBS, 0.1% Tween 20 (PBS/T), blocked with PBS/T containing 0.1% gelatin for 2 h, and incubated with anti-protamine P1 antisera for 2 h at 37 °C. Detection was performed with diluted peroxidase-conjugated anti-rabbit IgG (1:1000) and 4-chloro-1-naphtol as substrate. Chromatographic resolution of proteins in ion exchange columns was performed on CM-52 cellulose (Whatman). Protein samples were dissolved in 0.2 n NaCl, 50 mm acetate, pH 6.0, and loaded onto the column. Elution was carried out stepwise with 0.2 and 0.6 n NaCl, finally applying a linear gradient of NaCl concentrations from 0.6 to 2.0n in the same acetate buffer (25Chiva M. Kasinsky H.E. Mann M. Subirana J.A. J. Exp. Zool. 1988; 245: 304-317Crossref Scopus (27) Google Scholar). The collected fractions were dialyzed against 5 mm HCl and lyophilized. For reverse phase HPLC, 1The abbreviations used are: HPLC, high pressure liquid chromatography. a C4, 300-Å Delta-Pack column (25 × 0.46 cm) was used. Proteins were eluted applying a linear gradient of acetonitrile (0–25%) in 0.05% trifluoroacetic acid (26Buesa C. Del Valle L. Saperas N. Goethels M. Lloris D. Chiva M. Comp. Biochem. Physiol. 1998; 119B: 145-149Crossref Scopus (3) Google Scholar). One-dimensional polyacrylamide slab gel electrophoresis was performed according to Panyim and Chalkley (27Panyim S. Chalkley R. Arch. Biochem. Biophys. 1969; 130: 337-346Crossref PubMed Scopus (1968) Google Scholar) with the modifications described by Hurley (28Hurley C.K. Anal. Biochem. 1977; 80: 624-626Crossref PubMed Scopus (51) Google Scholar). Amino acid analysis of proteins was carried out after hydrolysis of the samples in 6 n HCl (29Chiva M. Mezquita C. FEBS Lett. 1983; 162: 324-328Crossref PubMed Scopus (16) Google Scholar). Sequencing of the M. brandaris proteins was performed on a Procise Sequenator/ABI-492 (Perkin-Elmer) using the Pulsed Liquid 2HL program. The molecular mass of proteins was determined by ion spray mass spectrometry. Samples were dissolved in 200 μl of an aqueous solution of 20% acetonitrile, 0.1% HCOOH. Ion spray mass spectra were recorded on a simple quadrupole mass spectrometer API I (Perkin-Elmer), equipped with an ion spray (nebulized-assisted electrospray) source (Sciex, Toronto, Canada). The solutions were continuously infused with a medical infusion pump (model 11, Harward Apparatus, South Natick, MA) at a flow rate of 5 μl/min. Polypropyleneglycol was used to calibrate the quadrupole. Ion spray mass spectra were acquired at unit resolution by scanning from m/z 400 to 1200 with a step size of 0.1 Da and a dwell time of 2 min. Ten spectra were summed. The potential of the spray needle was held at +4.5 kV. Spectra were recorded at an orifice voltage of +90 V. A Mac Bio Spec computer program was used to measure the molecular masses of the protein samples. Protein alignments were done using the method of Lipman and Pearson (30Lipman D.J. Pearson W.R. Science. 1985; 227: 1435-1441Crossref PubMed Scopus (2554) Google Scholar). The x-ray diffraction patterns were obtained from samples of nuclei or fibrous complexes (reconstituted complexes). In both instances, samples were sealed in capillaries containing a drop of a saturated salt solution used as a control of the relative humidity. The patterns were recorded with nickel-filtered copper radiation on Kodak film. Either a modified Philips microcamera or a Statton camera (W. R. Warhus, Wilmington, DE) was used. Complexes of M. brandaris protamines with DNA were prepared by mixing both components in a proportion to achieve complete charge neutralization. The mixing buffer used was 2 m guanidine hydrochloride, 1 mm Tris-HCl, pH 8.0, 1 mmEDTA. Mixtures were sequentially dialyzed against solutions of 2, 1, 0.8, 0.4, and 0.2 m guanidine hydrochloride, 1 mm Tris-HCl, pH 8.0, 1 mm EDTA, followed by extensive dialysis against 1 mm Tris-HCl, 1 mmEDTA. Complexes started to precipitate when the guanidine hydrochloride concentration was about 0.8 m. The fibrous precipitates were then pulled with tweezers and allowed to dry under tension in order to yield fibers suitable for x-ray diffraction analysis (31Fornells M. Campos J.L. Subirana J.A. J. Mol. Biol. 1983; 166: 249-252Crossref PubMed Scopus (17) Google Scholar,32Fita I. Campos J.L. Puigjaner L.C. Subirana J.A. J. Mol. Biol. 1983; 167: 157-177Crossref PubMed Scopus (60) Google Scholar). Electron microscopy analysis was performed as described previously (18Càceres C. Ribes E. Muller S. Cornudella L. Chiva M. Mol. Reprod. Dev. 1994; 38: 440-452Crossref PubMed Scopus (17) Google Scholar). Either gonadal