A Sperm Nuclear Basic Protein from the Sperm of the Marine Worm Chaetopterus variopedatus with Sequence Similarity to the Arginine-Rich C-Termini of Chordate Protamine-Likes

Abstract

The sperm nuclear basic proteins (SNBPs) of the marine annelid worm Chaetopterus variopedatus have been shown previously to consist of a mixture of two SNBPs: histone H1-like (CvH1) and C.variopedatus protamine-like (CvPL). Here, we report the structural characterization of CvPL. The protein has a molecular weight of 8370.5 Da, a K/R ratio of 0.34, and a secondary structure, which are intermediate between those of protamine (P) and protamine-like (PL) SNBPs. The N-terminal sequence of CvPL shows a high extent of similarity with the arginine-rich C-terminal domain of chordate PL-type SNBPs. Furthermore, the protein binds to DNA in a similar fashion as vertebrate PLs and their own CvH1, but in a way that is different from that of the lysine-rich somatic H1 histones. We have experimentally determined the molar ratio CvH1:CvPL to be ∼1:6 in C. variopedatus sperm. Based on all of these, a model is proposed for the organization of the sperm chromatin by CvH1 and CvPL.

Introduction

D uring the postmeiotic maturation of sperm (spermiogenesis), the chromatin becomes increasingly condensed and transcriptionally inert (Braun, 2001). Highly compacted sperm chromatin is needed for a more hydrodynamic sperm head and may also protect the sperm's haploid genome from a physical and chemical damage. In sperm, DNA is associated with highly basic proteins that often differ from somatic histones. The sperm nuclear basic proteins (SNBPs) are very different from the nucleosomal proteins of somatic cells and can be grouped into three categories: protamine (P-type), protamine-like (PL-type), and histones (H-type) (Ausió, 1999). P- and PL-type SNBPs have higher amount of arginine residues than the somatic histones, which they replace, lending to their ability to more densely compact the spermatozoa DNA. The high levels of compaction are in part because arginine residues have a higher DNA-binding affinity than lysine and can bind tighter to DNA. The PL-type is an intermediate type between histones and P-type (Ausió, 1999). Although initially described and characterized in the bivalve mollusks (Ausió, 1986, 1992), they are pervasive across the animal kingdom, having been identified in such phylogenetically diverse organisms as Cnidarians (Rocchini et al., 1995, 1996), as well as in chordates (Saperas et al., 1992, 1994). It consists of a highly heterogeneous group of SNBPs that are rich in both arginine and lysine and are phylogenetically related to somatic-type linker histones (histone H1 family) (Eirín-López et al., 2006b). There is a surprising variability in the size and number of different PL components present in the mature sperm of even closely related organisms, especially considering that the common function of these proteins is the chromatin condensation. In mollusks, the PL proteins are generally subclassified into four basic categories according to their relative electrophoretic mobilities: PL-I, PL-II, PL-III, and PL-IV (Ausió, 1986). PL proteins are highly basic, with an arginine+lysine content of at least 35–50 mol% (8–30 mol% arginine and 22–30 mol% lysine), with the notable presence of cysteine in a number of them (Zhang et al., 1999). They can vary in molecular weight from 6500 in the case of PL-IV of the blue mussel, Mytilus californianus, up to 200,000 for the high molecular weight basic nuclear proteins of winter flounder (Watson and Davies, 1998). For the majority of organisms studied thus far, PL-type proteins coexist in the mature sperm with a full histone complement, amounting to ∼30%–40% of the total SNBPs (Giancotti et al., 1983).

However, the little information available to date on the PL protein-mediated organization of chromatin derives mainly from invertebrate organisms (Ausió and van Holde, 1987; Casas et al., 1993). In this sense, the marine worm Chaetopterus variopedatus is the only invertebrate described to date with the sperm chromatin organized by a histone H1-like (CvH1) (Salvati et al., 2008) and a molecule defined as a protamine (De Petrocellis et al., 1983). The latter contains arginine and lysine, and therefore it could now be considered a PL-type protein. However, its arginine+lysine content is higher than 50 mol% (56.1 mol%) with arginine and lysine corresponding to 41.7 mol% and 14.4 mol%, respectively, and it does not contain cysteine. On these bases, it cannot be classified into one of the four basic categories (PL-I, PL-II, PL-III, and PL-IV) described in mollusks. Further, this molecule forms stable oligomers, and we have already shown, by specific amino acid side chain modifications, that the oligomeric structure depends on anion-mediated lysine–arginine interactions (Piscopo et al., 1993).

In this article, we structurally characterize the SNBP protamine component of C. variopedatus. Quite unexpectedly, the partial N-terminal primary structure of the protein reveals a strong similarity to the C-terminal tail of the highly arginine-rich PLs that are present in the sperm of several chordates (Lewis et al., 2004a; Saperas et al., 2006). The secondary structure of this protein is reminiscent of that exhibited by SNBPs of the P-type and short PL proteins (Toniolo et al., 1979; Frehlick et al., 2007).

Materials and Methods

Materials

All chemical reagents were obtained from Sigma-Aldrich. Annelid worms C. variopedatus were kindly provided by the Zoological Station of Naples (Italy). Specimens were collected in the bay of Naples in July and August, when they are sexually active. Sperm cells were obtained by cutting the parapods of the worms as described in De Petrocellis et al. (1983).

Extraction and purification

Sperm cells of individual males were purified by several centrifugation washes in Millipore-filtered sea water. C. variopedatus protamine-like (CvPL) was prepared following the procedure of De Petrocellis et al. (1983) using acid extraction and CM-cellulose column chromatography, and purity was determined according to De Petrocellis et al. (1980) and reverse phase–high-performance liquid chromatography (RP-HPLC).

Extinction coefficient of CvPL and CvH1

The extinction coefficient of CvH1 and CvPL at 230 nm was determined by an amino acid analysis using Norleucine as an internal standard (Frehlick et al., 2007). The values thus obtained were 1.74 and 4.32 cm² mg⁻¹ for CvPL and CvH1, respectively.

