Jacqueline Chauvet

The neurohypophysial endocrine regulatory cascade: precursors, mediators, receptors, and effectors.

The neurohypophysial endocrine regulatory cascade has been described as a molecular model of neuroendocrine control of organismal functions. Any physiological function can be analyzed in molecular terms as a succession of interactions occurring either in a solution or in a membrane system. The key mechanism in the ordering of the cascade is the conformational recognition of the two partners at each step. Each interaction results in a change of conformation of a recognized protein that in turn becomes a recognizer for the following molecule. The cascade starts within the secretory cell by the processing of the expressed precursor along the secretory pathway until the storage of the mature mediator in vesicles and its subsequent exocytic secretion in blood. The circulating mediator recognizes the target cell through specific membrane receptors that transduce the message within this target cell. A second intracellular cascade leads to activation of the effector, the protein fulfilling the physiological function. The complexity of the messages is, in part, due to the duplication propensity of the genomic DNA, the frequent occurrence of multiple copies for precursors, mediators, receptors, and effectors, and therefore, a combinatorial diversity that increases during the course of evolution. Vertebrate neurohypophysial hormones can be ordered in two main evolutionary lineages, culminating in oxytocin and vasopressin in placental mammals. In this field, diversification of the messages was made by differential processing of the precursors, secondary gene duplications, the emergence of several types of receptors for each hormone, and a variety of effectors triggered by the second messengers within differentiated target cells. This review is an attempt to integrate neurohypophysial functions at the molecular, cellular, and organismal levels.

L'ocytocine et la vasopressine du mouton: Reconstitution d'un complexe hormonal actif

Abstract From sheep posterior hypophysis a complex was obtained containing 90% of the oxytocic and vasopressic activity of the gland. The complex is an association of oxytocine and vasopressine with a protein, neurophysine. This complex was dissociated by TCA and the three components were purified by chromatography. The reconstitution of the complex from the elements was realised under specified conditions. Analogous complexes were obtained from glands of cow, horse and pig. It is possible to bring about associations between the hormones and neurophysines of different species.

Phylogeny of neurophysins: partial amino acid sequence of a sheep neurophysin.

Neurophysins [ 1 ] are proteins which are apparently associated with neurohypophysial hormones in the posterior pituitary gland and stable protein-hormone complexes have been isolated [2,3]. The components can be separated and reassociated for giving a complex similar to the native one [3] . In the mammalian species so far investigated, several neurophysin components have often been disclosed by electrophoresis but the real number of native neurophysins can hardly be determined by this procedure because of the possible presence of partially degraded forms [4]. Up to now the complete or partial amino acid sequences of two bovine [5-71, two porcine [8,9] and one human [ 10,l l] neurophysins have been determined. Our research on sheep neurophysins has revealed that a major neurophysin (about 70% of crude neurophysin) is present in the gland (M. T. Chauvet, G. Coffe, J. Chauvet and R. Acher unpublished results). This report deals with the determination of the Nterminal sequence of the major ovine neurophysin.

Structure, processing and evolution of the neurohypophysial hormone-neurophysin precursors.

Neurohypophysial hormones and neurophysins are derived from common precursors processed during the axonal transport from the hypothalamus to the neurohypophysis. Two neurohormones, an oxytocin-like and a vasopressin-like, on one hand, two neurophysins, termed VLDV-and MSEL-neurophysins according to residues in positions 2, 3, 6 and 7, on the other, are usually found in vertebrate species. In contrast to placental mammals that have oxytocin and arginine vasopressin, marsupials have undergone a peculiar evolution. Two pressor peptides, lysipressin and vasopressin for American species, lysipressin and phenylpressin for Australian macropods, have been identified in individual glands and it is assumed that the primordial vasopressin gene has been duplicated in these lineages. On the other hand, the reptilian mesotocin is still present in Australian species instead of the mammalian oxytocin, while the North American opossum has both hormones and South American opossums have only oxytocin. The neurophysin domain of each precursor is encoded by 3 exons and different evolutionary rates have been found for the 3 corresponding parts of the protein. The central parts, encoded by the central exons, are evolutionarily very stable and nearly identical in the 2 neurophysins of a given species. Recurrent gene conversions have apparently linked the evolutions of the 2 precursor lineages. In mammals, the 3-domain precursor of vasopressin is processed in 2 stages: a first cleavage splitting off vasopressin and a second cleavage separating MSEL-neurophysin from copeptin. Two distinct enzymatic systems seem to be involved in these cleavages. Processing is usually complete at the level of the neurohypophysis, but an intermediate precursor encompassing MSEL -neurophysin and copeptin linked by an arginine residue has been characterized in guinea pig. In vitro processing of this intermediate through trypsin--Sepharose reveals cleavages only in the interdomain region. In non-mammalian tetrapods, such as birds and amphibians, mesotocin and vasotocin are associated with neurophysins in precursors similar to those found in mammals. However, processing of the vasotocin precursor seems to be different from the processing of the vasopressin precursor, with a single cleavage leading to the hormone release.