tissue or sperm cell sediments were fixed in 2.5% glutaraldehyde, 0.1 mcacodylate buffer and postfixed in 1% osmium tetroxide in the same buffer. The samples were next dehydrated and soaked in Spurr's resin. Sample sections were stained with uranyl acetate and lead citrate and examined under a Hitachi H-600 transmission electron microscope. Sperm cells ofM. brandaris contain electrodense cylindrical nuclei grooved by axonemes (Fig. 1, inset). Acid extraction of purified nuclei with 0.4 n HCl yielded three basic proteins (Fig. 1, lane w). Previous chemical reduction of chromatin prior to extraction did not solubilize any additional proteins. Nucleoprotein components were purified by reverse-phase HPLC eluting in the order P3 → P2 → P1 (Fig. 1). Protamine P1 (peak 3 and lane 3 in Fig. 1) was repurified by reverse-phase HPLC in the same conditions to eliminate a small amount of contaminating P2 protamine. The amino acid analysis of M. brandaris protamines revealed a set of simple molecules of very basic nature (Table I). P1 is made up of six different aminoacyl types, P2 by only four types, whereas P3 appears to be the simplest protamine known to date, involving just three amino acid residues (Arg, Lys, and Gly). Arginine, lysine, and glycine are the major components of all three protamines (94.9% in P1, 96.7% in P2, and 100% in P3). Another relevant feature revealed by the amino acid analyses was the low proportion of phosphorylatable residues in all of the molecules (2% in P1, 3.2% in P2, and 0% in P3). This is a striking fact if it is considered that phosphorylation and dephosphorylation processes regulate the interactions with DNA in vertebrate protamines. All protamines were repeatedly analyzed by automated Edman degradation with consistent results. Due to the yields and purity of the protein samples together with the powerful methodology employed, it was possible to obtain complete and unambiguous sequences in single sequencing runs (Fig.2C).Table IAmino acid composition of M. brandaris protamines P1, P2, and P3P1P2P3abcabcabcmol %mol %mol %mol %mol %mol %Lys34.424.82537.121.52138.220.621Arg34.024.52436.521.22136.319.619Ser2.01.413.21.92Gly26.519.11923.113.41425.313.614Ala1.10.81Cys1.91.42Total residues725854Shown are the mol % determined from the compositional analysis (a), corresponding residue contents (b), and number of residues derived from the amino acid sequence (c). Open table in a new tab Shown are the mol % determined from the compositional analysis (a), corresponding residue contents (b), and number of residues derived from the amino acid sequence (c). Molecular masses of protamines P1, P2, and P3 as established by ion spray mass spectrometry were 8416 ± 1 Da, 6962 Da, and 6474 Da, respectively (Fig. 2 A). The molecular mass of protamine P1 was also determined in mature gonads containing partially ripe spermatozoa (Fig. 2 B). In this case, a molecular mass of 8497 Da was obtained for a fraction of the protamine, consistent with the phosphorylated form (8417 + 80 Da). This result suggests that protamine P1 is monophosphorylated in the stages preceding full ripening of the sperm and is coincident with the presence of only one serine residue in the P1 molecule. The analytical results so far reported are wholly congruent. First, the molecular masses derived from the amino acid analyses (P1, 8415 Da; P2, 6960 Da; P3, 6473 Da) are totally coincident with the masses determined. Second, the number of amino acid residues obtained from the compositional analyses is practically identical to that afforded by the primary structures (see Table I). It is relevant to note the simplicity of the M. brandaris protamine sequences whose organization will be dealt with later. Protamine P3 appears exclusively constituted of Arg, Lys, and Gly amino acids, while both P1 and P2 contain a few additional residues. This structural simplicity together with the low proportion of phosphorylatable residues (only three serines out of 184 amino acid residues) contrasts with the elaborated forms of spermiogenic chromatin condensation illustrated in Fig. 3. We have previously shown that unripe spermiogenic nuclei of M. brandaris contain a substantial number of proteins with an electrophoretic mobility intermediate between histones and protamines in denaturing polyacrylamide gels (18Càceres C. Ribes E. Muller S. Cornudella L. Chiva M. Mol. Reprod. Dev. 1994; 38: 440-452Crossref PubMed Scopus (17) Google Scholar). This abundant subset of intermediate proteins disappears in very advanced stages of spermiogenesis and becomes wholly absent from