Trypsin digestion

CvPL proteolitic digestion was carried out with trypsin (type III) (Sigma-Aldrich). Aliquots of 333 μL (1 mg/mL) of CvPL in 10 mM Tris-HCl buffer, pH 8.0, were incubated at room temperature with 5 μL of trypsin (40 ng/μL), for a final E/S ratio of 1:5000 (w/w), in presence or in absence of 1 M NaCl. Aliquots of the digestion mixture were collected at different times, immediately frozen in an alcohol and a solid-CO₂ bath, and stored in a freezer at −20°C, until used for the electrophoresis study (see below). An overnight digest in the presence of 1 M NaCl was carried out using 280 μg of CvPL, and the enzyme conditions were described above. The reaction was stopped by addition of a twofold (w/w) excess of tosyl-L-lysine chloromethyl ketone (TLCK). After addition of TLCK, the sample was diluted to a final NaCl concentration of 0.3 M and immediately subjected to HPLC fractionation.

Electrophoresis

Protein samples were analyzed on acetic acid–urea polyacrylamide gels (AU-PAGE). Polyacrylamide gels were prepared according to the method of Panyim and Chalkley (1969), but with 15.0% (w/v) acrylamide (acrylamide:bisacrylamide 30:0.8). Gels (83×58×0.75 mm) were run for 50 min at 150 V. High-resolution urea-acetic acid-PAGE (HrAU-PAGE) was carried out according to Frehlick et al. (2007). Proteins were fixed on gel; stained using 0.2% Amido Black, 30% methanol, and 10% acetic acid; and destained with 30% methanol and 10% acetic acid.

Tricine sodium dodecyl sulfate (SDS)-PAGE was carried out as described by Schägger (2006), using the stacking gel at 5%, the spacer gel at 10%, and the separating gel at 16.5% in 6 M urea. Aliquots of 20 μg of proteolytic mixture were incubated in the presence of a sample buffer in a ratio 2:1 (v/v) at 37°C for 15 min. Electrophoresis was carried out in a vertical gel apparatus mini-protean (20×20×0.12 mm) (Biorad); an initial voltage step of 100 V was applied until the sample has completely entered the stacking gel, and a next voltage step of 200 V was applied; at the end of the run, voltage was gradually increased to 300 V to shorten the total time of electrophoresis. Samples analyzed on Tricine SDS-PAGE were silver stained (Oakley et al., 1980).

HPLC analysis

RP-HPLC was carried out using a Vydac C₁₈ 4.6×250-mm column. In the case of the trypsin-resistant peptides, the column was eluted using a linear gradient from 0.1% trifluoroacetic acid to 40% acetonitrile in 80 min at a flow rate of 1 mL/min. Under these conditions, the peak corresponding to the trypsin-resistant peptide eluted at 12% acetonitrile. For the fractionation of CvH1 and CvPL, the column was eluted under the same conditions, but using a linear acetonitrile gradient to 60% in 60 min.

Far-ultraviolet circular dichroism spectroscopy

The circular dichroism analysis was carried out using a model J-710 spectropolarimeter (Jasco), which was equipped with a Neslab RTE-110 temperature controller (Neslab Instruments) and calibrated with a standard solution of (+)-10-camphorsulfonic acid. Circular dichroism measurement in the far-UV was performed on samples of CvPL at a protein concentration of 0.1 mg/mL in 50 mM sodium phosphate buffer, pH 7.0, using cuvettes of 0.1-cm optical path length (Hellma). Photomultiplier absorbance did not exceed 500 V in the spectral regions measured. Each spectrum was signal-averaged at least five times and smoothed with circular dichroism (CD) software Spectra Manager Ver. 1.53 (Jasco). All measurements were performed at the temperatures of 20°C under a nitrogen flow (3 L/h).

Mass spectrometry

Positive Reflectron Matrix-Assisted-Laser Desorption/Ionization (MALDI) spectra were recorded on a Voyager DE STR Pro instrument (Applied Biosystems). The Matrix-Assisted-Laser Desorption/Ionization matrices were prepared by dissolving 20 mg of 2,5-Dihydroxybenzoic acid (DHB) or 10 mg of α-cyano (α-cyano-4-hydroxycinnamic acid) in 1 mL of acetonitrile/0.1% trifluoroacetic acid (70:30 v/v). Typically, 1 mL of the matrix was applied to the metallic sample plate, and 1 mL of analyte was then added. Acceleration and reflector voltages were set up as follows: target voltage at 25 kV, first grid at 92% of target voltage, and delayed extraction at 500 ns. The individual spectra were analyzed using Applied Biosystems software.

Sequence analysis

Amino acid sequence determination was performed with the Applied Biosystems Procise 492 automatic sequencer, equipped with on-line detection of phenylthiohydantoin amino acids. Protein sequence analyses were performed using software available on the ExPASy Proteomics Server (www.expasy.org). The computational analysis of CvPL N-terminal sequence and other known protamine sequences was performed with multiple sequence alignment software such as ClustalX (Larkin et al., 2007) and SeaView (Gouy et al., 2009).

Plasmid DNA gel mobility retardation assay

The reported amounts (see Fig. 4) of C. variopedatus SNBP mixture were added to 600 ng of circular and linearized pGEM3 DNA in 30 μL of 89 mM Tris-borate buffer pH 8.0 and 2 mM ethylenediaminetetraacetic acid (TBE). After 10-min incubation at room temperature, the samples were loaded on 1.8% or 1% agarose gel in TBE. Electrophoretic conditions and visualization of DNA were carried out as described in Piscopo et al. (2010).

Results

CvPL has a molecular weight that is intermediate between that of P- and PL-types

CvPL has an oligomeric structure in solution that depends on the presence of both free lysine amino groups and arginine guanidino groups, as shown when comparing gel filtration patterns of the native to that of the modified molecules (Piscopo et al., 1993). Furthermore, as it is the characteristic of SNBPs of the P- and PL-types, the protein precipitates upon addition of SDS (De Petrocellis et al., 1983), making it difficult to estimate its molecular mass by SDS-PAGE. Therefore, the molecular weight of CvPL was carried out by mass spectrometry.

The molecular weight of 8370.5 Da determined by MALDI for the CvPL (Fig. 1A) is intermediate between those of P- and PL-types (Table 1). Moreover, the K/R ratio of this protein determined from its amino acid analysis composition exhibits a value of 0.34, which is similar to that of the ancestral protamine precursors observed in arginine-rich chordate PL-type SNBPs (Eirín-López and Ausió, 2009) (Table 1).

