Protamine-like Research Articles

Toxicological effects and potential reproductive risk of microplastic-induced molecular changes in protamine-like proteins and their DNA binding

Today, plastic pollution is a widespread problem in all ecosystems and has a particularly severe impact on marine ecosystems and external fertilisers such as the mussel Mytilus galloprovincialis. The present study aims to assess the toxicological reproductive health effects in this organism following exposure to two concentrations of polystyrene microplastics (PS-MPs) (0.5 and 1 μg/L), representative of conditions in the Mediterranean Sea. After exposure, the electrophoretic pattern of protamine-like (PL) proteins, the major basic protein component of Mytilus galloprovincialis sperm chromatin, was analysed. Compared to the unexposed condition, differences were observed by SDS-PAGE and an increased ability of PL to bind and protect DNA from oxidative damage was then measured, particularly for PL from mussels exposed to 1 μg/L PS-MPs. At this dose of PS-MPs, a reduced release of all PLs from the sperm nuclei was also observed, whereas the digestion by micrococcal nuclease did not show any significant differences between the exposed and the unexposed conditions. Finally, the possibility of poly(ADP)-ribosylation of the PLs was investigated. PL-II showed an increase in poly(ADP)-ribosylation after PS-MPs exposure, which may account for the difference in the ability of the PLs to bind DNA. In conclusion, while all the results might suggest a molecular mechanism of gametic plasticity occurring upon exposure of mussels to PS-MPs 1 μg/L, they also indicate that this dose of exposure could be extremely detrimental to the reproductive health of Mytilus galloprovincialis because it could prevent the release of basic nuclear proteins from the sperm DNA at fertilisation.

Molecular and toxicological mechanisms behind the effects of chromium (VI) on the male reproductive system of Mytilus galloprovincialis: First evidence for poly-ADP-ribosylation of protamine-like II

Studies on the molecular mechanisms of heavy metal toxicity in invertebrate reproduction are limited. Given that PARP-catalysed ADP-ribosylation is also involved in counteracting heavy metal toxicity and maintaining genomic integrity, and that PARylation is implicated in chromatin remodelling but its role in sperm chromatin remains to be elucidated, we investigated the effects of chromium(VI) at 1, 10 and 100 nM on the reproductive health of Mytilus galloprovincialis. The damage to the gonads was assessed by morphological analyses and the damage indices PARP and ɣH2A.X were measured. Changes in the binding of protamine-like (PL) to DNA and the possibility of poly(ADP-ribosyl)ation of PL proteins were also investigated. Gonadal chromium accumulation and morphological damage were found, especially when the mussels were exposed to the highest dose of chromium(VI). In addition, the maximum expression of gonadal ɣH2A.X and PARP were obtained at 100 and 10 nM Cr(VI), respectively. Interestingly, for the first time in all exposed conditions, poly(ADP)-ribosylation was detected on PL-II, which, together with PL-III and PL-IV, are the major nuclear basic proteins of Mytilus galloprovincialis sperm chromatin. Since PL-II is involved in the final high level of sperm chromatin compaction, this post-translational modification altered the binding of the PL protein to DNA, favouring the action of micrococcal nuclease on sperm chromatin. This study provides new insights into the effects of chromium(VI) on Mytilus galloprovincialis reproductive system and proposes a molecular mechanism hypothesis describing the toxic effects of this metal on PL-DNA binding, sperm chromatin and gonads.

Possible Molecular Mechanisms Underlying the Decrease in the Antibacterial Activity of Protamine-like Proteins after Exposure of Mytilus galloprovincialis to Chromium and Mercury.

Natural bioactive compounds represent a new frontier of antimicrobial molecules, and the marine ecosystem represents a new challenge in this regard. In the present work, we evaluated the possibility of changes in the antibacterial activity of protamine-like (PL) proteins, the major nuclear basic protein components of Mytilus galloprovincialis sperm chromatin, after the exposure of mussels to subtoxic doses of chromium (VI) (1, 10, and 100 nM) and mercury (1, 10, and 100 pM) HgCl2, since these metals affect some properties of PL. After exposure, we analyzed the electrophoretic pattern of PLs by both acetic acid-urea polyacrylamide gel electrophoresis (AU-PAGE) and SDS-PAGE and determined the MIC and MBC of these proteins on different gram+ and gram- bacteria. PLs, particularly after mussels were exposed to the highest doses of chromium and mercury, showed significantly reduced antibacterial activity. Just at the highest doses of exposure to the two metals, changes were found in the electrophoretic pattern of PLs, suggesting that there were conformational changes in these proteins, which were confirmed by the fluorescence measurements of PLs. These results provide the first evidence of a reduction in the antibacterial activity of these proteins following the exposure of mussels to these metals. Based on the results, hypothetical molecular mechanisms that could explain the decrease in the antibacterial activity of PLs are discussed.