ripe sperm nuclei. Considering both the electrophoretic behavior and amino acid composition of these intermediate proteins, we surmised that they might direct the complex transitions in the condensation of the M. brandaris spermiogenic chromatin, modulating the interaction with DNA through a series of posttranslational intranuclear modifications and acting as precursors of the ripe sperm protamines. In order to verify the preceding assertion these proteins were analyzed in detail. First, the largest intermediate molecule Pr-P1, displaying the slowest electrophoretic mobility, was purified, and the sequence of its N terminus comprising the initial 52 amino acid residues was determined by automated Edman degradation together with the assessment of its molecular mass by ion spray mass spectrometry (Fig.4). Moreover, purified Pr-P1 protein reacted positively with anti-protamine P1 antisera (Fig.5, lane e). TableII shows the actual amino acid composition of the putative precursor molecule and its comparison with the composition estimated under the assumption that the molecule is a true precursor of protamine P1. The results indicate that this intermediate protein (Pr-P1) indeed corresponds to a monophosphorylated precursor of protamine P1. Thus, the molecular mass of the monophosphorylated form of protamine P1 (8497 Da in Fig. 2) plus that corresponding to the 35 initial residues of the N terminus of Pr-P1 yielded a value (12,657 Da) almost identical to the molecular mass of Pr-P1 obtained by ion spray mass spectrometry (12,662 Da). In addition, the determined amino acid composition is very similar to the composition estimated (Table II). Finally, the last 17 sequenced residues of Pr-P1 are identical to the 17 initial amino acid residues of P1. The presence of an alanine in position 48 of Pr-P1 confirms that this molecule truly encompasses protamine P1, since both P2 and P3 lack the former residue.Figure 5Immunoreactivity of protein Pr-P1 to anti-P1 antisera. Western blot analysis of M. brandarisproteins with polyclonal antibodies after acetic acid-urea-polyacrylamide gel electrophoresis is shown. Lane a, blot of the nucleoproteins extracted from whole gonads stained with Amido Black following electrotransfer onto nitrocellulose filter.Lane b, same strip as in A immunoreacted with anti-P1. Lanes c and d, electrophoretically resolved gonadal nucleoproteins (lane c) and purified protein Pr-P1 (lane d), blotted to membranes and stained with Amido Black. Lane e, strip containing purified protein Pr-P1 reacted with anti-P1 antibodies. Migration ranges are indicated for histones (h), intermediate proteins (i), and protamines (p).View Large Image Figure ViewerDownload Hi-res image Download (PPT)Table IICompositional analysis of Pr-P1abmol %Lys26.126.2His1.40.9Arg27.127.1Asx4.03.8Asp1.9Asn1.9Thr1.20.9Ser1.30.9Glx6.69.3Glu8.4Gln0.9Pro2.92.8Gly20.819.6Ala2.32.8Cys0.91.9Val2.12.8MetIleLeu1.20.9Tyr0.20.9Phe1.91.9TrpNDaNot determined.Shown are the amino acid composition (mol %) determined for protein Pr-P1 (a) and estimated composition considering Pr-P1 a direct precursor form of protamine P1 (b).a Not determined. Open table in a new tab Shown are the amino acid composition (mol %) determined for protein Pr-P1 (a) and estimated composition considering Pr-P1 a direct precursor form of protamine P1 (b). It can be seen in Fig. 5 that the anti-protamine P1 antiserum reacts positively with the bulk of intermediate proteins present in spermiogenic nuclei (see lane b). This result suggests that these proteins do contain also the protamine P1 sequence and therefore can be considered precursor molecules. To confirm this assumption, the material under the individual peaks in Fig.4 A was subfractionated by HPLC, and the resulting proteins were directly sequenced by automated Edman degradation. In some cases, protein yields allowed for additional determinations of the molecular mass by ion spray mass spectrometry (TableIII). The sequences obtained (Fig.6) clearly show that the intermediate proteins analyzed arise from the gradual trimming of the N-terminal residues of protein Pr-P1. Moreover, the molecular weights determined (Table III) indicate that these molecules are monophosphorylated. Nine major precursor forms have been identified; however, the presence of additional minor components cannot be discounted. These results show that the M. brandaris Pr-P1 precursor is processed by means of serial deletions each involving one or a few more amino acid residues. Although this processing mechanism is not common to other molluscs (33Wouters-Tyrou D. Martin-Ponthieu A. Ledoux-Andula N. Kouach M. Jaquinod M. Subirana J.A. Sautière P. Biochem. J. 1995; 309: 529-534Crossref PubMed Scopus (12) Google Scholar), it stands comparison with the processing of the P2 precursor protamine in some mammals (34Chauvière M. Martinage A. Sautière P. Chevaillier P. Eur. J. Biochem. 