FIG. 1.

Structural characterization of CvPL. (A) Matrix-Assisted-Laser Desorption/Ionization spectrum for the determination of molecular weight. Spectrum was recorded in the 5,000–15,000 m/z range. The protonated molecular ion (M+H⁺) was the dominant species, indicating a MW of 8370.5 for CvPL. (B) Circular dichroism spectrum carried out in the far-ultraviolet range from 190 to 240 nm, in 50 mM sodium phosphate buffer, pH 7.0, at 20°C. CvPL, Chaetopterus variopedatus protamine-like.

Table 1.

Comparison Between Histone H1 and Protamine- and Protamine-Like-Type Sperm Nuclear Basic Proteins

Histone H1					Protamine					Protamine-like
	Mr (Da)	K ^*	R ^#	K/R		Mr (Da)	K ^*	R ^#	K/R		Mr (Da)	K ^*	R ^#	K/R
Mytilus californianus ^a	19,559	55	5	10.0	Doryteuthis opalescens ⁱ	10,650	4	45	0.09	Ostrea edulis ^q	21,932	37	23	1.61
H1					P					PL-I
Strongylocentrotus purpuratus ^b	22,043	59	5	11.8	Octopus vulgaris ^j	7429	0	39	-	Ostrea edulis ^r	13,007	29	4	7.25
H1					P					PL-II
Bos taurus ^c	22,022	63	3	21.0	Homo sapiens ^k	6823	0	24	-	Mytilus californianus ^s	11,653	22	28	1.00
CT–H1					P1					PL-III
Gallus gallus ^d	20,602	44	22	2.0	Mus musculus ^l	6958	3	28	0.11	Mytilus californianus ^t	22,352	61	14	4.36
H5					P1					PL-II/PL-IV
Homo sapiens ^e	21,711	57	4	14.2	Bos taurus ^m	6758	0	26	-	Chaetopterus variopedatus ^u	8370	-	-	0.34
H1.1					P1					PL
Homo sapiens ^f	20,863	56	6	9.33	Oncorhynchus keta ⁿ	4236	0	21	-	Chaetopterus variopedatus ^v	(?)	-	-	2.03
H1⁰					Salmine					H1+PL(?)
Homo sapiens^g	22,356	41	13	3.15	Clupea pallasii ^o	4165	0	21	-	Styela plicata ^w	21,603	28	58	0.48
H1x					Clupeine					P1
Homo sapiens^h	22,019	40	14	2.86	Thunnus thynnus ^p	4641	0	22	-	Styela plicata ^x	11,539	16	49	0.33
H1t					Thinnin					P2
										Styela montereyensis ^y	22,138	32	56	0.57
										P1
										Branchiostoma floridae ^z	12,068	27	27	1.00
										PL
										Mullus surmuletus ^α	20,391	41	37	1.11
										PL-I

The values relative to Cv PL and Cv H1+Cv PL are in bold.

Number of lysine residues; ^#number of arginine residues; ^aCAC94904.1; ^bNP_999714.1; ^cAAI22614.1; ^dNP_001038138.1; ^eNP_005316.1; ^fAAH00145.1; ^gNP_006017.1; ^hNP_005314.2; ⁱAAP32169.1; ^jP83214.1; ^kAAC50486.1; ^lNP_038665.1; ^mAAA30741.1; ⁿB02669; ^oP69011.1; ^pTYTUZ2; ^qQ86QH9.2; ^rQ86QI0.1; ^sABC18201.1; ^tABC18200.1; ^uB3EWJ1; ^vThis publication; ^w(Lewis et al., 2004a); ^x(Lewis et al., 2004a); ^yAQ01227.1; ^zABY89927.1; ^αQ08GK9.2.

CvPL has a CD spectrum similar to that observed for P- and PL-types

The purified CvPL was structurally characterized by circular dichroism. The CvPL spectrum carried out under pH and ionic-strength buffer conditions close to physiological values (Fig. 1B) showed similarity with spectra obtained for SNPBs from other PL- and P-types, including human protamines (Ausió and Subirana, 1982a, 1982b; Gatewood et al., 1990). With a negative band at about 200 nm, this spectrum is very close to those that have long been observed for charged polypeptides presumed to be in the so-called random coil conformation (Johnson, 1988). The secondary structure estimation using the method described by Yang (Chen and Yang, 1971) indicated the presence of a high content in random (64.6%) and β-turn (28.0%) structures, the absence of α-helix, and a slight content in β-sheet (7.4%). Generally, in solution, most protamines have CD spectra similar to the CD presumed to correspond to denatured proteins (Toniolo et al., 1979). Although these proteins could have relatively well-defined topologies, they do not have the regular repeating pattern or defined geometry as those observed in any of the canonical secondary structure features found in other proteins (Janes and Wallace, 2009).

Therefore, like most SNBPs of P- and PL-types, CvPL appears to belong to the family of intrinsically disordered proteins (Hansen et al., 2006) that, while exhibiting a low secondary structure in solution, they may acquire an ordered secondary structure conformation upon interacting with DNA as it has been shown for the N- and C-terminal tails of H1 histone (Vila et al., 2001; Roque et al., 2005).

CvPL has an electrophoretic mobility and an amino acid sequence similar to PL-type and consists of discrete short domains that are resistant to trypsin digestion under high ionic strength conditions

Figure 2A shows a HrAU-PAGE of CvPL in comparison to other SNBPs from invertebrates and chordates. Under these electrophoretic conditions, CvPL migrates in the gel in the region between that of PL-II and PL-III types of bivalve mollusks (Ausió, 1986), and far from the region of P-type proteins, suggesting its closeness to SNBPs of the PL-type family. In agreement with this, the N-terminal sequence determined for CvPL (which comprises ∼55% of its whole sequence) (Fig. 2B) did not show similarity with the known P-type sequences (data not shown), while it appeared to have similarity with the C-terminal domain of some PL-type proteins (Fig. 2B). In particular, and quite unexpectedly, the closest similarity was with those from the PLs of the tunicates Styela plicata and S. montereyensis (Lewis et al., 2004a) and the fish Mullus surmuletus (Saperas et al., 2006). All of these suggest a potential structural relatedness of CvPL to the PL-type of invertebrate and chordate SNBPs.

