Arlette Martinage

Purification and characterization of nuclear basic proteins of human sperm

Highly purified nuclei were obtained from human sperm without protein loss through the use of CHAPS (3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate), a newly available detergent. The basic protein complement of these nuclei is highly heterogeneous and comprises histones (some of which are testis-specific), protamines and proteins of intermediate basicity and molecular size. The protamines belong to two different classes of protein. Microheterogeneity observed in some of these protamines originates from slight variations in their amino acid composition as well as from post-synthetic modifications. Two of these protamines previously considered as two different proteins are in fact the same protein with different degrees of phosphorylation. All these protamines and intermediate basic proteins are characterized by high amounts of arginine and cysteine. Three of the protamines and all five intermediate basic proteins are also histidine-rich.

Nuclear basic proteins in spermiogenesis

In animal species, spermiogenesis, the late stage of spermatogenesis, is characterized by a dramatic remodelling of chromatin which involves morphological changes and various modifications in the nature of the nuclear basic proteins. According to the evolution of species, three situations can be observed: a) persistence of somatic histones or appearance of sperm-specific histones; b) direct replacement of histones by generally smaller and more basic proteins called protamines; and c) occurrence of a double nuclear basic protein transition: histones are not directly replaced by protamines but by intermediate basic proteins which are themselves replaced by one or several protamines. However, in some species, two kinds of intermediate basic proteins can be distinguished in spermatid nuclei: transition proteins and protamine precursors. Whereas transition proteins are not structurally related either to histones or to protamines, protamine precursors are further processed at the end of spermiogenesis to give rise to the mature protamine. The molecular characteristics of the protamines as well as number of protamine types present in the spermatozoon vary from species to species. In some cases, protamine-encoding genes, although present, are not expressed to a significant level. The diversity and the precise function of intermediate basic proteins remain open to discussion. Some of them are the precursors of protamines but the mechanism, sequential or not, as well as the enzyme(s) involved in the proteolytic processing, remain to be discovered.

Post-translational chemical modification(s) of proteins.

1. The role played by the modification of protein in determining its fate is reported by us. 2. Post-translational modifications such as acetylation, phosphorylation, sulfation, methylation, hydroxylation, ADP-ribosylation, maturation, amidation, carboxylation, adenylylation, glycosylation, ubiquitination, and prenylation are extensively reviewed. 3. Each post-translational modifications significance and its role played in biological function(s) is summarized in the general discussion and the conclusions remark is directed at the problems left to solve (e.g. post-translational modification reactions in recombinant protein in modern genetic engineering).

Histone phosphorylation in native chromatin induces local structural changes as probed by electric birefringence

In order to understand how the phosphorylation of histones affects the chromatin structure, we used electron microscopy, sedimentation velocity, circular dichroism and electric birefringence to monitor the salt-induced filament reversible solenoid transition of phosphorylated and native chromatin. Phosphorylation in vitro of chicken erythrocyte chromatin by cyclic-AMP-dependent protein kinase from porcine heart led to the modification of the histones H3 and H5 only, which were modified at a level of one phosphate and about three phosphate groups per molecule, respectively. In contrast to circular dichroism and sedimentation studies, which tend to suggest that phosphorylation of H3 and H5 does not affect chromatin structure, electron microscopy reveals that phosphorylation causes a relaxation of structure at low ionic strength. Electric birefringence and relaxation time measurements clearly prove that local structural changes are induced in chromatin: we observe a decrease of the steady-state birefringence with the appearance of a negative contribution in the signal and a marked increase of the flexibility of fibres. The component with the negative birefringence presents very short relaxation times, like those exhibited by small DNA fragments or individual nucleosomes. Two possibilities are then suggested. First, the conformational change is consistent with what would be expected from the presence of DNA segments loosely associated with the core histone H3. That the length of such segments could correspond to about one to two base-pairs per nucleosome strongly suggests that phosphorylation induces changes affecting some specific H3-DNA interactions only. This result could corroborate previous observations indicating that the N-terminal region of H3, where the site of phosphorylation is located, plays a decisive role in maintaining the superstructure of chromatin. Second, phosphorylation could introduce hinge points between each nucleosome. In this case, the negative birefringence results from partial orientation of the swinging nucleosomes. A possible mode of action of phosphorylation might be to weaken structural restraints imposed by histone H3, thus facilitating further condensation of chromatin.

Amino acid sequence of a cysteine-rich, arginine-rich sperm protamine of the dog-fish Scylliorhinus caniculus

Abstract Scylliorhinine Z2 is a small basic protein of 46 residues which has been purified from sperm nuclei of the dog-fish Scylliorhinus caniculus . It contains a high proportion of basic residues and of cysteine. When, compared to other protamines, it is characterized by the diversity of the amino acids represented in the molecule. The amino acid sequence shows that the N-terminal half is highly basic and that the hydrophobic residues are preferentially localized in the C-terminal region. The three residues with a hydrophilic side-chain are clustered and serine can occur in a phosphorylated forms; in mature sperm, the major fraction of the protamine is monophosphorylated, whereas mono- and diphosphorylated molecules are found during the terminal differentiation of the gamete within the testis. Scylliorhinine Z2 does not share common structural properties either with the other two scylliorhinines or with other fish protamines whose sequences have been previously determined.

Primary structure of scylliorhinine S4, a protamine isolated from sperm nuclei of the dog-fish Scylliorhinus caniculus

Abstract The primary structure of scylliorhinine S4, a second protamine extracted from sperm nuclei of the dog-fish Scylliorhinus caniculus has been determined. This protein contains 32 residues, 65.6% being basic, and the reduced form of the molecule has an Mr of 3877. The amino-acid composition of the protein is: Pro2, Gly1, Ala3, Cys4, Val1, Lys14, Arg7. The unique glycine residue of the molecule represents its N-terminal end and one of the four cysteine residues is C-terminal. Compared to other protamines previously sequenced, scylliorhinine S4 is characterized by high levels of lysine and cysteine. The amino-acid sequence of scylliorhinine S4 reveals no clear homology with other protamines but two identical nonapeptides are tandemly repeated within the molecule.

Phosphorylation ofToxoplasma gondii major surface antigens

The phosphorylation of the major surface proteins ofToxoplasma gondii tachyzoites was investigated. Metabolic labeling of intracellular tachyzoites with [32P]-orthophosphate followed by immunoprecipitation with specific monoclonal antibodies showed that all five major surface proteins were labeled as expected for GPI-anchored proteins. More detailed analysis of the phosphorylated sites revealed that surface proteins P30 and P22 contained phosphoserine residues in addition to the phosphorylated molecules that are presumably localized in the GPI-membrane anchors

Amino-acid sequence of scylliorhinine Z1 and comparison of the primary structure of the protamines of the dogfish Scylliorhinus caniculus

Abstract Scylliorhinine Z1 is one of the four protamines of mature sperm nuclei of the dogfish. This protein has a high content of arginine and cysteine and the polypeptide chain contains 50 residues. These molecular characteristics are also found in mammalian protamines, but differ from those of teleost protamines. The amino-acid sequence reveals that the N-terminal half is enriched in hydrophobic amino acids, whereas the C-terminal domain is more basic and contains a cluster of four hydroxylated amino acids. A sequence of eight amino acids is duplicated. The protein remains monophosphorylated in mature sperm nuclei. The amino-acid sequence reveals no clear homology with teleost protamines. Moreover, the four scylliorhinines could play specific functions in DNA packaging, as they do not appear to be structurally related. Therefore, they cannot be considered as being derived from a unique ancestral gene, as has been suggested for teleost protamines.