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Sperm nuclear basic proteins of tunicates and the origin of protamines.

Abstract. Sperm nuclear basic proteins (SNBPs) are the chromosomal proteins that are found associated with DNA in sperm nuclei at the end of spermiogenesis. These highly specialized proteins can be classified into three major types: histone type (H-type), protamine-like type (PL-type), and protamine type (P-type). A hypothesis from early studies on the characterization of SNBPs proposed a mechanism for the vertical evolution of these proteins that involved an H1 [right arrow] PL [right arrow] P transition. However, the processes and mechanisms involved in such a transition were not understood. In particular, it was not clear how a molecular transition from a lysine-rich protein precursor (H1 histone) to the arginine-rich protamines might have taken place. In deuterostomes, the presence of SNBPs of the H-type in echinoderms and of protamines in the higher phylogenetic groups of vertebrates had long been known. The initial work on the characterization of tunicate SNBPs attempted to define the types and range of SNBPs that characterize this phylogenetically intermediate group. It was found that tu-nicate SNBPs belong to the PL-type. In this work we discuss how the study of SNBPs in the tunicates has been key to providing support to the H1 [right arrow] PL [right arrow] P transition. Most significantly, it was in tunicates that a potential molecular mechanism to explain the lysine-to-arginine transition was first reported.

Marine Invertebrates and SNBP Types

The compositional heterogeneity and potential structural variability of SNBPs were first envisaged from the early comparative studies of the SNBPs from marine molluscs in what has become a seminal paper on this topic (Subirana et al., 1973). At the time, a thorough descriptive analysis of SNBPs from different taxa including molluscs had been provided by Bloch (1969, 1976) through a compilation of cytochemical staining methods and partial amino acid analysis characterization. On the basis of these early studies, Bloch proposed an initial classification of SNBPs that distinguished five major groups: (1) The Salmo type consisting of relatively small arginine-rich "monoprotamines" found in fish such as salmon; (2) the mouse/grasshopper type consisting of cysteine-containing arginine-rich "stable protamines"; (3) the Mytilus type, with proteins intermediate between those of protamines and histones with respect to composition of arginine and lysine; (4) the Rana type in which mature sperm retains somatic-like chromosomal proteins; and (5) the crab type in which the DNA in the mature sperm apparently lacks any chromosomal proteins and as a result the nucleus appears large and swollen. However, it was the comparative work on different species within more closely related groups of molluscs (Subirana et al., 1973), which included SNBPs belonging to each of these first four groups, that allowed the first hypothesis to be formulated on the potential evolutionary relationships between the various types of SNBPs. The hypothesis was put forward that the first three groups of this classification could have had a histone origin.

The structural characterization of the SNBPs of the surf clam Spissula solidissima, one of the species studied in Subirana et al. (1973), followed several years later and allowed H1 to be identified as the putative chromosomal protein precursor to SNBPs of groups 1-3 of Bloch's classification (Ausio et al., 1987). Such an evolutionary relationship has now been thoroughly substantiated (Ausio, 1995, 1999; Eirin-Lopez et al., 2006a, Ellin-Lopez and Ausio, 2009). These studies also allowed Bloch's classification to be revised on the basis of protein structure and phylogeny, in which Bloch's first four groups were rearranged as P (protamine), PL (protamine-like) and H (histone type) (Ausio, 1999) (Table 1).

Table 1

Sperm nuclear basic proteins (SNBPs) cited in
this paper: R/K ratio and representative species

References: (1) Strickland et al., 1976; (2) Lewis et
al., 2004b; (3) Lewis et al. 2004c; (4) Lewis et
al., 2004a.

SNBP     SNBP      R/K  Species name               Reference

type"    name    ratio

H type   Hl sp    0.37  Parechinus angulosus (sea        (1)

PL type  PL-1     0.95  Spisula solidissima (surf        (2)

         P1       0.51  Ciona inleslinolis               (3)

         p1 (b)    2.3  Styela montereyensis             (3)

         p2 (c)    2.5  Styela monterevensis             (3)

P type   P       all R  Loligo opalescens (squid)        (4)

(a) HI-like proteins can be extracted separately with 5%
perchloric acid, histones (including HI) can be extracted
with 35% acetic acid, and proteins with a higher R/K ratio
need to be extracted with stronger acids (0.4 HCI).
(b) In the Styela genus, this PL has evolved to a
high R/K ratio (see text).
(C) This protein corresponds to the C-terminal end of
P1 but already has the structural features of a protamine
(see text).

