Human sperm protamines. Amino-acid sequences of two forms of protamine P2

Human protamine P2 was purified to homogeneity by solubilizing whole spermatozoa in guanidinium X HCl containing 2-mercaptoethanol, alkylating the resulting protamine thiols with vinylpyridine, removing acid-insoluble material by acid dialysis and using CM-cellulose chromatography to remove non-protamine basic proteins and separate protamines P1 and P2. The P2 preparation contained two components, P2a and P2b, which were sequenced completely without being separated. The peptides obtained from thermolysin and endoproteinase Lys-C digestions were purified by reverse-phase high-pressure liquid chromatography and sequenced using a gas-phase sequencer. P2a contains 57 amino acids and has a relative molecular mass of 7636 while P2b contains 54 amino acids, which are identical to residues 4-57 of P2a, and has a relative molecular mass of 7242. Protamine P2a is approximately 50% homologous with human protamine P1. The amino acid sequence of P2a is: (sequence; see text)     

Phosphorylation state of protamines 1 and 2 in human spermatids and spermatozoa

The basic nuclear proteins of a fraction of elongating spermatids from human tests and of a fraction of motile spermatozoa from the ejaculate, separated by ion-exchange chromatography, were compared. Analysis by acetic acid-urea polyacrylamide gel electrophoresis (PAGE) showed that, in both fractions, four proteins of lower mobility were coeluted with protamine 1 by 23% guanidinium chloride (GuCI) while protamine 2 alone was eluted by 50% GuCI. Treatment with alkaline phosphatase identified those four proteins as phosphorylated protamines, and cyanogen bromide (CNBr) treatment of the dephosphorylated protamines distinguished them as variants of protamine 2 and not of protamine 1. Thus far, phosphorylated forms of protamine 1 have not been detected in either spermatids or spermatozoa. Those observations indicate that protamine 2 functions in the cycle of phosphorylation-dephosphorylation, which is essential to the process of sperm chromatin condensation, while the role of protamine 1 in human spermiogenesis is not yet defined. The presence of phosphorylated protamine in motile, presumably mature spermatozoa appears to be characteristic of human sperm but not of the sperm of other mammals and is probably the basis for the heterogeneity of chromatin condensation frequently observed in human spermatozoa.     

Synthesis and processing of mammalian protamines and transition proteins

Mouse and rat seminiferous tubule fragment cultures were used to examine synthesis and processing of mammalian protamines and transition proteins. The tubule fragments were incubated with [³H]-arginine, [³H]-histidine, [³⁵S]-cysteine, or [³²P]-PO₄, and radiolabeled proteins were analyzed by acid/urea polyacrylamide gel electrophoresis and fluorography or autoradiography. Newly synthesized protamines were recovered from sonication-resistant nuclei (SRN) and could not be detected in cytoplasmic fractions, indicating that protamines are deposited into nuclei immediately after synthesis. Newly synthesized mouse protamine 1 (mP1) and the precursor to mouse protamine 2 (pre-mP2) migrated more slowly during electrophoresis than their predominant testicular forms, identified by staining with Coomassie blue R-250. Within 1 hour of synthesis, the electrophoretic mobilities of mP1 and pre-mP2 increased to match those of their predominant forms. These changes are consistent with initial charge-neutralizing modifications of the newly synthesized protamines, followed by removal of at least some of the modifying ligands, to unmask protamine basicity. Steady-state phosphorylation rates were high for rat protamine 1 (rP1) and were independent of phosphate content; both rP1 molecules of low and high phosphate content were rapidly phosphorylated. Pre-mP2-3, a major processing intermediate derived by proteolysis of pre-mP2, was also rapidly phosphorylated. Like the protamines, transition protein 2 (TP2) was rapidly phosphorylated and increased in electrophoretic mobility soon after synthesis. In contrast, transition protein 1 (TP1) was not phosphorylated and did not exhibit multiple electrophoretic forms. © 1994 Wiley-Liss, Inc.     

Analysis of hamster protamines: primary sequence and species distribution.