Altered Expression of Protamine-like and Their DNA Binding Induced by Cr(VI): A Possible Risk to Spermatogenesis?

Chromium (VI) is the most dangerous oxidation state among the stable forms of chromium. In this work, we evaluated the effect of exposing Mytilus galloprovincialis for 24 h to 1, 10, and 100 nM chromium (VI) on the properties of Protamine-like (PLs) and their gene levels in the gonads. Specifically, we analyzed, by AU-PAGE and SDS-PAGE, PLs extracted from unexposed and exposed mussels. In addition, via EMSA, we evaluated the ability of PLs to bind DNA and also verified their potential to protect DNA from oxidative damage. Finally, we assessed possible alterations in gonadal expression of mt10, hsp70, and genes encoding for PLs-II/PL-IV and PL-III. We found that for all experimental approaches the most relevant alterations occurred after exposure to 1 nM Cr(VI). In particular, a comigration of PL-II with PL-III was observed by SDS-PAGE; and a reduced ability of PLs to bind and protect DNA from oxidative damage was recorded. This dose of chromium (VI) exposure was also the one that produced the greatest alterations in the expression of both mt10 and PL-II/PL-IV encoding genes. All of these changes suggest that this dose of chromium (VI) exposure could affect the reproductive health of Mytilus galloprovincialis.

New Insights into Alterations in PL Proteins Affecting Their Binding to DNA after Exposure of Mytilus galloprovincialis to Mercury-A Possible Risk to Sperm Chromatin Structure?

Mercury (Hg) is a highly toxic and widespread pollutant. We previously reported that the exposure of Mytilus galloprovincialis for 24 h to doses of HgCl2 similar to those found in seawater (range 1–100 pM) produced alterations in the properties of protamine-like (PL) proteins that rendered them unable to bind and protect DNA from oxidative damage. In the present work, to deepen our studies, we analyzed PL proteins by turbidimetry and fluorescence spectroscopy and performed salt-induced release analyses of these proteins from sperm nuclei after the exposure of mussels to HgCl2 at the same doses. Turbidity assays indicated that mercury, at these doses, induced PL protein aggregates, whereas fluorescence spectroscopy measurements showed mercury-induced conformational changes. Indeed, the mobility of the PLII band changed in sodium dodecyl sulphate-polyacrylamide gel electrophoresis, particularly after exposure to 10-pM HgCl2, confirming the mercury-induced structural rearrangement. Finally, exposure to HgCl2 at all doses produced alterations in PL-DNA binding, detectable by DNA absorption spectra after the PL protein addition and by a decreased release of PLII and PLIII from the sperm nuclei. In conclusion, in this paper, we reported Hg-induced PL protein alterations that could adversely affect mussel reproductive activity, providing an insight into the molecular mechanism of Hg-related infertility.

Spermatozoa Transcriptional Response and Alterations in PL Proteins Properties after Exposure of Mytilus galloprovincialis to Mercury.

Mercury (Hg) is an environmental pollutant that impacts human and ecosystem health. In our previous works, we reported alterations in the properties of Mytilus galloprovincialis protamine-like (PL) proteins after 24 h of exposure to subtoxic doses of toxic metals such as copper and cadmium. The present work aims to assess the effects of 24 h of exposure to 1, 10, and 100 pM HgCl2 on spermatozoa and PL proteins of Mytilus galloprovincialis. Inductively coupled plasma–mass spectrometry indicated accumulation of this metal in the gonads of exposed mussels. Further, RT-qPCR analyses showed altered expression levels of spermatozoa mt10 and hsp70 genes. In Mytilus galloprovincialis, PL proteins represent the major basic component of sperm chromatin. These proteins, following exposure of mussels to HgCl2, appeared, by SDS-PAGE, partly as aggregates and showed a decreased DNA-binding capacity that rendered them unable to prevent DNA damage, in the presence of CuCl2 and H2O2. These results demonstrate that even these doses of HgCl2 exposure could affect the properties of PL proteins and result in adverse effects on the reproductive system of this organism. These analyses could be useful in developing rapid and efficient chromatin-based genotoxicity assays for pollution biomonitoring programs.