Histones are a defined set of basic chromosomal proteins that are responsible for the organization of somatic chromatin in all eukaryote organisms. They can be grouped into two major families: core histones (H2A, H2B, H3, and H4) and linker histones, or histones of the H1 family. Core histones are rich in arginine and lysine, whereas somatic linker histones are rich in lysine. Structurally, histones are relatively simple proteins. Core histones contain a dimerizing histone fold domain (Arents and Moudrianakis, 1995) consisting of three a-helices connected by two loops and flanked by unstructured N- and C-terminal domains (commonly referred to as tails) (Fig. 1.1). Core histones interact to form a heterotypic octamer [2(H2A-H2B)-[(H3-H4).sub.2]] that serves as a core around which approximately 146 bp of DNA are wrapped, forming the most basic repetitive subunit of chromatin, the nucleosome core particle (van Holde, 1988). Linker histones have a winged helix domain (WHD) (Ramakrishnan et al., 1993) (Fig. 1.1) that is flanked by unstructured N- and C-terminal tails. The WI-ID specifically recognizes four-way-junction organization of DNA and is responsible for the specificity of the binding of the histones to DNA at the entry and exit sites of DNA into the nucleosome. As their name indicates, these histones bind to the linker DNA regions of the chromatin fiber connecting adjacent nucleosomes. Indeed, the loss of nucleosome organization in the sperm of those organisms in which the somatic-type histones are replaced by other SNBP types could account for the structural and evolutionary relationship of these types to histone Hl. Many PL SNBPs (such as PL-I) retain the WHD and hence maintain their preference for elongated four-way junction-like structures resulting from an intertwined DNA organization (Saperas et al., 2006; Frehlick et al., 2007). At the chromatin level, the transition from H-type to PL-type to P-type (Fig. 1.2) is associated with a more relaxed topological organization of DNA (Fig. 1.3) (Ausio, 1995). In all instances, regardless of the SNBP composition, sperm chromatin adopts a complex, densely packed organization (Casas et al., 1981, 1993; Balhorn et al., 1999; Balhom, 2007; Ausio et al., 2007; Kurtz et al., 2009; Kasinsky et al., 2012), being the most densely packed in the sperm containing SNBPs of the PL-and P-types (Ausio et al., 2007). As first described in the SNBP characterization of molluscs (Subirana et al., 1973), SNBPs of the PL- and P-type consist of proteins of heterogeneous size and high lysine and arginine content that tend to become predominantly arginine-rich in P-type SNBPs (Ausio, 1999) (Fig. 1.2). Both PL- and P-type SNBPs belong to the intrinsically disordered family of proteins (Dunker et al., 2001) that can acquire secondary structure upon interaction with DNA (Roque et al., 2012).

Tunicate Sperm Nuclear Basic Proteins

Initial characterization of the SNBPs of tunicates

By the end of the 1980s, the SNBPs of several echinoderms and many vertebrate species had been characterized. Vertebrate species, including mammals and fish, typically have sperm composed of protamines (Kasinsky, 1989; Oliva and Dixon, 1991). The sperm of echinoderms, in contrast, contains both typical somatic histones and special sperm-specific histone variants of the H1 and H2B classes (von Holt et al., 1984; Poccia et al., 1987; Poccia and Green, 1992). Although several hypotheses were postulated to explain the origin of protamines in vertebrates (see the following), there were no studies at that time on the SNBPs of other zoological groups with close affinity to the vertebrates, such as chordates or cephalochordates, to help understand the problem. Thus, the first attempts to characterize the SNBPs of tunicates emerged from the need to contribute information about the origin of vertebrate protamines.

The first tunicate SNBPs characterized were from the stalked ascidian Styela montereyensis (Chiva et al., 1990). The elongated sperm cells of S. montereyensis, as well of those from S. plicata, fall into the category of "ascidian type" (Franzen, 1956, 1977, 1987). Cytochemical staining of mature testis from S. montereyensis showed that its SNBPs fell into the Mytilus type 3 intermediate category of Bloch, indicating that they were rich in both arginine and lysine (Chiva et al., 1990). Electrophoretic analysis of proteins extracted with 0.4 N HC1 from nuclei of mature testis of S. montereyensis showed that the SNBPs in this species were distributed mainly into two major groups: about 40% of the extracted proteins migrated in the region of core histones, while 40%-50% of the extractable proteins were of electrophoretic mobility intermediate between histones and fish protamines (Chiva et al., 1990). To further characterize S. montereyensis SNBPs, a sequential acid extraction was performed. Very lysine-rich histones H1 and other related proteins were first solubilized with 5% perchloric acid (PCA), then core histones were solubilized with 35% acetic acid, and finally arginine-rich sperm proteins with dilute HCI (Subirana et al., 1973). Following this procedure, about 5%-10% and 10%-15% of the nuclear protein content was solubilized with 5% PCA and 35% acetic acid, respectively, while the 0.4 N HC1 extract (after acetic acid extraction) contained most of the sperm basic proteins (75%-80% of the entire protein complement) and consisted mainly in two major components: one with an electrophoretic mobility similar to that of core histones and another faster moving component consisting in two bands that migrated very close together, with an electrophoretic migration between histones and salmine (Chiva et al., 1990) (see Fig. 2A). Although a purification of the separate components was not possible at that time, an amino acid analysis of the mixture of these proteins showed a highly basic amino acid composition, with about 50% of arginine and 17% of lysine, in good agreement with the previous cytochemical results (Chiva et al., 1990).

Hence, although a small quantity of somatic histones and other lysine-rich basic proteins remain in the sperm nuclei from mature testis, arginine-rich SNBPs were found to replace most (if not all) of the histones during the spermiogenesis of the solitary ascidian tunicate S. montereyensis. Sperm nuclear basic proteins of other Ascidiacea

To determine if the SNPB pattern found in Styela montereyensis was representative of the group, the SNBPs of eight more ascidian species from different groups were electrophoretically analyzed. Four species belonged to the order Enterogona, suborder Phlebobranchia--Chelyosoma productum (family Corellidae), Ascidia callosa, Ascidiella aspersa, and Phallusia mammillata (family Ascidiidae); four species belonged to the order Pleurogona, suborder Stolidobranchia--Styela plicata, Cnemidocarpa finmarkiensis (family Styelidae), Pyura haustor, and Boltenia villosa (family Pyuridae)-(Chiva et al., 1992).

Figure 2B shows the SNBPs obtained in the HCI (post-acetic acid and post-PCA) extract from mature testes of one member of each of the four families. Although the PCA and acetic acid post-PCA extracts were either devoid of proteins (A. callosa and C. productum) or contained very small amounts of solubilized proteins (B. villosa and C. finmarkiensis), the HCI arginine-rich extract contained most of the basic proteins associated with sperm-cell DNA. In all cases, a major component with an electrophoretic mobility similar to that of the H4 histone was found (Chiva et al., 1992). Despite this similarity, this protein could not be interpreted as a histone because it could not be solubilized by 35% acetic acid and because it was insoluble in SDS-containing buffers, as shown previously for the histone-like migrating component of S. montereyensis (Chiva et al., 1990).