1995; 204: 759-765Crossref Scopus (38) Google Scholar, 35Debarle M. Martinage A. Sautière P. Chevaillier P. Mol. Reprod. Dev. 1995; 40: 84-90Crossref PubMed Scopus (18) Google Scholar).Table IIIPrecursor forms of protamine P1ProteinNSMwoMweDaDaPr-P15212,66212,657Pr85012,09212,090Pr75411,82011,819Pr65211,55011,549Pr54011,07411,075Pr47NDaND, not determined.10,082Pr329ND9798Pr243ND9635Pr12592489249Shown are the number of amino-terminal residues sequenced for each intermediate molecule (NS), molecular masses determined by ion spray mass spectrometry (Mwo), and molecular mass estimated under the assumption that these molecules are precursors of the monophosphorylated form of protamine P1 (Mwe) (see also Fig. 6).a ND, not determined. Open table in a new tab Shown are the number of amino-terminal residues sequenced for each intermediate molecule (NS), molecular masses determined by ion spray mass spectrometry (Mwo), and molecular mass estimated under the assumption that these molecules are precursors of the monophosphorylated form of protamine P1 (Mwe) (see also Fig. 6). To determine the capacity of M. brandarisprotamines to compact DNA, protamine-DNA complexes were subjected to x-ray diffraction analysis. Two sets of experiments were carried out. In the first series, x-ray diffraction patterns were obtained from purified sperm nuclei, whereas in the second the analyses were performed with reconstituted complexes of DNA with purified M. brandaris protamines (see “Materials and Methods”). Nuclei or reconstituted complexes were analyzed at different values of relative humidity. Fig. 7 shows the x-ray diffraction patterns obtained from reconstituted complexes at 76% relative humidity (Fig. 7 A) and from sperm nuclei at 92% relative humidity (Fig. 7 B). The complex in Fig.7 A may be interpreted as a B form of DNA packed in an orthorhombic lattice with two molecules per unit cell, with approximate cell parameters as follows: a = 33.2 Å,b = 24.1 Å, and c = 33.4 Å. The values reported in Table IV show that distances between DNA strands observed in reconstituted complexes and sperm nuclei are highly coincident at each value of relative humidity.Table IVDNA packing by M. brandaris protaminesRelative humidityabÅÅ3319.519.35419.519.47620.520.19222.822.8Shown are the distances between neighbor DNA strands of the x-ray diffraction patterns obtained from nuclei (a) and reconstituted (M. brandaris protamine-DNA) complexes (b) at different values of relative humidity. Open table in a new tab Shown are the distances between neighbor DNA strands of the x-ray diffraction patterns obtained from nuclei (a) and reconstituted (M. brandaris protamine-DNA) complexes (b) at different values of relative humidity. These experiments show that DNA becomes packaged by M. brandaris protamines in a very tight and compact manner as deduced from the distance between axes of the neighbor DNA strands, which varies between 19.5 and 22.8 Å (see Table IV). These values are similar to those found in squid nucleoprotamine, which contains 79% arginine (36Suau P. Subirana J.A. J. Mol. Biol. 1977; 117: 909-926Crossref PubMed Scopus (47) Google Scholar). It is likely that the charge distribution and large percentage of glycine (an amino acid with a minimal volume) in the protamines of M. brandaris generate a compactness of the DNA to such extremes that the resulting volume of the mature sperm nucleus becomes comparable with that of the DNA alone when considered as a cylinder of 20 Å in diameter. Three protamines of a very simple composition (P1, P2, and P3) are present in the ripe sperm nuclei of the mollusc M. brandaris. One of them (P3) is the simplest protamine known to date, being made up of only three types of amino acids (Gly, Lys, and Arg) and completely lacking phosphorylatable residues. The most relevant features of these three molecules are their extreme basicity and the presence of arginine clusters interspersed with very rich GK tracts. Arginine clusters have been held responsible for the cooperative interaction of protamines with DNA (37Willmitzer L. Wagner R.G. Biophys. Struct. Mech. 1980; 6: 95-110Crossref PubMed Scopus (38) Google Scholar), but GK-rich regions are absent in other known protamines. Among the M. brandaris protamines, the