Seasonal dependence of cadmium molecular effects on Mytilus galloprovincialis (Lamarck, 1819) protamine-like protein properties.

Mussels have a seasonal reproduction and cadmium is a common stressor in estuarine and coastal environments. In previous studies, we have shown that exposure to subtoxic doses of cadmium produced alterations in the properties of winter Mytilus galloprovincialis sperm protamine-like (PL) proteins. In this study, it was analyzed the possibility that these cadmium effects may be seasonal. Winter and summer mussels were exposed to CdCl2 , and it was tested the PL-proteins for cadmium bioaccumulation, electrophoretic pattern, DNA binding, and potentiality to induce DNA oxidative damage. It was found that cadmium exposure did not produce the same effects on PL-proteins of summer mussels that were produced on PL-proteins of winter mussels, that is: cadmium bioaccumulation, alterations in the acetic acid-urea polyacrylamide gels (AU-PAGE) and sodium dodecyl sulfate-PAGE pattern, a reduced DNA binding affinity and the ability to induce DNA oxidative damage. PL-proteins from summer mussels, apart from not being affected by all the abovementioned effects of cadmium, also showed a very low DNA binding affinity, independent of cadmium exposure. This study reveals clock-associated seasonal responses to cadmium in M. galloprovincialis. Understanding the mechanisms through which environmental signals guide biological rhythms is fundamental to understanding the seasonal sensitivity of this bioindicator, to use M. galloprovincialis in appropriate seasonal periods.

A Sperm Nuclear Basic Protein from the Sperm of the Marine WormChaetopterus variopedatuswith Sequence Similarity to the Arginine-Rich C-Termini of Chordate Protamine-Likes

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.

Origin and evolution of chromosomal sperm proteins

In the eukaryotic cell, DNA compaction is achieved through its interaction with histones, constituting a nucleoprotein complex called chromatin. During metazoan evolution, the different structural and functional constraints imposed on the somatic and germinal cell lines led to a unique process of specialization of the sperm nuclear basic proteins (SNBPs) associated with chromatin in male germ cells. SNBPs encompass a heterogeneous group of proteins which, since their discovery in the nineteenth century, have been studied extensively in different organisms. However, the origin and controversial mechanisms driving the evolution of this group of proteins has only recently started to be understood. Here, we analyze in detail the histone hypothesis for the vertical parallel evolution of SNBPs, involving a "vertical" transition from a histone to a protamine-like and finally protamine types (H --> PL --> P), the last one of which is present in the sperm of organisms at the uppermost tips of the phylogenetic tree. In particular, the common ancestry shared by the protamine-like (PL)- and protamine (P)-types with histone H1 is discussed within the context of the diverse structural and functional constraints acting upon these proteins during bilaterian evolution.

The Sperm Proteins from Amphioxus Mirror Its Basal Position among Chordates and Redefine the Origin of Vertebrate Protamines

The sperm nuclear basic proteins (SNBPs) that participate in chromatin condensation in spermatozoa belong to 3 groups: histone (H), protamine-like (PL), and protamine (P) type. They share a common origin with histone H1 resulting from the segregation of PL components, corresponding to different regions of an H1 precursor molecule (N-terminal, winged-helix, C-terminal domains), becoming independent and following a subsequent process of parallel vertical evolution (H <--> PL <--> P). In the present work, we describe the sequence and primary structure of the main SNBP component in the sperm of the cephalochordate Branchiostoma floridae (amphioxus), revealing that it represents the deuterostome counterpart of the PL-III SNBP component from molluscs corresponding to the H1 N-terminal region. Until now, this has been a missing piece needed to complete the evolutionary history of SNBPs in metazoan genomes. The discovery of this PL lineage in deuterostomes definitively validates the parallel vertical evolution of SNBPs across metazoans, giving further support to the "basal" position of amphioxus among chordates, with respect to tunicates. Sequence analyses suggest that later on in evolution, the appearance of positively selected arginine-rich protamines, derived from the H1 C-terminal region, led to the extinction of this PL lineage in the genomes of early protostomes and deuterostomes. Given that tunicates are now viewed as a sister group of vertebrates, the lysine to arginine transition responsible for the origin of vertebrate protamines must be set a step back from tunicates.