When additional members of the same families were analyzed, a consistent SNBP pattern was found except in the family Styelidae. While C. finamarkiensis showed the same pattern as the other families--that is, one main component that migrates similar to H4 (referred to as P1 hence-forth)--S. montereyensis and S. plicata presented a more complex pattern. As already observed, in addition to the P1 component, S. montereyensis SNBPs include faster migrating proteins as well (referred to as P2 henceforth) (Fig. 2A). The P2 component is also present in S. plicata, together with a more slowly migrating protein (X in Fig. 2A). Neither of these proteins was extracted with 35% acetic acid.

Therefore a general model in which a protamine-like protein P1 replaces most of the histones in the sperm cell of ascidian tunicates can be defined. Although protein P1 can be considered the characteristic SNBP of ascidian tunicates (Chiva et al., 1992, 1995), a shorter protein (P2) is also found in the genus Styela.

Characterization of the PI and P2 proteins from Styela

To further characterize both the typical ascidian SNBP PI and the Styela P2 component, a more detailed study of the SNBPs from Styela plicata was performed. Thus PI and P2 proteins, as well as the X protein also observed in this species (Fig. 2A), were purified. Amino acid analysis of PI showed a very rich content in both lysine (14.3%) and arginine (32.5%), consistent with data that had already been reported (Chiva et al., 1992). Protein X showed a very similar amino acid composition. Electrophoretic behavior of protein P1 during purification revealed the formation of multiple aggregated forms that disappeared with addition of p-mercaptoethanol. Amino acid analysis of P1 confirmed the presence of cysteine, in an amount consistent with the presence of two residues of cysteine per molecule of P1 (Saperas et al., 1992). Although cysteine had not been detected in previous cytochemical studies (Chiva et al., 1990, 1992), its presence in the PI component of another tunicate, Chelyosoma productum, has been reported (Zhang et al., 1999).

The amino acid composition of protein P1 is reminiscent of that of the PL-I of marine invertebrates (Ausio, 1986). A characteristic feature of all PL-I molecules is the presence of a histone H1-like trypsin-resistant core (Ausio et al., 1987). Significantly, a single highly trypsin-resistant peptide can be obtained from S. plicata PI as well. This trypsin-resistant peptide shows both an electrophoretic mobility and an amino acid composition very similar to that of PL-I of molluscs. Thus, P1 is not only compositionally but also structurally related to PL-1 of molluscs (Saperas et al., 1992), indicating that the P1 protein of S. plicata is related to histone H1.

P2, the shorter protein, displayed microheterogeneity and was found in three forms with an almost identical amino acid composition. Its arginine content (50.4%) was much higher than that of P1, and similar to that of many vertebrate protamines. However, in contrast to vertebrate protamines, S. plicata P2 has a high content of lysine (17.4%) (Saperas et al., 1992). Of special interest was the fact that P2 amino acid composition was very similar to that of S. plicata P1 C-terminal tail (Chiva et al., 1995). This suggested a close relationship between these two molecules--that is, the possibility that P2 originated from P1.

Sequencing of S. plicata P1 and P2 proteins confirmed this relationship (Lewis et al., 2004c) (Fig. 3). Sequencing of PI and P1 -trypsin-resistant peptide showed that PI presented a very short N-terminal tail consisting of only two arginines, a globular domain (the trypsin-resistant core) that contained the two cysteine residues of the molecule, and a C-terminal tail where the basic amino acids were mostly found (Fig. 3A). Although the C-terminal tail of P1 protein could not be completely sequenced, it was indirectly determined by comparison with the analogous sequence obtained from the P2 component. When P2 was sequenced, its sequence matched that of the C-terminal part of the P1 protein from position 75 (Lewis et al., 2004b) (Fig. 3A). Therefore, the origin of the shorter protein P2 from P1 could be clearly established. As already predicted, the Pl C-terminal tail contained most of the arginine residues, and the arginine residues were found in tracts of four to eight residues each. Thus, while P2 contains an unusually high lysine content compared to other protamines (20.4% according to the sequence data), it also has a high amount of arginine (51.6%) and repeated arginine clusters, which are characteristic of many invertebrate and vertebrate canonical protamines.

Another issue concerned the N-terminal tail of S. plicata P1. The fact that it was unusually short suggested that protein PI may have originated from a larger precursor with a more extended N-terminal domain, such as the X component that is also present in the sperm cell. Although this possibility could not be unequivocally determined in the case of S. plicata, it was subsequently demonstrated in the related species S. montereyensis by combining both amino acid sequencing and DNA cloning. A genomic fragment of the larger sperm nuclear protein (P1) was obtained from S. montereyensis by using degenerate primers based on the amino acid sequence from S. plicata. This partial sequence was then used to design polymerase chain reaction primers for rapid amplification of cDNA ends (RACE) to obtain the full-length cDNA of the 131 from S. montereyensis. The sequence thus obtained showed an additional I 6-amino acid leading peptide (Fig. 3) not present in the mature PL protein (Lewis et al., 2004c).

Therefore, a relationship between histone H1 [right arrow] P1 [right arrow] P2 (i.e., H1 [right arrow] PL-Ptype [right arrow] P-type) proteins was demonstrated.

Presence of PL/P-Type Sperm Nuclear Basic Proteins in Tunicates Provides Additional Support for the Vertical Evolution of SNBPs

The unique arginine-rich composition of SNBPs of the protamine type (up to 80% arginine in the squid Loligo sp.) (Lewis et al., 2004a) and their relatively small size of not larger than 100 amino acids (Daban et al., 1995) make these proteins very different from histones. This casts doubt on the evolutionary origin of these proteins.