GK-alternating residues are particularly evident near the N terminus of protamine P1 (see residues 8–22 in Fig.2). It is worth indicating that some other protamines contain substantial clusters of alternating basic/nonbasic residues close to their N termini, notably the (RS)n repeat (5Oliva R. Dixon G.H. Prog. Nucleic Acids Res. and Mol. Biol. 1991; 40: 25-94Crossref PubMed Scopus (363) Google Scholar, 45Daban M. Martinage A. Kouach M. Chiva M. Subirana J.A. Sautière P. J. Mol. Evol. 1995; 40: 663-670Crossref PubMed Scopus (17) Google Scholar). Interestingly, a (RS)n motif adjacent to a tract of basic amino acid residues has been observed in some splicing factors, having been implicated in intranuclear location (38Hedley M.L. Amrein H. Maniatis T. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 11524-11528Crossref PubMed Scopus (126) Google Scholar). The repeating GK dipeptide might also represent a novel protamine-DNA element, although further structural studies are required to unambiguously establish its specific role in the highly efficient and organized packaging of DNA in the sperm chromatin. In addition to the amino acids Arg, Lys, and Gly, protamine P1 also contains a phosphorylatable residue of serine, one alanine and two cysteine residues. The high content of cysteine usually found in protamines from mammals and some other species is thought to stabilize the nuclear structure of sperm by intermolecular disulfide bridging (39Balhorn R. Corzett M. Mazrimas J. Watkins B. Biochemistry. 1991; 30: 175-181Crossref PubMed Scopus (52) Google Scholar, 40Balhorn R. Adolph K.W. Molecular Biology of Chromosome Function. Springer-Verlag, New York1982: 366-420Google Scholar). This notwithstanding, the fact that protamine P1 fromM. brandaris can be extracted from nuclei with no need of previous chemical reduction of the sperm chromatin strongly supports the notion that the cysteine residues present in this protein do not form intermolecular covalent linkages. The compositional and structural simplicity of the M. brandaris protamines suggests that these molecules have acquired a defined specialization to perform a tight and compact packaging of DNA (Table IV). It has been established in the present work that protamine P1 fromM. brandaris appears as a precursor molecule in the spermiogenic nuclei encompassing the mature protamine in a phosphorylated form preceded by an N-terminal precursor peptide. The precursor sequence is made up of 35 amino acid residues of which nine are basic (five Arg, three Lys, one His) and seven are acidic (two Asp and five Glu, four of them in a row, Glu4). The presence of basic and acidic residues allows the peptide to interact with both DNA and basic proteins (histones or protamines). It is noticeable that nucleoplasmin, a protein specialized in remodeling chromatin structure during fertilization, also interacts with histones or protamines by means of its clusters of acidic residues (41Dilworth S.M. Black S.J. Laskey R.A. Cell. 1987; 51: 1009-1018Abstract Full Text PDF PubMed Scopus (186) Google Scholar, 42Laskey R.A. Mills A.D. Philpott A. Leno G.H. Dilworth S.M. Dinwall C. Philos. Trans. R. Soc. Lond-Biol. Sci. 1993; 339: 263-269Crossref PubMed Scopus (58) Google Scholar, 43Leno G.H. Philpott A. Laskey R.A. Heslop-Harrison J.S. Flavele R.B. The Chromosome. BIOS Scientific Publishers Ltd., Oxford1993: 135-147Google Scholar, 44Ruiz-Lara S. Cornudella L. Rodriguez Campos A. Eur. J. Biochem. 1996; 240: 186-194Crossref PubMed Scopus (14) Google Scholar). The successive deletions of the precursor peptide (see Fig. 6) might modulate the interactions of the proteins with DNA and might also be instrumental in the structural transitions undergone by the spermiogenic chromatin (see Fig. 3). Likewise, the dephosphorylation observed may have a significant role, although most probably restricted to the final step of chromatin condensation (coalescence of lamellae). The protamine P1 precursor contains a single serine residue in the sequence. The latter residue appears phosphorylated throughout the processing of the molecule (see Table III), undergoing dephosphorylation only in the fully ripe spermiogenic nuclei. Finally, the analysis of the internal repeats in M. brandaris protamines (Fig. 8) suggests a particular evolution for these proteins, which may have arisen from ancestral peptides having undergone a Lys4indel (insertion or deletion) together with some duplications. Ancestral peptides might be represented by R6(GK)8 tracts in protamine P1 and R6(GK)3K4(GK)5stretches in protamines P2 and P3, respectively.