Common Phylogenetic Origin of Protamine-like (PL) Proteins and Histone H1: Evidence from Bivalve PL Genes

Sperm nuclear basic proteins (SNBPs) can be grouped into three main categories: histone (H) type, protamine (P) type, and protamine-like (PL) type. Protamine-like SNBPs represent the most structurally heterogeneous group, consisting of basic proteins which are rich in both lysine and arginine amino acids. The PL proteins replace most of the histones during spermiogenesis but to a lesser extent than the proteins of the P type. In most instances, PLs coexist in the mature sperm with a full histone complement. The replacement of histones by protamines in the mature sperm is a characteristic feature presented by those taxa located at the uppermost evolutionary branches of protostome and deuterostome evolution, while the histone type of SNBPs is predominantly found in the sperm of taxa which arose early in metazoan evolution; giving rise to the hypothesis that protamines may have evolved through a PL type intermediate from a primitive histone ancestor. The structural similarities observed between PL and H1 proteins, which were first described in bivalve molluscs, provide a unique insight into the evolutionary mechanisms underlying SNBP evolution. Although the evolution of SNBPs has been exhaustively analyzed in the last 10 years, the origin of PLs in relation to the evolution of the histone H1 family still remains obscure. In this work, we present the first complete gene sequence for two of these genes (PL-III and PL-II/PL-IV) in the mussel Mytilus and analyze the protein evolution of histone H1 and SNBPs, and we provide evidence that indicates that H1 histones and PLs are the direct descendants of an ancient group of "orphon" H1 replication-dependent histones which were excluded to solitary genomic regions as early in metazoan evolution as before the differentiation of bilaterians. While the replication-independent H1 lineage evolved following a birth-and-death process, the SNBP lineage has been subject to a purifying process that shifted toward adaptive selection at the time of the differentiation of arginine-rich Ps.

Sperm nuclear basic proteins of two closely related species of scorpaeniform fish (Sebastes maliger, Sebastolobus sp.) with different sexual reproduction and the evolution of fish protamines

In this paper, we present a review of sperm nuclear basic proteins (SNBPs) in teleost fish. The distribution of the three basic groups of SNBPs [histone (H)-type, protamine-like (PL)-type and protamine (P)-type], their evolution and possible relation to the mode of fertilization are described. In this regard, we have characterized the SNBPs from two closely related species of Scorpaeniform fish: internally fertilizing Sebastes maliger and externally fertilizing Sebastolobus sp., both in the family Scorpaenidae. Despite the different reproductive behavior of these two closely related rockfish species, in both instances the SNBP consists of protamines. However, there is a significant increase in the arginine content of the protamine in the internally fertilizing rockfish. The relevance of this observation is discussed within the context of the P-type SNBP in teleosts. The rapid evolution of teleost protamines, including those in rockfish, has also allowed us to obtain a molecular phylogeny for this group of bony fish that is almost indistinguishable from that currently available from the use of conventional anatomical/paleontological markers.

Basic nuclear protein pattern and chromatin condensation in the male germ cells of a tropical abalone, Haliotis asinina

The basic nuclear proteins (BNPs) in spermatozoa of a tropical abalone, Haliotis asinina, were composed of a majority of protamine-like (PL) protein and a small amount of histones H1 and H4. Abalone H1 and PL proteins exhibited strong immunological cross reactivities among themselves as well as with chick H5 and calf thymus H1. Thus, all these proteins may belong to the same family. Immunolocalization by indirect immunofluorescence and immunoelectron microscopy indicated that H1 and H4 were present in all steps of the male germ cells, however, with decreasing amount in late stage cells, particularly spermatids and spermatozoa. On the other hand, PL was present in middle step cells (secondary spermatocytes) with increasing amount in spermatids and spermatozoa when the chromatin became tightly packed. Thus, PL may be involved in the condensation of chromatin in the spermatozoa of this species.

PL-I of Spisula solidissima, a highly elongated sperm-specific histone H1.