Basic nuclear proteins were isolated from the sperm of the Syrian hamster Mesocricetus auratus and characterized by gel electrophoresis, amino acid analysis, and sequencing. Analyses of the proteins by gel electrophoresis show that sperm of this species contain both protamines 1 and 2. The two proteins were purified by HPLC and the complete primary sequence of hamster protamine 1 was determined by automated amino acid sequence analysis. The protein sequence was subsequently confirmed by sequencing the PCR-amplified protamine 1 gene. The first forty-two residues of the hamster protamine 2 sequence were obtained by amino acid sequence analysis of the isolated protein, and this sequence was also confirmed and extended by sequencing the gene. Total basic nuclear protein was also isolated from sperm of six other species of hamsters, the protamines were identified by HPLC and amino acid analysis, and the proportion of protamines 1 and 2 in each species was determined. Marked differences in the protamine 2 content of sperm were observed among the different species of hamster. This variation and the high level of sequence similarity between mouse and hamster protamines provide insight into how the two protamines may be organized in sperm chromatin. Mol. Reprod. Dev. 54:273-282, 1999. Published 1999 Wiley-Liss, Inc.     

Rainbow trout protamines. Amino acid sequences of six distinct proteins from a single testis

All the protamines present in detectable amounts in a single mature testis from rainbow trout have been purified to homogeneity using acid extraction, gel filtration chromatography on Bio-Gel P-10, ion-exchange chromatography on carboxymethylcellulose and reverse-phase high-pressure liquid chromatography. Each of the six purified protamines was completely sequenced using automated gas-phase Edman degradation. Each protamine is two-thirds arginine and also contains proline, serine, valine and glycine. Three protamines also contain alanine while two contain isoleucine. Four of the protamines have 32 amino acids while the remaining two have 30. The six protamines have been classified into three families on the basis of their amino acid sequences.     

Zinc-induced secondary structure transitions in human sperm protamines

Using CD we show that human group II protamines undergo novel zinc-dependent secondary structure transitions. The CD spectra of protamine is characteristic of random coil proteins with a large minima at 197 nm. Upon the addition of 1 mM zinc, the magnitude of this minima is decreased by 44%. This spectral change is not induced by 1 mM calcium or magnesium. Cadmium, which has chemical properties similar to zinc, can also induce the structural transition although not as effectively as zinc. The spectral changes that accompany zinc binding are indicative of an increase in beta-turn and anti-parallel beta-sheet structures. This is consistent with the predicted secondary structure for protamines which is dominated by beta-turns. Our data support a model in which protamine adopts a folded structure in the presence of zinc. We propose that a zinc-modulated structure is physiologically significant considering the relatively high levels of zinc in human sperm.     

Nucleoplasmin interaction with protamines. Involvement of the polyglutamic tract

Different recombinant forms of nucleoplasmin including truncations at the carboxyl-terminal end of the molecule (r-NP121, r-NP142) have been expressed and purified. All of them were found to oligomerize, forming pentameric complexes which, according to their hydrodynamic properties, can be modeled by oblate ellipsoids of constant width. In this model, the highly charged carboxyl ends appear to be arranged around a pentameric core along the plane defined by the major axes of the ellipsoid. Importantly, all the recombinant forms appear to be able to decondense protamine-containing sperm nuclei. However, although the stoichiometry with which protamines bind to these forms appears to be constant (2.5 mol of protamine/mol of nucleoplasmin pentamer), the efficiency with which they remove protamines from the sperm DNA decreases in the following order: o-NP > r-NP142 > or = r-NP > r-NP121. Therefore, the main polyglutamic tract of nucleoplasmin (which is absent in r-NP121), while enhancing the efficiency of protamine removal, is not indispensable for sperm chromatin decondensation in the biological model we have used.     

High-performance liquid chromatographic separation and partial characterization of human protamines 1, 2, and 3

A method for separating the three human protamines by HPLC of underivatized, total protamine extracts on a Nucleosil RP-C18 column is described. The identities of the three proteins have been confirmed by a combination of disc gel electrophoresis, amino acid composition, and primary sequence analysis. The results show that human protamine 3 elutes first, closely followed by protamine 2. Protamine 1 elutes later. The amino acid compositions and partial amino terminal sequences of human protamines 2 and 3 indicate that these two proteins are very closely related and suggest that they differ only by three amino-terminal amino acids.     