FIG. 2.

CvPL N-terminal is related to the C-terminal end of arginine-rich PL-type SNBPs. (A) HrAU [urea (2.5 M)–acetic acid (5%), polyacrylamide (15%)] gel electrophoresis of the SNBPs extracted from different organisms. Lane 1: PL from sperm of C. variopedatus (CvPL). Lane 2: Histone H1 from C. variopedatus (CvH1). Lane 3: SNBPs from C. variopedatus (marine worm). Lane 4: SNBPs from Mytilus edulis (mussel) (Ausió and Subirana, 1982b). Lane 5: SNBPs from Paracentrotus lividus (sea urchin). Lane 6: SNBPs from Branchiostoma floridae (amphioxus, lancelet) (Eirín-Lopez et al., 2008). Lane 7: SNBPs from Styela montereyensis (tunicate) (Lewis et al., 2004a). Lane 8: SNBPs from M. surmuletus (red mullet, fish) (Saperas et al., 2006). Lane 9: salmine (protamine from salmon, Oncorhynchus keta). Arrowhead indicates CvPL. CM: histones from chicken erythrocyte used as a marker. The roman numerals refer to the PL protein designations (PL-I, PL-II, PL-III, and PL-IV) (Ausió, 1986). P: P-type SNBPs. Direction of electrophoresis was from top (+) to bottom (−). (B) Amino acid sequence alignment of SNBPs from Ostrea edulis (PL-I, GI: 44887996) (Agelopoulou et al., 2004); Mytilus californianus (PL-II/PL-IV, GI: 83416329, PL-III GI: 83416331) (Lewis et al., 2004a); C. variopedatus PL (this article); M. surmuletus (PL-I, GI: 118572479) (Saperas et al., 2006); Styela montereyensis (P1, GI: 33243113) (Lewis et al., 2004a); Styela plicata (P1, P2) (Lewis et al., 2004a); and Branchiostoma floridae (P, EU 271675) (Eirín-López et al., 2008). The boxes highlight the regions of sequence identity, and the purple box highlights the high extent of similarity between CvPL and M. surmuletus PL-I. Alignment was generated using Seaview and ClustalX. (C) HPLC separation of CvPL and CvH1 extracted from C. variopedatus sperm. SNBPs, sperm nuclear basic proteins; HrAU, high-resolution urea–acetic acid; HPLC, high performance liquid chromatography.

As shown in Figure 3A, the CvPL protein is quite unique in that, in the presence of 1 M NaCl, it exhibits the presence of short protein domains, with an average molecular weight of 850 Da (Fig. 3B), that are resistant to trypsin digestion. The protein is completely digested under the same conditions in the absence of NaCl. Mass spectrometry of the HPLC-purified-resistant peptide reveals the presence of a complex mixture of peptides of molecular weight at 820–870 Da as well as other smaller components at 650 Da. The presence of these small salt-dependent trypsin-resistant fragments, in this highly basic protein, is intriguing, but is possibly related to the lysine–arginine-mediated oligomerization exhibited by CvPL (Piscopo et al., 1993) that upon association probably results in small segments (6–8 amino acids) that are refractory to trypsin digestion.

FIG. 3.

CvPL contains salt-dependent trypsin-resistant domains. (A) Acetic acid–urea (AU)-PAGE analysis of the time course of trypsin digestion of CvPL carried out in the presence of 1 M NaCl (lanes 2–6) or in its absence (lanes 7–8). The protein was digested at room temperature. Lane 1: undigested CvPL. The digestion times are indicated on top of the lanes (O.N., overnight). Digestion without salt at 60 min, O.N., lanes 7 and 8, respectively. (B) Tricine SDS-PAGE analysis of the time course of trypsin digestion of CvPL carried out in the presence of 1 M NaCl for the times indicated in the figure. Three peptides (972, 1684, and 4000 Da) were used as markers for the determination of the molecular weight. SDS, sodium dodecyl sulfate; PAGE, polyacrylamide gel.

SNBPs in C. variopedatus consist of a mixture of approximately six molecules of CvPL per each molecule of CvH1

The relationship of CvPL to PL-types is intriguing, especially in regard to the potential chromatin organization resulting from its interaction and that of CvH1 with DNA in the sperm of C. variopedatus. An insight into this question can be gained from the determination of the molar ratio between these two proteins. We used two different methods for such determination. In the first approach, the ratio was determined by scanning densitometry of the two electrophoretic bands corresponding to these two proteins (see Fig. 2A, lane 3). The analysis was performed using an electrophoretic titration with different amounts of the purified CvPL and CvH1 (Fig. 2A, lanes 1 and 2). A value of 5.6 molecules of CvPl per molecule of CvH1 was obtained in this way. In a second approach, the areas of the peaks corresponding to each of the proteins in the HPLC chromatogram (shown in Figure 2C) were determined. These values were then used in conjunction with the molecular weights (CvPL: 8370 Da; CvH1 20,270 Da) of the proteins and their corresponding extinction coefficients at 230 nm (determined as indicated in section 2.3) to calculate a molar ratio of 5.8 molecules of CvPL per molecule of CvH1. Therefore, it appears from these two independent methods that ∼6 molecules of CvPL coexist with one molecule of CvH1 in the sperm chromatin of C. variopedatus.

C. variopedatus SNBPs bind to DNA in the same way as CvH1 and differently from somatic histone H1

We have previously studied the interaction of CvH1 with linear and circular DNA (Piscopo et al., 2010), and we have shown that CvH1 binds in a way that is different from canonical lysine-rich histone H1 (such as calf thymus H1) (Cole, 1987). We have also shown that this differential binding is dependent on the arginine presence. If CvH1 and CvPL are structurally related to commonly organize the C. variopedatus sperm chromatin, one would expect that these two proteins would bind to DNA in a similar fashion. Therefore, we decided to study the interaction of the C. variopedatus SNBP mixture (see Fig. 2A, lane 3).

