Philippe Chevaillier

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.

Extraction, purification and characterization of the sperm protamines of the dog-fish Scylliorhinus caniculus.

Dog-fish sperm nuclei contain four low molecular weight basic proteins called scylliorhinines. Protein Z3 is a typical arginine-rich protamine, whilst the three other components, Z1, Z2 and S4, are characterized by high arginine and cysteine contents. In contrast to protamine Z3, which can be directly solubilized by 0.25 M HCl, the three other protamines must be reduced and alkylated before acid extraction. They were further purified by ion-exchange chromatography on carboxymethyl-cellulose. The amino acid compositions and the N-terminal sequences reveal significant differences between scylliorhinines, particularly in their molecular size and amino acid diversity. Moreover, they show no common feature with other sperm-specific protamines previously described.

Phosphorylation of human sperm protamines HP1 and HP2: identification of phosphorylation sites

Human sperm is characterized by a high heterogeneity of its basic nuclear protein complement of pro-protamines, protamines and histones. This heterogeneity is increased by the persistence of phosphorylated protamines in mature spermatozoa. Alkaline phosphatase treatment of whole protein indicated that protamines HP1 and HP2 were phosphorylated to various degrees. Presence of non-phosphorylated and phosphorylated protamines HP1 and HP2 was further demonstrated by electrospray mass spectrometry. Phosphorylation sites of mono- and di-phosphorylated protamine HP1 were identified by automatic Edman degradation of the protein after phosphoserine derivatization to S-ethylcysteine. In both phosphorylated forms, Ser-10 was found phosphorylated; in the di-phosphorylated form, Ser-8 was identified as the second site of phosphorylation. In protamine HP2, the unique site of phosphorylation (Ser-14) was located after limited acid hydrolysis of enzymic peptides and thin-layer electrophoresis.

Chromosomal localization of the human protamine genes, PRM1 and PRM2, to 16p13.3 by in situ hybridization.

SummaryProtamines are sperm-specific proteins that replace histones in the nuclear chromatin of mature spermatozoa. A chromosomal localization of the genes coding for human protamines has been achieved by in situ hybridization. Two cDNA probes of 423 bp and 397 bp containing the entire coding sequence for human protamine 1 (HP1) and human protamine 2 (HP2), respectively, have been used. The genes, called PRM1 and PRM2, have been found, tightly linked, on band 16p13.3. Arguments are given for the existence of these two genes as single copies, PRM1 coding for the unique HP1 protamine and PRM2 coding for a precursor of several proteins belonging to the HP2 family.

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.

Purification and characterization of two basic spermatid‐specific proteins isolated from the dog‐fish Scylliorhinus caniculus

In dog‐fish spermatid nuclei two intermediate proteins S1 and S2 replace histones before the setting down of protamines. These spermatid‐specific proteins were isolated by carboxymethyl‐cellulose chromatography and purified by high pressure liquid chromatography. S1 and S2 are characterized by a high content of basic residues and by the lack of cysteine and phenylalanine. The determination of their amino acid composition and of their N‐ and C‐terminal sequences prove that each protein corresponds to a specific molecule which can be considered neither as a histone hydrolytic product nor as a protamines precursor.

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.

Basic homopolyamino acids, histones and protamines are potent antagonists of angiogenin binding to ribonuclease inhibitor

A radio‐ribonuclease inhibitor assay based on the interaction of 125I‐angiogenin with ribonuclease inhibitor (RI) was used to detect pancreatic‐type ribonucleases and potential modulators of their action. We show that highly basic proteins including the homopolypeptides poly‐arginine, poly‐lysine and poly‐ornithine, core histones, spermatid‐specific S1 protein and the protamines HP3 and Z3 were strong inhibitors of angiogenin binding to RI. A minimum size of poly‐arginine and poly‐lysine was required for efficient inhibition. The inhibition likely resulted from direct association of the basic proteins with the acidic inhibitor, as RI bound to poly‐lysine and protamines while 125I‐angiogenin did not. Antagonists of the angiogenin‐RI interaction are potential regulators of either angiogenin‐triggered angiogenesis and/or intracellular RI function, depending on their preferential target.