The presence of additional TATA boxes in the flanking regions of the trout protamine genes as well as the presence of conserved AATAAA poly-adenylation signals. CATCG promoter sequences, and short inverted repeats suggested a relationship to the retroviral long terminal repeat sequences--in particular, to those of the avian sarcoma virus (Jankowski et al., 1986). This led to the retroviral evolutionary hypothesis for the origin of protamines through a mechanism of horizontal transmission (infection) that would account for the sporadic distribution of protamines that was observed within different organisms of distantly related phylogenetic groups (Jankowski et al., 1986).

However, the evolutionary relationship between the H-, PL-, and P-types of SNBPs implies a vertical process of evolution that is difficult to reconcile with the retroviral origin hypothesis. Very strong support for a vertical process of evolution came from a detailed comparative study of the SNBP distribution within different fish taxa (Saperas et al., 1993, 1994). Representatives of the three SNBP types can be found within this group (Saperas et al., 1993; Ausio et al., 2011). The study conclusively showed that the sporadic distribution of SNBPs of the protamine P-type within this group of vertebrates was not random. It was shown that the SNBP transition from H to PL to P had occurred several times within the bony fish during the diversification of the orders of this group (Saperas et al., 1994).

The evolutionary relationship of PL- and P-type SNBPs to a histone H1 through a process of vertical evolution has now been extensively documented (Erin-Lopez et al., 2006b; Eirin-Lopez and Ausio, 2009). The seemingly sporadic distribution of the different SNBP types is the result of this transition having occurred repeatedly in the course of evolution (Kasinsky, 1995) within different groups of both the deuterostome and protostome branches (Ausio, 1999; Eirin-Lopez et al., 2006a; Erin-Lopez and Ausio, 2009), with the P-type always present in organisms at the tips of the phylogenetic branches (Hunt et al., 1996).

Echinoderms constitute a good example of SNBPs of the H-type. Their sperm chromatin consists of highly specialized somatic-like core and linker histones (Hamer, 1955; Paoletti and Huang, 1969; Johnson et al., 1973). Sea urchin sperm histone HI contains extended N- and C-terminal regions consisting largely of multiple repeats of the tetrapeptide "SPBB" (where B = R and/or K) (Suzuki, 1989) that are also present at the extended N-terminal end of H2B (Poccia and Green, 1992). These repeats are also found in many somatic H1 histones as well as in many other proteins (Poccia and Green, 1992), including SNBPs of the PL-type. SPBB tetrapeptides are target sites of phosphorylation by cdc2 (Poccia and Green, 1992), and they may subsequently become a target of (S/TP)-phosphorylation-dependent pep-tidyl-prolyl isomerases such as [peptidyl-prolyl cis-trans isomerase NIMA (never in mitosis gene a)-interacting ii Pini (Shen et al., 1998) before a final unique, highly ordered chromatin condensation takes place in the mature sperm of these organisms (Athey et al., 1990; Woodcock, 1994). Such condensation leads to the highly homogeneous 30-40-nm folded chromatin fiber organization that has been used for many years as a model for the higher order organization of the chromatin fiber (Fussner et al., 2011).

As described in the previous section, the analyses of SNBPs in tunicates indicate that the phylogenetic transition from echinoderms to this group involved the appearance of PL/P-type SNBPs. This entailed a progressive decrease in lysine content at the expense of an increase in arginine content that, in some species such as Styela, became compositionally prominent, providing yet another good example of the vertical evolutionary transition between the H-, PL-, and P-type SNBPs. Nevertheless, this still left unsolved an important question as to how the highly arginine-rich SNBPs (P-type) could have had their origin in the lysinerich family (Cole, 1984, 1987) of histone HI and how the transition from lysine to arginine could have taken place.

A Potential Molecular Mechanism for the Lysine-to-Arginine Transition in Tunicates

As has already been mentioned, a major conceptual problem with the vertical evolution of the PL- and P-types of SNBPs from a histone HI ancestor has to do with the high lysine content of the somatic version of this histone family. The number 1 assigned to this chromosomal protein came from being the first fraction (F1) obtained in the first attempts of chemical fractionation of histones by Johns and Butler (1962). Not only was histone HI the first fraction obtained, it was uniquely soluble in 5% PCA, a feature most likely due to its lysine-rich composition. It was later found that in somatic tissues histone Hi was not a single protein but a microhetereogeneous group of closely related proteins (Cole, 1984, 1987) that were also given the name of lysine-rich histones (Cole, 1984, 1987). From a structural perspective, the name histone given to this family of chromatin-associated proteins may be a misnomer. The only structured part of the molecule, in the absence of other interacting partners, is the winged helix domain (WHD). which is common to some transcription factors (Ramakrishnan et al., 1993) but bears no similarity to the histone fold (Arents and Moudrianakis. 1995) that is characteristic of core histones (H2A, H2B. H3, and H4). It was actually the sequence of this domain, and not the lysine content of the molecule, that uncovered the potential relationship between histone H1 and PL-I from S. solidissima, which was compositionally rich in both lysine and arginine (Ausi6 et al., 1987).

The increase in arginine content observed in PL SNBPs was likely driven by a transitional improvement of the interaction of SNBPs with DNA that, in the absence of a transcriptional activity, is better achieved by arginine-rich protamines. Acquisition of internal fertilization may have also played an important role in irreversibly locking the H-to-PL-to-P transition at the protatnine stage within different groups (Kasinsky et al., 1999, 2005).