Figure 4 shows the interaction of C. variopedatus SNBP mixture with linear and circular pGEM3 DNA, in comparison to CvH1 and calf thymus H1 (Piscopo et al., 2010). As it can be seen in Fig. 4C, the mixture of C. variopedatus SNBPs binds to DNA in a way that is almost indistinguishable from that of CvH1, reinforcing the hypothesis that these proteins are probably related. Interestingly, the linear DNA-binding saturation takes place at a (w/w) protein/DNA ratio of 1.2 for both CvH1 and C. variopedatus SNBP mixture. The molecular mass per positive charge of CvH1 and of C. variopedatus SNBPs (CvH1-CvPL considered as a joint molecule) determined from their amino acid analysis composition (De Petrocellis et al., 1983) was 288.5 and 242, respectively. Since the complete shift takes place at a 1.2 protein/DNA (w/w) ratio, this corresponds very closely to charge saturation [1 mole positive charge/1 mole of nucleotide (negative charge)] as it has been observed with Xenopus laevis SNBPs (Frehlick et al., 2007).

FIG. 4.

Interaction of C. variopedatus SNBPs with DNA. (A) Gel shift analysis of the interaction of the C. variopedatus SNBP mixture with linear pGEM3 DNA (LP) on 1.8% agarose gel. The numbers in the upper part of the figure denote the (w/w) SNBP/DNA ratios. (B) Same as in (A), but using circular pGEM3 DNA (CP). (C) Percentage of mobility of linear pGEM3 DNA in the presence of increasing amounts of the C. variopedatus SNBP mixture (black diamonds) in comparison to C. variopedatus H1 (light-gray squares) and calf thymus (C.T.) histone H1 (dark-gray triangles) (Piscopo et al., 2010) expressed as a w/w protein/DNA ratio. The insert shows a HrAU-PAGE of C. variopedatus SNBPs (lane 1), CvH1 (lane 2), and calf thymus H1 (H1 C.T.) (lane 3).

Discussion

CvPL has similarity to arginine-rich C-terminal of chordate PL-types

Annelida and Mollusca derive from a common protostome ancestor. The Polychates, among the Annelida phylum classes, have many typical features of ancestral annelids. Then, the general opinion is that the worms are closer to the Mollusca phylum. The nuclei of the sperm of the marine bivalve mollusks contain a complex protein mixture in which PL-type and histone-like proteins coexist with a complete set of somatic histones (Ausió, 1986; Ausió et al., 1987). The nuclear protein composition consists of about 20% histones and about 80% sperm-specific protein component. The persistence of a small amount of the somatic-type histones seems to be a common feature of sperm of the class Bivalvia (Ausió, 1986) that contains only PL-type proteins. Evidence also exists for the coexistence of P-type proteins and histones in some mammals (Ausió et al., 2011), including humans (Hammoud et al., 2009). The sperm chromatin of C. variopedatus does not contain somatic-type histones, but only CvPL and a sperm histone H1 (De Petrocellis et al., 1983). However, the coexistence or not with remnant somatic histones at the end of spermiogenesis is not a good criterion for the classification of SNBPs; the CvPL is highly likely a PL-type SNBP. Moreover, the electrophoretic mobility in HrAU-PAGE of CvPL (see Fig. 2A) as well as its primary sequence (Fig. 2B) suggests that this protein shares features with PL-type proteins. In fact, the mobility of the CvPL is between those of PL-II and PL-III subtypes of bivalve mollusks.

There are basically two main subtypes of PLs: those whose structure consists of a winged-helix fold domain (WHD) (Ramakrishnan et al., 1993), like PL-I from the surf clam Spisula solidissima (mollusk) (Ausió and Subirana, 1982a; Lewis et al., 2004b), and those that lack this domain, like Mytilus (a bivalve mollusk) PL-III (Eirín-López et al., 2008). Although CvPL is certainly a PL-type protein, its high arginine content (see Table 1) reveals some common features with P-type proteins such as it happens in some chordate PLs (Ausió et al., 1997; Lewis et al., 2004a; Saperas et al., 2006). In fact, based on the compositional amino acid analysis, it was hypothesized by Subirana et al. (1973) that P-type had evolved from a primitive somatic-like histone precursor via a protamine-like intermediate through a mechanism of vertical evolution. This is supported by the observation that these organisms that replace histones with P-type in the mature sperm are always found at the furthermost tips of the evolutionary branches (Ausió, 1999), whereas sperm-specific germinal histones are found in the sperm of more primitive organisms such as the sponge Neofibularia (Ausió et al., 1997) and the sea urchin Paracentrotus lividus (De Petrocellis et al., 1980). Recent evidence has identified histone H1 as the putative precursor of protamines (Lewis et al., 2004a; Eirín-López and Ausió, 2009). A detailed systematic analysis of the distribution of SNBPs in fish provided additional support for the vertical evolution hypothesis of SNBPs by revealing that the sporadic distribution of P-type was not random and could be traced phylogenetically (Saperas et al., 1994). A recent publication has provided an important insight into potential molecular mechanisms by which specialized lysine-rich PLs of low-molecular weight could have evolved into the arginine-rich SNBPs of the P-type (Lewis et al., 2004a).

What is really surprising is the similarity observed between CvPL and the C-terminal of the arginine-rich PL of some chordates (Fig. 2B), both in sequence and in electrophoretic mobility (Fig. 2A, compare lanes 1, 6, and 8). The potential implications from this are that the lysine-to-arginine transition observed in the PL-types in deuterostomes (Eirín-López and Ausió, 2009) has also taken place in a similar fashion in protostomes likely as a result of a parallel evolution.