The major chromosomal protein of the mature sperm of the surf clam, Spisula solidissima, is a histone H1-related protamine-like (PL-I) protein of low electrophoretic mobility. We report here the complete sequence of two isoforms of its encoding genes. These genes encode a protein of 453 and 454 amino acids, respectively. The predicted mass of the larger isoforms (51,437 Da) was confirmed using electrospray ionization mass spectrometry. The amino-terminal tail of the S. solidissima PL-I is greatly elongated because of the presence of 39 tandem hexapeptide repeats of the motif (K/R)KRSAS with a few semiconservative amino acid substitutions. These repeats are very closely mirrored by their encoding DNA sequence, which indicates that an expansion because of sequence duplication most likely occurred. The C-terminal domain consists of a histone H1-related core with a predicted winged-helix tertiary structure, which is followed by an unstructured lysine-rich tail. This information provides additional molecular support for the classification and underlying evolution of sperm nuclear basic proteins in bivalve molluscs.

Protamine-like proteins: evidence for a novel chromatin structure.

Protamine-like (PL) proteins are DNA-condensing proteins that replace somatic-type histones during spermatogenesis. Their composition suggests a function intermediate to that of histones and protamines. Although these proteins have been well characterized at the chemical level in a large number of species, particularly in marine invertebrates, little is known about the specific structures arising from their interaction with DNA. Speculation concerning chromatin structure is complicated by the high degree of heterogeneity in both the number and size of these proteins, which can vary considerably even between closely related species. After careful examination and comparison of the protein sequences available to date for the PL proteins, we propose a model for a novel chromatin structure in the sperm of these organisms that is mediated by somatic-type histones, which are frequently found associated with these proteins. This structure supports the concept that the PL proteins may represent various evolutionary steps between a sperm-specific histone H1 precursor and true protamines. Potential post-translational modifications and the control of PL protein expression and deposition are also discussed.

Chromosomal Proteins of the Sperm of a Cephalochordate (Branchiostoma floridae) and an Agnathan (Petromyzon marinus): Compositional Variability of the Nuclear Sperm Proteins of Deuterostomes.

We have isolated and characterized for the first time the chromosomal proteins from the nucleus of the sperm of a lancelet (amphioxus) Branchiostoma floridae (Hubbs, 1922) (Phylum Chordata: Subphylum Cephalochordata) and of a lamprey Petromyzon marinus (Linnaeus, 1758) (Phylum Chordata: Subphylum Vertebrata: Class Agnatha). In the first case, the major protein component of the sperm-chromatin of a lancelet is a highly specialized protamine-like (PL) protein that has structural and compositional features similar to those of PL-III from bivalve mollusks. In contrast, the chromatin of the sperm of the lamprey has a structural arrangement and protein composition (histones) very similar to that found in the somatic cells of all eukaryotic organisms. Among the deuterostomes, chromosomal protein variability is considerably greater in representatives of the Phylum Chordata than in echinoderms. The possible evolutionary significance of these findings is discussed.

Microheterogeneity and interspecific variability of the nuclear sperm proteins from Mytilus

We have used acetic acid-urea-triton (AUT) gel electrophoresis and ionic exchange chromatography in order to analyze the interspecific, variability and microheterogeneity pattern of the protamine-like (PL) protiens of the sperm or 4 different species or the bivalve molluse, Mytilus. We have found that based upon these 2 criteria, it is possible to unambiguously distinguish each species from the rest. We have thus been able to corroborate the identity or M. irossulus, We have also analyzed the amino acid composition of some of the PL components for each different species, In the case of the PL-11* fraction, we have analyzed the composition of its major protein subcomponents.

An unusual cysteine-containing histone H1-like protein and two protamine-like proteins are the major nuclear proteins of the sperm of the bivalve mollusc Macoma nasuta.

The sperm-specific protamine-like (PL) components PL-I, PL-II, and PL-III from the sperm of the bent-nose clam Macoma nasuta have been isolated and characterized for the first time. These proteins coexist in the sperm nuclei with a small percentage of a full histone complement. All of them have a very similar amino acid composition, following what seems to be the general composition prototype for the class Bivalvia (Ausió, J. (1986) Comp. Biochem. Physiol. B Comp. Biochem. 85, 439-449). Nevertheless, they have different molecular weights (PL-I = 23,500, PL-II = 15,600, and PL-III = 7,900) as measured by sedimentation equilibrium in the analytical ultracentrifuge. Furthermore, the PL-I component shares common features with the proteins of the histone H1 family. Yet, it is very unusual, for it contains 2 cysteine residues that are located in the trypsin-resistant core of this protein. The protamine-like fraction PL-III exhibits intraspecific microheterogeneity which is reflected by the presence of two protein variants which most probably are the result of an allelic polymorphism.