Biophysical characterization by nuclear magnetic resonance of the interaction of Mytilus SNBPs of the PL-type with DNA has shown that the strength of this interaction increases with the increase in arginine composition of the proteins (Puigdomenech et al., 1976). The strength increase is due to the stronger interaction of this amino acid with DNA (Helene and Lancelot, 1982; Ausi6 et al., 1984). This arises from the greater hydrogen bonding flexibility of arginine as a result of the three-dimensional organization of the three asymmetrical N and N112) of its guanidinium group (Cheng et al., 2003) when compared to the unidimensional constraint of the amino group of lysine (Borders et al., 1994; Donald et al., 2011). In addition, the early studies on the interaction of poly-lysine and polyarginine with DNA provide evidence for a preference of the interaction of poly-lysine with A-T rich DNA in contrast to the sequence indifference of poly-arginine (Leng and Felsenfeld, 1966; Olins et al., 1967). These studies were in good agreement with the more physiologically relevant preference of histone Hl for the A-T rich regions of DNA in native nucleohistones described by Johns and Butler (1964) at the time. Hence, the lysine-rich nature of the somatic members of the histone HI family may represent an adaptation of these molecules to the interaction with the rigid poly(dA:dT) tract--containing intern ucleosomal regions of somatic chromatin (Suter et al., 2000; Segal and Widom, 2009; Hughes et al., 2012). This preferential interaction with A-T rich DNA regions may no longer be needed in the absence of a nucleosome organization in the chromatin of the sperm of organisms consisting of PL- and P-type SN BPs.

However, none of these arguments provides any information as to how the lysine-to-arginine replacement took place during the transition to the PL- and P-SNBP types. Our study of the SNBPs in tunicates described in the preceding section has proven to be highly informative in this regard. It provided insight into one of the rather unexpected molecular mechanisms among the several that are possibly involved.

The presence of two arginine-rich P1 and P2 SNBPs in the sperm of different species of the genus Styela (see Fig. 4A) (Chiva et al., 1990, 1995; Saperas et al., 1992) in tunicates, at the interface between echinoderms and vertebrates, fostered interest in the sequencing of these proteins (Lewis et al., 2004c), which was achieved by a combination of Edman degradation N-terminal sequencing and DNA cloning, as already explained. The analysis came at a time when the genome sequence of the related tunicate Ciona intestinal/is, which contains only a PI SBP (Lewis et al., 2004c), had been recently determined (Dehal et al., 2002). Therefore, it was of interest to use the sequence determined for S. montereyensis (Lewis et al., 2004c) to try to identify the corresponding sequence of PI within this genus. Surprisingly, although the P1 protein precursor of both species shared a highly conserved N-terminal sequence, the C-terminal end was very lysine-rich in C. intestinalis (Fig. 4A). This finding was in sharp contrast to what was observed in different species of Styela, which all had an exceedingly rich arginine composition (Lewis et al., 2004c). Although the result was initially puzzling, close inspection of the nucleotide composition corresponding to the C-terminal domain of both proteins revealed a significant codon usage bias in each. While each utilized an almost equivalent number of AAA codons to encode the lysines commonly shared by their sequences, the lysines at the C-terminal end of C. intestinalis were encoded by AAG, a codon that is commonly used by the lysine-rich histones of the HI family. Moreover, of the six possible codons that encode arginine, 63% of the arginines within the corresponding C-terminal domain of S. montereyensis PI were AGA (Lewis et al., 2004c). This led to the hypothesis that although C. intestinalis is not a direct ancestor of S. montereyensis, its sperm-specific PI was most likely representative of a common ancestor to both organisms (Lewis et al., 2004c).

For the above hypothesis to be correct, it would require two point mutations for each AAG-preferred lysine codon in C. intestinalis to transition to the AGA-preferred arginine codon in Styela. This is a highly unlikely repetitive event. especially if it had to be selectively directed to the C-terminal domain of the molecule. Further inspection of the nucleotide sequences indicated that it was more likely that a frameshift mutation took place at the C-terminal domain of a lysine-rich sperm-specific histone H1 that led to the appearance of the arginine-rich Styela Pl. It was observed that deletion of a single nucleotide at position 342 and two nucleotides at position 437 (see Fig. 4B), corresponding to the coding region of C. intestinalis Pl, creates a frameshift mutation that alters the relative K/R composition in a way that mimics the compositional differences between the two species of tunicates. This could account for one of the modes of rapid protein evolution required for the K-to-R changes involved in the H1-to-PL-to-P-type transition that has repeatedly occurred in the course of SNBP evolution. Such a unique frameshift mutation not only increases the arginine content of the protein, it also introduces in two instances (see Fig. 4B) the SPRR motif that is present in some of the vertebrate PL proteins (Saperas et al., 2006), in the early vertebrate protamines (Lewis et al., 2003), and in the sperm-specific histones of echinoderms (Poccia and Green, 1992).

There is no doubt that the phenomenon described here is not the only mechanism involved in the transition from lysine to arginine during the SNBP type transition. Yet, it provides another useful example of how the study in marine invertebrates has been key to the phylogenetic and mechanistic understanding of SNBPs and their evolutionary transitions.


We dedicate this article to the memory of our good friend and colleague Manel Chiva Royo who over the years made seminal contributions to the field of sperm nuclear chromosomal proteins. Manel passed away on May 24, 2011.

We are thankful to Elsa Fonfria for helping us during the final stages of preparation of the figures. This work has been supported in part by grants BFU2009-10380 from the Spanish Ministerio de Ciencia e Innovacion and 2009 SGR 1208 from the Generalitat de Catalunya to N.S., and in part by a grant from the Natural Science and Engineering Research Council of Canada (NSERC-46399-12) to J.A.