CvPL/CvH1 and the structure of the sperm chromatin

How can a histone H1 and a relatively short PL protein (Fig. 2A, lane 3) organize the sperm chromatin of C. variopedatus? The presence of only two proteins with a low secondary structure poses the problem as to how these can contribute to the organization of the chromatin in the sperm of C. variopedatus. Another important related question is: Are these SNBPs deposited on DNA sequentially or independently during spermiogenesis? A similar situation to that observed in C. variopedatus is observed in the mussel (Mytilus sp.) in which a histone H1-like (PL-II/PL-IV) coexists with a PL-III of smaller molecular weight (see Fig. 2A, lane 4). PL-III is encoded by its own gene (Ruiz-Lara et al., 1993; Eirín-López et al., 2006a), and it is sequentially incorporated onto the sperm chromatin after initial deposition PL-II/PL-IV during spermiogenesis (Bloch, 1966). In other instances, a single arginine-rich histone H1-like related protein (PL-I) consisting of the characteristic WHD is present in the sperm as is the case of the fish M. surmuletus (Fig. 2A, lane 8) (Saperas et al., 2006). This protein, in some instances, is post-translationally cleaved (Fig. 2B), presumably after deposition onto DNA into two fractions: one consisting of the WHD and another one corresponding to the highly arginine-rich C-terminal domain as in the tunicates from the genus Styela (Fig. 2A, lane 7) (Saperas et al., 1992). The need for the presence of an SNBP component with a WHD in all these organisms is not clear, but it is presumably the result of the mechanism involved in the displacement of histones by these SNBPs during spermiogenesis. The presence of a WHD may result from the need of targeting them to the DNA exit and entry sites of the nucleosomes whose histones are being displaced in order to maintain the DNA superhelicity (Ausió, 1995). Indirect evidence for this comes from the experimentally observed chromatin organization in the sperm of the PL-containing fish M. surmuletus (Saperas et al., 2006). Therefore, it is likely that C. variopedatus SNBPs, consisting of an H1 and a PL protein, organize the sperm chromatin in a similar fashion as other PL-containing sperm. The high similarity of CvPL to the C-terminal of M. surmuletus PL-I comes in support of this notion. Furthermore, both CvH1 and CvPL bind to DNA in a way that is very similar to that of vertebrate SNBPs (Frehlick et al., 2007) and very different from that of somatic H1 histone (Fig. 4) (Piscopo et al., 2010). The reasons for this observed difference could reflect the higher ability of arginine versus lysine (or their low K/R ratio [Piscopo et al., 2010], see Table 1) to form hydrogen bonding with the DNA (Ausió, 1995) in addition to the electrostatic interactions (Ichimura et al., 1982; Vila et al., 2001), achieving a more efficient chromatin packing favored by the sperm of highly evolved organisms with internal fertilization (Kasinsky et al., 1985) in both the protostome and deuterostome branches. Another contributing factor could be the observed potential of CvPL that could also be able to form aggregated structures. Indeed, we have reported that the oligomeric state of CvPL depends on the presence of both lysine amino groups and arginine guanidine groups (Piscopo et al., 1993) that also determine the DNA-binding mode (Piscopo et al., 2010).

The answer to the two questions posed at the beginning of this section will ultimately depend on whether CvH1 and CvPL share a precursor relationship (Fig. 5A) as that observed in tunicates or whether they are the product of two independent genes as in the case of PL-II/PL-IV and PL-III in Mytilus.

FIG. 5.

Two hypothetical models to explain the relationship between CvPl and CvH1 and their chromatin deposition. (A) CvPL and CvH1 are the post-translational cleavage products of a common protein precursor as it is observed in PL-II/PL-IV of Mytilus (Eirín-López et al., 2006a) or in P1 and P2 in tunicates (Eirín-López et al., 2006b). (B) CvPL is a PL product of an independent gene as it occurs with PL-III of Mytilus (Eirín-López et al., 2006a). In the model proposed here, 6 molecules of CvPL and one molecule of CvH1 would replace the canonical nucleosome histones during the transition from somatic to sperm-specific chromatin organization.

In the first scenario (Fig. 5A), CvH1 and CvPL will be the products of the protein precursor after a putative post-translational cleavage as it has been observed for the PL-II and PL-IV in the mussel Mytilus (Carlos et al., 1993) or for P1 and P2 in the tunicates of the genus Styela (Fig. 2A, lane 7) (Lewis et al., 2004a). It is important to notice that in this latter group, a transition leading from the characteristic lysine-rich nature of the histone H1 C-terminal tail to arginine was observed. Such transition has been ascribed to one of the potential molecular mechanisms involved in the transition from PL- to P-types starting from a primitive H1 histone precursor (Lewis et al., 2004a). Given the aforementioned similarities between the C-terminal tails of vertebrate PLs (Fig. 2B) and those of CvPL, it is possible that a similar mechanism may have occurred in lower invertebrates.

Alternatively (Fig. 5B), CvH1 and CvPL could be the result of two independent genes related to an ancestral H1 histone gene as it is the case of PL-II/PL-IV and PL-III of Mytilus (Fig. 2A, lane 4) (Erín-López et al., 2006a). In an attempt to elucidate whether this could be the case, the molar ratio between these two proteins, as they are obtained from acid extraction of the sperm chromatin, was determined. The analysis indicated that there are ∼6 molecules of CvPL per each molecule of CvH1. This result clearly supports this second alternative and suggests a sperm chromatin organization pattern such as the one shown in Fig. 5B. In this model, the interaction of these proteins with DNA will be mainly electrostatic as supported by their in vitro interaction analysis (Fig. 4). Taking into consideration their molecular mass as well as their amino acid composition (De Petrocellis et al., 1983), it is possible to estimate that such arrangement would correspond to ∼340 positively charged amino acids for every nucleosome. Assuming an average value of 200 bp of DNA per nucleosome in the somatic chromatin at the onset of spermiogenesis, this corresponds to a value of 85% charge neutralization, a value that is very close to that observed for other P- and PL-type sperm chromatin.

The sequence of the genes for CvPL and CvH1 and how these two proteins are sequentially deposited onto DNA during the replacement of somatic histones are the objectives of future research in our lab.

Conclusion

We report evidence of a peculiar sperm protein from the annelid C. variopedatus. The electrophoretic mobility in HrAU-PAGE of CvPL as well as its primary structure suggests that this protein shares features with PL-type proteins. Because of this evidence, we could classify CvPL as a PL-type protein. However, its high arginine content led us to suppose that this protein shares some common features with P-type proteins, such as it happens in some chordate PLs.

It is now well established that P-type proteins had evolved from a primitive somatic-like histone precursor via a PL-type intermediate through a mechanism of vertical evolution. The similarity observed between CvPL and the C-terminal of the arginine-rich PL proteins of some chordates, both in sequence and in electrophoretic mobility, is the possible evolutionary evidence that the lysine-to-arginine transition observed in the PL-type in deuterostomes has also taken place in a similar way in protostomes as a result of parallel evolution.