Factors affecting nucleosome disassembly by protamines in vitro. Histone hyperacetylation and chromatin structure, time dependence, and the size of the sperm nuclear proteins

Histone displaced in vitro from nuclei by protamine competition display a higher degree of hyperacetylation than the residual histones. In addition, hyperacetylated core particle pools are disassembled in vitro with a higher efficiency than control or nonacetylated core particles and when analyzed by electron microscopy display an elongated shape (length/width ratio = 1.52 +/- 0.19) instead of the round compact shape of control nucleosomes (length/width ratio = 1.06 +/- 0.06). In the absence of histone hyperacetylation, the fish protamines, salmine and iridine (32-33 residues), are relatively inefficient in disassembling nucleosomal core particles in vitro as compared to the large (65-70 residues), tyrosine-containing protamines from rooster (galline), squid, and cuttlefish which disassemble nucleosomes in a range of protamine concentrations close to physiological. The fact that an artificially cross-linked salmine dimer acquires the ability of the large protamines from rooster, squid, and cuttlefish to disassemble core particles in vitro and also binds more tightly to the DNA, suggests that the size of the sperm nuclear protamines is a critical factor in this process. Even when the core histones of spermatid chromatin are hyperacetylated in the trout testis, the replacement process by iridine or salmine is slow and time-dependent in vitro. However, since spermiogenesis in trout occurs over several weeks, the slow in vitro nucleosome disassembly process by salmine is sufficient to allow complete displacement, thus supporting the hypothesis that a protamine-mediated displacement of the histones from DNA in vivo may take place in the salmonid fishes by a mechanism similar to that in the rooster, squid, and cuttlefish.     

Phosphorylation of protamines by protein kinase C: involvement of sites which are phosphorylated in vivo and are not affected by cAMP-dependent protein kinase

Most fish protamines contain two phosphorylatable sites both of which incorporate phosphate in vivo. Here we show that in two protamines (salmine A1 and clupeine Y1) the site more distant from the N-terminus (residues 20-21) is unaffected by cAMP-dependent protein kinase while it represents the main target for protein kinase C. Such a phosphorylation is typically independent of Ca2+ and phospholipids: responsiveness to these effectors however is conferred by previous fragmentation of protamine with thermolysin. These results suggest that Ca2+, phospholipid-independent phosphorylation of protamine by protein kinase C might have physiological relevance and shed light on the structural basis for the specificity of such an unique process.     

Cuttlefish sperm protamines. 1. Amino acid sequences of two distinct variants

The amino acid sequences of two cuttlefish protamine variants Sp1 and Sp2 have been established from automated sequence analysis and mass spectrometry data. Sp1 (57 residues) and Sp2 (56 residues) have molecular masses of 8410 and 8253 Da, respectively. They are almost identical proteins which differ only by one residue of arginine and the position of two of the serine residues (14 and 37 in Sp1; 13 and 35 in Sp2). With an arginine content of about 77%, cuttlefish protamine is one of the most basic proteins which have ever been characterized and the first typical protamine sequenced in invertebrates. It is closely similar to sperm basic proteins identified in squids but strongly differs from the protamine-like components isolated from the sperm of bivalve molluscs.     

Isolation and amino-acid sequence analysis of human sperm protamines P1 and P2. Occurrence of two forms of protamine P2

The two protamines of human sperm cell nuclei, P1 and P2, were isolated in pure form after extraction with 6M guanidine/5% mercaptoethanol and alkylation with vinyl pyridine by reversed-phase high-performance liquid chromatography. The amino-acid sequence of protamine P1 was determined by analysing the intact protein and the fragments obtained by cyanogen bromide cleavage. Out of the 50 amino-acid residues 24 are arginines and 6 are cysteines. The sequence of protamine P2 was determined by analysing the intact protein and the fragments resulting from cleavage with endoproteinase Lys-C and thermolysin. Protamine P2 was found to occur in two forms which only differ in their N-terminal regions. The form P2' is three amino-acid residues longer at the N-terminus than the form P2'. Out of the 57 amino-acid residues in the longer form 27 are arginines and 5 are cysteines. Human protamine P1 is highly homologous with the protamines isolated from bull, boar, ram and mouse sperm cells, but human protamine P2 shows a novel type of structure, although also here the dominant amino acids are arginine and cysteine.     