Received 11 January 2013; accepted 22 May 2013.

* To whom correspondence should be addressed. E-mail:

Abbreviations: H-type, histone type; P-type, protamine type; PCA, per-chloric acid; PL-type, protamine-like type; SNBPs, sperm nuclear basic proteins; WM), winged helix domain. 1:

Literature Cited

Arents, G. and E. N. Moucirianakis. 1995. The histone fold: a ubiquitous architectural motif utilized in DNA compaction and protein dimerization. Proc. Natl. Acad. Sci. USA 92: 11170-11174.

Athey, B. D., M. F. Smith, D. A. Rankert, S. P. Williams, and J. P. Langmore. 1990. The diameters of frozen-hydrated chromatin fibers increase with DNA linker length: evidence in support of variable diameter models for chromatin. J. Cell Biol. 111: 795-806.

Ausio, J. 1986. Structural variability and compositional homology of the protamine-like components of the sperm from the bivalve molluscs. Comp. Biochem. Physiol. 8511: 439-449.

Ausio J. 1995. Histone H1 and the evolution of the nuclear sperm-specific proteins. Pp. 447-462 in Advances in Spermatozoa! Phylogeny and Taxonomy. B. G. M. Jamieson. J. Ausio, and J.-L. Justine, eds. Memoires de Museum National d'Histoire Naturelle, Vol. 166, Paris.

Ausio, J. 1999. Histone HI and evolution of sperm nuclear basic proteins. J. Biol. Chem. 274: 31115-31118.

Ausio, J., K. 0. Greulich, E. Haas. and E. Wachtel. 1984. Characterization of the fluorescence of the protamine thynnine and studies of binding to double-stranded DNA. Biopohmers 23: 2559-2571.

Ausio, J., A. Toumadje. R. McParland. R. R. Becker, W. C. Johnson, and K. E. van HoIde. 1987. Structural characterization of the trypsin-resistant core in the nuclear sperm-specific protein from Spisula solidissima. Biochemistry 26: 975-982.

Ausio, J., J. M. Eirin-Lopez, and L. J. Frehlick. 2007. Evolution of vertebrate chromosomal sperm proteins: implications for fertility and sperm competition. Soc. Reprod. Fertil Suppl. 65: 63-79.

Ausio, J., N. Saperas, and M. Chiva. 2011. Sperm nuclear basic proteins and sperm chromatin organization in fish. Pp. 80-91 in Cryopreservation of Aquatic Species. T. Tirersch and C. Green, eds. World Aquaculture Society. Baton Rouge. LA.

Balhorn. R. 2007. The protamine family of sperm nuclear proteins. Genome Biol. 8: 227.

Balhorn, R., M. Cosman, K. Thornton, V. V. Krishnan, M. Corzett, G. Bench, C. Kramer, J. Lee IV, N. V. Hud, M. Allen, et al. 1999. Protamine Mediated Condensation of DNA in Mammalian Sperm. Cache River Press, Vienna. IL.

Bloch, D. P. 1969. A catalog of sperm histones. Generics Suppl. 61: 93-111.

Bloch. D. P. 1976. Histones of Sperm. Plenum Press. New York.

Borders, C. L., Jr., J. A. Broadwater, P. A. Bekeny, J. E. Salmon, A. S. Lee, A. M. Eldridge, and V. B. Pett. 1994. A structural role for arginine in proteins: multiple hydrogen bonds to backbone carbonyl oxygens. Protein Sci. 3: 541-548.

Casa.% M. T.. S. Munoz-Guerra, and J. A. Subirana. 1981. Preliminary report on the ultrastructure of chromatin in the histone containing spermatozoa of a teleost fish. Biol. Cell 40: 87-92.

Casas, M. T., J. Ausio, and J. A. Subirana. 1993. Chromatin fibers with different protamine and histone compositions. Exp. Cell. Res. 204: 192-197.

Cheng, A. C., W. W. Chen, C. N. Fuhrmann, and A. D. Frankel. 2003. Recognition of nucleic acid bases and base-pairs by hydrogen bonding to amino acid side-chains. J. Mol. Biol. 327: 781-796.

Chiva, M., E. Rosenberg, and H. E. Kasinsky. 1990. Nuclear basic proteins in mature testis of the ascidian tunicate Styela montereyensis. J. Exp. Zool. 253: 7-19.

Chiva, M., F. Lafargue, E. Rosenberg, and H. E. Kasinsky. 1992. Protamines, not histones, are the predominant basic proteins in sperm nuclei of solitary ascidian tunicates. J. Exp. Zool. 263: 338-349.

Chiva, M., N. Saperas, C. Caceres. and J. Ausio. 1995. Nuclear basic proteins from the sperm of tunicates. cephalochordates. agnathans and fish. Pp. 501-514 in Advances in Spermatocoal Phylogeny and Taxonomy. B. G. M. Jamieson, J. Ausio. and J. L. Justine, eds. Museum National d'Histoire Naturelle. Vol. 166. Paris.

Cole, R. D. 1984. A minireview of microheterogeneity in HI histone and its possible significance. Anal. Biochem. 136: 24-30.

Cole, R. D. 1987. Microheterogeneity in HI histones and its consequences. hit. J. Pept. Protein Res. 30: 433-449.

Daban, M., A. Martinage. M. Kouach, M. Chiva, J. A. Subirana, and P. Sautiere. 1995. Sequence analysis and structural features of the largest known protamine isolated from the sperm of the archaeogas-tropod Monodonta turbinata. J. Mol. Evol. 40: 663-670.