Both CvH1 and CvPL bind to DNA in a way that is very similar to that of vertebrate SNBPs and different from that of somatic histones H1 (Fig. 4). The reasons for this observed difference could reflect the higher ability of arginine versus lysine to form hydrogen bonding with the DNA, in addition to the electrostatic interactions. In addition, a particular chromatin organization must be present in the sperm of C. variopedatus that is not found in other organisms. A histone H1 and a relatively short PL protein are involved (Fig. 5). It is likely that C. variopedatus SNBPs organize the sperm chromatin in a similar fashion as other PL-containing sperm (Lewis and Ausió, 2002).

Footnotes

Acknowledgments

We thank Dr. Maria Malanga, prematurely passed away, in 2011 November, for providing the peptides used in the Tricine SDS-PAGE as a molecular weight marker.

Disclosure Statement

No competing financial interests exist.

References

Agelopoulou

, Cary

P.D.

, Pataryas

, Aleporou-Marinou

, Crane-Robinson

2004. The sperm-specific proteins of the edible oyster (European flat oyster (Ostrea edulis)) are products of proteolytic processing. Biochim Biophys Acta, 1676:12–22.

Ausió

1986. Structural variability and compositional homology of the protamine-like components of the sperm from bivalve molluscs. Comp Biochem Physiol, 85:439–449.

Ausió

1992. Presence of a highly specific histone H1-like protein in the chromatin of the sperm of the bivalve mollusks. Mol Cell Biochem, 115:163–172.

Ausió

1995. Histone H1 and the Evolution of the Nuclear Sperm Specific Proteins. 166. Memoires de Museum National d'Histoire Naturelle: Paris.

Ausió

1999. Histone H1 and evolution of sperm nuclear basic proteins. J Biol Chem, 274:31115–31118.

Ausió

, Brewer

L.R.

, Frehlick

2011. Genome organization by vertebrate sperm nuclear basic proteins (SNBPs) Epigenetics and Reproduction. Rousseaux

, Khochbin

Springer: NY.

Ausió

, Subirana

J.A.

1982a. A high molecular weight nuclear basic protein from the bivalve mollusc Spisula solidissima. J Biol Chem, 257:2802–2805.

Ausió

, Subirana

J.A.

1982b. Nuclear proteins and the organization of chromatin in spermatozoa of Mytilus edulis. Exp Cell Res, 141:39–45.

Ausió

, Toumadje

, McParland

, Becker

R.R.

, Johnson

W.C.

, van Holde

K.E.

1987. Structural characterization of the trypsin-resistant core in the nuclear sperm-specific protein from Spisula solidissima. Biochemistry, 26:975–982.

10.

Ausió

, van Holde

K.E.

1987. A dual chromatin organization in the sperm of the bivalve mollusc Spisula solidissima. Eur J Biochem, 165:363–371.

11.

Ausió

, Van Veghel

M.L.

, Gomez

, Barreda

1997. The sperm nuclear basic proteins (SNBPs) of the sponge Neofibularia nolitangere: implications for the molecular evolution of SNBPs. J Mol Evol, 45:91–96.

12.

Bloch

D.P.

1966. Cytochemistry of the histones. Protoplasmatologia, 5:1a–56.

13.

Braun

R.E.

2001. Packaging paternal chromosomes with protamine. Nat Genet, 28:10–12.

14.

Carlos

, Hunt

D.F.

, Rocchini

, Arnott

D.P.

, Ausió

1993. Post-translational cleavage of a histone H1-like protein in the sperm of Mytilus. J Biol Chem, 268:195–199.

15.

Casas

M.T.

, Ausió

, Subirana

J.A.

1993. Chromatin fibers with different protamine and histone compositions. Exp Cell Res, 204:192–197.

16.

Chen

Y.H.

, Yang

J.T.

1971. A new approach to the calculation of secondary structures of globular proteins by optical rotatory dispersion and circular dichroism. Biochem Biophys Res Commun, 44:1285–1291.

17.

Cole

R.D.

1987. Microheterogeneity in H1 histones and its consequences. Int J Pept Protein Res, 30:433–449.

18.

De Petrocellis

, De Petrocellis

, Lancieri

, Geraci

1980. Species specificity and individual variability of sea urchin sperm H2B histones. Cell Differ, 9:195–202.

19.

De Petrocellis

, Parente

, Tomei

, Geraci

1983. An histone H1 and a protamine organize the sperm chromatin of the marine worm C. variopedatus. Cell Differ, 12:129–135.

20.

Eirín-López

J.M.

, Ausió

2009. Origin and evolution of chromosomal sperm proteins. Bioessays, 31:1062–1070.

21.

Eirín-López

J.M.

, Frehlick

L.J.

, Ausió

2006b. Protamines, in the footsteps of linker histone evolution. J Biol Chem, 281:1–4.

22.

Eirín-López

J.M.

, Frehlick

L.J.

, Chiva

, Saperas

, Ausió

2008. The sperm proteins from amphioxus mirror its basal position among chordates and redefine the origin of vertebrate protamines. Mol Biol Evol, 25:1705–1713.

23.

Eirín-López

J.M

, Lewis

J.D.

, Howe le

, Ausió

2006a. Common phylogenetic origin of protamine-like (PL) proteins and histone H1: evidence from bivalve PL genes. Mol Biol Evol, 23:1304–1317.

24.

Frehlick

L.J.

, Prado

, Calestagne-Morelli

, Ausió

2007. Characterization of the PL-I-related SP2 protein from Xenopus. Biochemistry, 46:12700–12708.

25.

Gatewood

J.M.

, Schroth

G.P.

, Schmid

C.W.

, Bradbury

E.M.

1990. Zinc-induced secondary structure transitions in human sperm protamines. J Biol Chem, 265:20667–20672.

26.

Giancotti

, Russo

, Gasparini

, Serrano

, Del Piero

, Thorne

A.W.

, Cary

P.D.

, Crane-Robinson

1983. Proteins from the sperm of the bivalve mollusc Ensis minor. Co-existence of histones and a protamine-like protein. FEBS, 136:509–516.

27.

Gouy

, Guindon

, Gascuel

2009. SeaView version 4: a multiplatform graphical user interface for sequence alignment and phylogenetic tree building. Mol Biol Evol, 27:221–224.

28.

Hammoud

S.S.

, Nix

D.A.

, Zhang

, Purwar

, Carrell

D.T.

, Cairns

B.R.

2009. Distinctive chromatin in human sperm packages genes for embryo development. Nature, 460:473–478.

29.