Sperm nuclei glutathione peroxidases and their occurrence in animal species with cysteine-containing protamines

The selenoenzyme sperm nuclei glutathione peroxidase (snGPx), also called the nuclear form of phospholipid hydroperoxide glutathione peroxidase (n-PHGPx), was found to be involved in the stabilization of condensed sperm chromatin, most likely by thiol to disulfide oxidation of the cysteine residues of the mammalian protamines, small nuclear basic proteins in the nuclei of sperm cells. By applying Acidic Urea-PAGE in combination with SDS-PAGE, snGPx with an apparent molecular mass of 34 kDa and a 24-kDa protein were purified from rat sperm nuclei. The 24-kDa protein was identified by means of mass spectrometry as a truncated form of snGPx produced by cleavage at the N-terminal end. After defined processing of spermatozoa and detergent treatment of the sperm nuclei fraction, snGPx and its truncated form were shown to be the only selenoproteins present in mature mammalian sperm nuclei. Both forms were found in mature rat and horse sperm nuclei but in man only snGPx was detected. In trout and chicken, species with sperm cells which likewise undergo chromatin condensation but do not contain cysteine in their protamines, the snGPx proteins were missing. This can be taken as an indirect proof of the function of snGPx to act as protamine cysteine thiol peroxidase in the mammalian species with cysteine-containing protamines.     

Phosphorylated protamines. I. Binding stoichiometry and thermal stability of complexes in DNA.

To decipher on a molecular level the role of protamine phosphorylation in spermiogenesis, clupeine Z species containing one, two or three serine phosphates were prepared utilizing a recently developed chemical procedure. The melting of complexes with calf thymus DNA showed that thermal stability decreases with increasing degree of phosphorylation. The stoichiometry of the nucleoprotamine complexes was investigated analyzing the melting curves and using the fluorescamine assay recently described. Phosphorylation significantly reduces binding stoichiometry defined as DNA-nucleotides covered by a protamine molecule. Thus, phosphorylated protamines are more densely packed along DNA; the implications on processes occurring in spermiogenesis as i. e. histone replacement, are discussed. A general discussion on the variability in protein-DNA stoichiometry values obtained by different procedures is included.     

Quail (Coturnix japonica) protamine, full-length cDNA sequence, and the function and evolution of vertebrate protamines

Using the chicken protamine gene as a probe, we have isolated and sequenced several positive clones from a quail testis cDNA library which reveal the complete sequence for the quail protamine cDNA. The predicted amino acid sequence for the quail protamine contains the N-terminal tetrapeptide ARYR present in the N-terminal region of the mammalian protamines as well as several conserved motifs and arginine clusters. In addition the size of the quail protamine (56 amino acids) is closer to that of mammals (50 amino acids) than that of the chicken (61 amino acids). Altogether this data strongly suggests the existence of an avian-mammalian protamine gene line during evolution. Southern blot analysis suggests a small number of copies (2) per haploid genome (similar to that of chicken). The reported quail protamine cDNA sequence is the second avian protamine for which the amino acid sequence is available so far and provides new insights into vertebrate protamine function and evolution.     

Marked differences in the ability of distinct protamines to disassemble nucleosomal core particles in vitro

In accordance with the results of classical experiments performed in vitro with calf thymus chromatin and the fish protamine salmine, we have observed that this highly basic, small molecular weight protamine cannot cause major displacement of histones from nucleosomal core particles at concentrations several times higher than physiological (arginine/nucleotide ratios 1-8) and that hyperacetylation of histones facilitates nucleosome disassembly. However, the avian protamine galline, with molecular weight and number of arginine residues almost twice those of common fish protamines, is able to displace the nucleosomal core histones from DNA in vitro at concentrations (arginine/nucleotide ratios 0.6-1.2) within the physiological range (0.8). Our results suggest that the binding of the avian protamine galline to chromatin could be directly involved in the rapid disassembly of nucleosomes that takes place during the nucleohistone nucleoprotamine transition in chicken spermiogenesis.     

Purification and analysis of the major components of chum salmon protamine contained in insulin formulations using high-performance liquid chromatography

A simple high-performance liquid chromatographic method has been developed for the rapid purification and analysis of protamine components contained in insulin formulations. Only a single step is needed to separate peptides whose compositions, sizes, and unusual isoelectric points (pI 13.8) are nearly identical. The method involves their isocratic separation on a reversed-phase column using a pH 2 phosphate buffer and a low acetonitrile content as an eluant. The purified chum salmon components were analyzed by amino acid analysis, solid-phase amino acid sequencing, carboxypeptidase B digests, insulin complexation analysis, and a mass spectrophotometric procedure which gives an accurate mass of the intact peptides. This HPLC purification technique may also be applicable to protamines and other highly basic peptides isolated from other sources.