Dehal, P., Y. Satou. R. K. Campbell, J. Chapman, B. Degnan, A. De Tomaso, B. Davidson, A. Di Gregorio, M. Gelpke, D. Ni. Goodstein et al. 2002. The draft genome of Ciona intestinalis: insights into chordate and vertebrate origins. Science 298: 2 I 57-2167.

Donald, J. E., D. W. Kulp. and W. F. DeGrado. 2011. Salt bridges: geometrically specific, designable interactions. Proteins 79: 898-915.

Dunker, A. K., J. D. Lawson, C. J. Brown, R. M. Williams. P. Romero, J. S. Oh, C. J. Oldfield. A. M. Campen, C. M. Ratliff, K. W. Hipps et al. 2001. Intrinsically disordered protein. J. Mol. Graph. Model. 19: 26-59.

Eirin-Lopez, J. M., and J. Ausio. 2009. Origin and evolution of chromosomal sperm proteins. Bioessays 31: 1062-1070.

Eirin-Lopez, J. M., L. J. Frehlick, and J. Ausio. 2006a. Protamines, in the footsteps of linker histone evolution. J. Biol. Chem. 281: 1-4.

Eirin-Lopez. J. M., J. D. Lewis, L. A. Howe, and J. Ausio. 2006b. Common phylogenetic origin of protamine-like (PL) proteins and histone H I: evidence from bivalve PL genes. Mol. Biol. Evol. 23: 1304-1317.

Franzen, A. 1956. On spermiogenesis. morphology of the spermatozoan, and biology of fertilization among invertebrates. Zool. Bidr. Upps. 31: 355-482.

Franzen. A. 1977. Sperm structure with regard to fertilization biology and phylogenetics. Verh. Dtsch. Zool. Ges. 70: 123-138.

Franzen, A. 1987. Spermatogenesis. Pp. 1-47 in Reproduction of Marine Invertebrates. Vol. 10. General Aspects: Seeking Unity in Diversity. R. C. King. J. S. Pearse. and V. B. Pearse. eds. Blackwell. Palo Alto. CA.

Frehlick, L. J., A. Prado, A. Calestagne-Morelli, and J. Ausio. 2007. Characterization of the PL-I-related SP2 protein from Xenopus. Biochemistry 46: 12700-12708.

Fussner, E., R. W. Ching. and D. P. Bazett-Jones. 2011. Living without 30nm chromatin fibers. Trends Biochem. Sci. 36: 1-6.

Hamer, D. 1955. The composition of the basic proteins of echinoderm sperm. Biol. Bull. 108: 35-39.

Helene, C., and G. Lancelot. 1982. Interactions between functional groups in protein-nucleic acid associations. Prog. Biophys. Mol. Biol. 39: 1-68.

Hughes, A. L., Y. Jin. 0. J. Rando. and K. Struhl. 2012. A functional evolutionary approach to identify determinants of nucleosome positioning: a unifying model for establishing the genome-wide pattern. Mot Cell 48: 5-15.

Hunt, J. G., H. E. Kasinsky, R. M. Elsey, C. L. Wright, P. Rice, J. E. Bell, D. J. Sharp, A. J. Kiss, D. F. Hunt, D. P. Arnott et al. 1996. Protamines of reptiles. J. Biol. Chem. 271: 23547-23557.

Jankowski, J. M., J. C. States, and G. H. Dixon. 1986. Evidence of sequences resembling avian retrovirus long terminal repeats flanking the trout protamine gene. J. Mol. Eval. 23: 1-10.

Johns, E. W., and J. A. Butler. 1962. Further fractionations of histones from calf thymus. Biochem. J. 82: 15-18.

Johns. E. W., and J. A. Butler. 1964. Specificity of the interactions between histones and deoxyribonucleic acid. Nature 204: 853-855.

Johnson, A. W., J. A. Wilhelm. D. N. Ward, and L. S. Hnilica. 1973. The composition of sea urchin sperm and embryo histones. Biochim. Biophys. Acta 295: 140-149.

Kasinsky, H. E. 1989. Specificity and distribution of sperm basic proteins. Pp. 73-163 in Histones and Other Basic Nuclear Proteins, L. S. Hnilica. G. S. Stein, and J. L. Stein. eds. CRC Press. Boca Raton. FL. Kasinsky, H. E. 1995. Evolution and Origins of Sperm Basic Proteins. Memoires du Museum d'Histoire Naturellc. Paris.

Kasinsky, H. E., L. Gutovich. D. Kulak, M. Mackay, D. M. Green, J. Hunt, and J. Ansio 1999. Protamine-like sperm nuclear basic proteins in the primitive frog Ascaphus truei and histone reversions among more advanced frogs. J. Exp. Zool. 284: 717-728.

Kasinsky, H. E., L. J. Frehlick, H. W. Su, and J. Ausio. 2005. Protamines in the internally fertilizing neobatrachian frog Eleutherodactylus coqui. Mol. Reprod. Dev. 70: 373-381.

Kasinsky, H. E., J. M. Eirin-Lopez, and J. Ausio. 2012. Protamines: structural complexity, evolution and chromatin patterning. Protein Pept. Lett. 18: 755-771.

Kurtz. K., N. Saperas, J. Ausio, and M. Chiva. 2009. Spermiogenic nuclear protein transitions and chromatin condensation. Proposal for an ancestral model of nuclear spermiogenesis. J. Exp. Zool. B 312: 149163.

Leng, M., and G. Felsenfeld. 1966. The preferential interactions of polylysine and polyarginine with specific base sequences in DNA. Proc. Natl. Acad. Sci. USA 56: 1325-1332.

Lewis. J. D., Y. Song, M. E. de Jong, S. M., Bagha, and J. Ansio 2003. A walk though vertebrate and invertebrate protamines. Chromosoma 111: 473-482.