Hansen

J.C.

, Lu

, Ross

E.D.

, Woody

R.W.

2006. Intrinsic protein disorder, amino acid composition, and histone terminal domains. J Biol Chem, 281:1853–1856.

30.

Ichimura

, Mita

, Zama

1982. Essential role of arginine residues in the folding of deoxyribonucleic acid into nucleosome cores. Biochemistry, 21:5329–5334.

31.

Janes

R.W.

, Wallace

B.A.

2009. An introduction to circular dichroism and synchroton radiation circular dichroism spectroscopyIOS Press1:1–18.

32.

Johnson

W.C.

1988. Secondary structure of proteins through circular dichroism spectroscopy. Annu Rev Biophys Phys Chem, 17:145–166.

33.

Kasinsky

H.E.

, Mann

, Lemke

, Huang

S.Y.

1985. Diversity of Sperm Basic Chromosomal Proteins in Vertebrates: A Phyologenetic Point of View. Reeck

G.R.

, Goodwin

G.H.

, Puigdomenech

Plenum Press: New York, 333–359.

34.

Larkin

M.A.

, Blackshields

, Brown

N.P.

, Chenna

, McGettigan

P.A.

, McWilliam

, Valentin

, Wallace

I.M.

, Wilm

, Lopez

2007. Clustal W and Clustal X version 2.0. Bioinformatics(Oxford, England).23:2947–2948.

35.

Lewis

J.D.

, Ausió

2002. Protamine-like proteins: evidence for a novel chromatin structure. Biochem Cell Biol, 80:353–361.

36.

Lewis

J.D.

, McParland

, Ausió

2004b. PL-I of Spisula solidissima, a highly elongated sperm-specific histone H1. Biochemistry, 43:7766–7775.

37.

Lewis

J.D.

, Saperas

, Song

, Zamora

M.J.

, Chiva

, Ausió

2004a. Histone H1 and the origin of protamines. Proc Natl Acad Sci USA, 101:4148–4152.

38.

Oakley

B.R.

, Kirsch

D.R.

, Morris

N.R.

1980. A simplified ultrasensitive silver stain for detecting proteins in polyacrylamide gels. Anal Biochem, 105:361–363.

39.

Panyim

, Chalkley

1969. High resolution acrylamide gel electrophoresis of histones. Arch Biochem Biophys, 130:337–346.

40.

Piscopo

, Conte

, Di Paola

, Conforti

, Rana

, De Petrocellis

, Fucci

, Geraci

2010. Relevance of arginines in the mode of binding of H1 histones to DNA. DNA Cell Biol, 29:339–347.

41.

Piscopo

, Tomei

, De Petrocellis

, Geraci

1993. Anion-mediated lysine-arginine interaction. Evidence in Chaetopterus variopedatus sperm protamine. FEBS Lett, 334:125–127.

42.

Ramakrishnan

, Finch

J.T.

, Graziano

, Lee

P.L.

, Sweet

R.M.

1993. Crystal structure of globular domain of histone H5 and its implications for nucleosome binding. Nature, 362:219–223.

43.

Rocchini

, Marx

R.M.

, Carosfeld

J.S.

, Kasinsky

H.E.

, Rosenber

, Sommer

, Ausió

1996. Replacement of histones by histone H1-like proteins during spermiogenesis in Cnidaria: evolutionary implications. J Mol Evol, 42:240–246.

44.

Rocchini

, Zhang

, Ausió

1995. Two highly specialized histone H1 proteins are the major chromosomal proteins of the sperm of the sea anemone Urticina (Tealia) crassicornis. Biochemistry, 34:15704–15712.

45.

Roque

, Iloro

, Ponte

, Arrondo

J.L.

, Suau

2005. DNA-induced secondary structure of the carboxyl-terminal domain of histone H1. J Biol Chem, 280:32141–32147.

46.

Ruiz-Lara

, Prats

, Casas

M.T.

, Cornudella

1993. Molecular cloning and sequence of a cDNA for the sperm-specific protein phi 1 from the mussel Mytilus edulis. Nucleic Acids Res, 21:2774.

47.

Salvati

, Conforti

, Conte

, Matassa

D.S.

, Fucci

, Piscopo

2008. Self-association of Chaetopterus variopedatus sperm histone H1-like. Relevance of arginine content and possible physiological role. Acta Biochim Pol, 55:701–706.

48.

Saperas

, Ausió

, Lloris

, Chiva

1994. On the evolution of protamines in bony fish: alternatives to the “retroviral horizontal transmission” hypothesis. J Mol Evol, 39:282–295.

49.

Saperas

, Chiva

, Ausió

1992. Purification and characterization of the protamines and related proteins from the sperm of a tunicate. Comp Biochem Physiol, 103 B:969–974.

50.

Saperas

, Chiva

, Casas

M.T.

, Campos

J.L.

, Eirin-Lopez

J.M.

, Frehlick

L.J.

, Prieto

, Subirana

J.A.

, Ausió

2006. A unique vertebrate histone H1-related protamine-like protein results in an unusual sperm chromatin organization. FEBS J, 273:4548–4561.

51.

Schägger

2006. Tricine-SDS-PAGE. Nat Protoc, 1:16–22.

52.

Subirana

J.A.

, Cozcolluel

, Palau

, Unzeta

1973. Protamines and other basic proteins from spermatozoa of molluscs. Biochim Biophys Acta, 317:364–379.

53.

Toniolo

, Bonora

G.M.

, Marchiori

, Borin

, Filippi

1979. Protamines. II. Circular dichroism study of the three main components of clupeine. Biochim Biophys Acta, 576:429–439.

54.

Vila

, Ponte

, Collado

, Arrondo

J.L.

, Suau

2001. Induction of secondary structure in a COOH-terminal peptide of histone H1 by interaction with the DNA: an infrared spectroscopy study. J Biol Chem, 276:30898–30903.

55.

Watson

C.E.

, Davies

P.L.

1998. The high molecular weight chromatin proteins of winter flounder sperm are related to an extreme histone H1 variant. J Biol Chem, 273:6157–6162.

56.

Zhang

, Lewis

J.D.

, Ausió

1999. Cysteine-containing histone H1-like (PL-I) proteins of sperm. Mol Reprod Dev, 54:402–409.