Lewis, J. D., M. E. de Jong, S. M. Bagha. A. Tang, W. F. Gilly. and J. Ansio 2004a. All roads lead to arginine: the squid protamine gene. J. Mol. Eval. 58: 673-680.

Lewis, J. D., R. McParland, and J. Ausio. 2004b. PL-1 of Spisula solidissima, a highly elongated sperm-specific histone HI. Biochemistry 43: 7766-7775.

Lewis, J. D., N. Saperas, Y. Song, M. J. Zamora, M. Chiva, and J. Ausio. 2004c. Histone HI and the origin of protamines. Proc. Natl. Acad. Sci. USA 101: 4148-4152.

Olins, D. E., A. L. Olins, and P. H. Von Hippel. 1967. Model nucleoprotein complexes: studies on the interaction of cationic homopolypeptides with DNA. J. Ma Biol. 24: 157-176.

Oliva, R., and G. H. Dixon. 1991. Vertebrate protamine genes and the histone-to-protamine replacement reaction. Prog. Nucleic Acid Res. Mol. Biol. 40: 25-94.

Paoletti. R. A., and R. C. Huang. 1969. Characterization of sea urchin sperm chromatin and its basic proteins. Biochemistry 8: 1615-1625.

Poccia, D. L., and G. R. Green. 1992. Packaging and unpackaging the sea urchin sperm genome. Trends Biochem. Sci. 17: 223-227.

Poccia, D. L., M. V. Simpson, and G. R. Green. 1987. Transactions in histone variants during sea urchin spermatogenesis. Dev. Biol. 121: 445-453.

Puigdomenech, P., P. Martinez, J. Palau, E. M. Bradbury, and C. Crane-Robinson. 1976. Studies on the role and mode of operation of the very-lysine-rich histones in eukaryote chromatin. Nuclear-magnetic-resonance studies on nucleoprotein and histone phi 1-DNA complexes from marine invertebrate sperm. Eur. J. Biochem. 65: 357-363.

Ramakrishnan, V., J. T. Finch, V. Graziano, P. L. Lee, and R. M. Sweet. 1993. Crystal structure of globular domain of histone H5 and its implications for nucleosome binding. Nature 362: 219-223.

Roque, A., 1. Ponte. and P. Suau. 2012. Secondary structure of protamine in sperm nuclei: an infrared spectroscopy study. BMC Struct. Biol. 11: 14.

Saperas, N. M., M. Chiva, and J. Ausi6. 1992. Purification and characterization of the protamines and related proteins from the sperm of a tunicate. Comp. Biochem. Physiol. B 103: 969-974.

Saperas, N., D. Lions, and M. Chiva. 1993. Sporadic appearance of histones, histone-like proteins and protamines in sperm chromatin of bony fish. J. Exp. Zool. 265: 575-586.

Saperas, N., J. Ausio, D. Lions, and M. Chiva. 1994. On the evolution of protamines in bony fish: alternatives to the "retroviral horizontal transmission" hypothesis. J. Mol. Evol. 39: 282-295.

Saperas, N., M. Chiva, M. T. Casas, J. L. Campos, J. M. Eirin-Lopez, L. J. Frehlick, C. Prieto, J. A. Subirana, and J. Ausio. 2006. A unique vertebrate histone H1-related protamine-like protein results in an unusual sperm chromatin organization. FEBS J. 273: 4548-4561.

Segal. E.. and J. Widom. 2009. Poly(dA:dT) tracts: major determinants of nucleosome organization. Curr. Opin. Struct. Biol. 19: 65-71.

Shen, M., P. T. Stukenberg, M. W. Kirschner, and K. P. Lu. 1998. The essential mitotic peptidyl-prolyl isomerase Pinl binds and regulates mitosis-specific phosphoproteins. Gene Dev. 12: 706-720.

Strickland, W. N., H. Schaller, M. Strickland, and C. von Holt. 1976. Partial amino acid sequence of histone HI from sperm of the sea urchin, Parechinus angulosus. FEBS Lett. 66: 322-327.

Subirana, J. A., C. Cozcolluela, J. Palau, and M. Unzeta. 1973. Protamines and other basic proteins from spermatozoa of molluscs. Biochim. Biophys. Acta 317: 364-379.

Suter. B., G. Schnappauf, and F. Thoma. 2000. Poly(dA.dT) sequences exist as rigid DNA structures in nucleosome-free yeast promoters in vivo. Nucleic Acids Res. 28: 4083-4089.

Suzuki, M. 1989. SPKK. a new nucleic acid-binding unit of protein found in histone. EMBO J. 8: 797-804.

van HoIde, K. E. 1988. Chromatin. Springer-Verlag, New York.

von Holt, C., P. de Groot, S. Schwager, and W. F. Brand. 1984. The structure of sea urchin histones and consideration of their function. Pp. 65-105 in Histone Genes: Structure, Organization and Regulation, G. S. Stein, J. L. Stein, and W. F. Marzluff, eds. John Wiley, New York.

Woodcock, C. L. 1994. Chromatin fibers observed in situ in frozen hydrated sections. Native fiber diameter is not correlated with nucleosome repeat length. J. Cell Biol. 125: 11-19.

Zhang, F., J. D. Lewis, and J. Ausio 1999. Cysteine-containing histone H1-like (PL-I) proteins of sperm. Mol. Reprod. Der. 54: 402-409.


(1) Departament d'Enginyeria Quimica, Universitat Politecnica de Cataluna, Barcelona, Spain; and

(2) Department of Biochemistry and Microbiology, University of Victoria, British Columbia, Canada V8W 3P6
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Author:Saperas, Nuria; Ausio, Juan
Publication:The Biological Bulletin
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Date:Aug 1, 2013
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