Dynamic processing of neuropeptides

Neurohypophysial preprohormones are single polypeptide chains folded into 3/4 domains, namely a signal prepeptide (18/20 residues), a hormone peptide (9 residues), and a propeptide neurophysin-copeptin (93/134 residues). Neuro-hormone and neurophysin contain 1 and 7 disulfide bridges, respectively, whose pairing depends on correct primordial folding within the endoplasmic reticulum (ER) compartment (pH 7.0) of hypothalamic magnocellular neurons. During intracellular travel in the secretory pathway from ER to secretory granules (SG), the precursor is submitted to successive processings (glycosylation, proteolysis, amidation) in distinct compartments, leading to domain separation and reshaping. In particular the hormone domain is subjected, in the SG, pH 5.5, to a 4-enzyme cascade in order to reach the bioactive conformation. We have purified SG from rat and ox neurohypophyses and characterized: 1) the processed domains (neurohormone, neurophysin, copeptin); 2) the four processing enzymes acting successively at the level of the processing sequence, namely a Lys-Arg calcium-dependent endopeptidase, a carboxypeptidase B-like enzyme, a peptidyl-glycine monooxygenase and a peptidyl-hydroxyglycine lyase (amidating enzyme).A reconstitution of the processing has been carried out in vitro using purified granular enzymes and synthetic nonactive prohormone peptides, vasopressinyl-Gly-Lys-Arg, vasotocinyl-Gly, and oxytocinyl-Gly. Vasopressin (yield 17% at pH 6.0, 30% at pH 8.0) has been identified by both coelution in high-performance liquid chromotography (HPLC) and bioactivity.In the homozygote mutant Brattleboro rats, a single nucleotide deletion in the gene entails a complete change in aminoacid sequence of neurophysin from residue 64 onwards. A misrouting in the ER or a misprocessing in the SG could occur so that neither vasopressin nor associated-neurophysin are found in the neurophypophysis, this lack determining diabetes insipidus. In addition there is a 50% decrease of the Lys-Arg-endoendopeptidase activity in the SG of the homozygote Brattleboro.

The complete amino acid sequence of the major ovine neurophysin (MSEL-neurophysin); comparison with a re-investigated bovine MSEL-neurophysin

Neurophysins [l] are proteins which have been subjected to extensive studies [2] because of their binding properties towards neurohypophysial hormones. Hormone . protein complexes have been purified from posterior pituitary powders of ox [3], pig [4], sheep [5], horse [6], whale [7], man [8] and cross associations between hormones of a species and neurophysins of another have been carried out [ 51. The neurophysins of the sheep have been purified through a hormone . protein complex [ 51 and recently isolated by molecular sieving and ion exchange chromatography [9]. A major neurophysin (MSELneurophysin) accounting for about 70% of the neurophysin material, has been partially sequenced [lo] . We present now data permitting the determination of the complete amino acid sequence of this protein. On the other hand a comparative investigation was carried out on bovine and porcine MSELneurophysins purified under similar conditions.

Structure primaire d'un inhibiteur pancréatique de la trypsine (inhibiteur de kunitz et northrop): II. Caractérisation des peptides résultant de l'hydrolyse trypsique

Abstract The pancreatic trypsin inhibitor, oxidized by performic acid, was submitted to tryptic hydrolysis. The polypeptide chain was split into 9 peptides which were purified by chromatography and electrophoresis and analysed. The sequence of amino acids in each tryptic unit was determined and the 9 peptides account for the 58 residues of the inhibitor. Three amide groups were localized. Sequences of identical residues occur quite frequently even though in some cases the total number of residues of the particular amino acid is low: Ile-Ile (2 residues), Pro-Pro (4 residues), Asn-Asn (5 residues) and two sequences Gly-Gly (6 residues).

Identification of rat neurophysins: Complete amino acid sequences of MSEL- and VLDV-neurophysins

Abstract Two rat neurophysins have been purified by salt precipitation, molecular sieving and ion-exchange chromatography. The proteins, performic-acid oxidized or reduced-alkylated, have been split either by trypsin or by staphylococcal proteinase and fragments have been separated by peptide mapping. Amino acid sequences of tryptic peptides have been determined either directly or after cleaving the large fragments by subtilisin, chymotrypsin, elastase or staphylococcal proteinase and characterizing the subfragments. Tryptic peptides have been ordered through the fragments given by staphylococcal proteinase. The N-terminal sequences of both proteins have also been established by automated degradation. The two usual types of mammalian neurophysins have been identified. One neurophysin belongs to the MSEL-neurophysin family and shows 11 substitutions and a 2-residue C-terminal truncation when compared with bovine MSEL-neurophysin. The other belongs to the VLDV-neurophysin family and shows 8 substitutions when compared with bovine VLDV-neurophysin. There are 23 differences between the MSEL- and VLDV-neurophysins of the rat.

Unique evolution of neurohypophysial hormones in cartilaginous fishes: possible implications for urea-based osmoregulation.

Most bony vertebrate species display a great evolutionary stability of their two neurohypophysial hormones, so that two molecular lineages, isotocin-mesotocin-oxytocin and vasotocin-vasopressin, have been traced from bony fishes to mammals. Chondrichthyes, in contrast, show a striking diversity of their oxytocin-like hormones, yet show a substantial decrease in vasotocin stored in neurohypophysis when compared to nonmammalian bony vertebrates. In the rays, glumitocin ([Ser(4),Gln(8)]-oxytocin) has been identified. In the spiny dogfish, aspargtocin ([Asn4]-oxytocin) and valitocin ([Val(8)]-oxytocin) have been characterized whereas in the spotted dogfish, asvatocin ([Asn(4),Val(8)]-oxytocin) and phasvatocin ([Phe(3),Asn(4),Val(8)]-oxytocin) have been found. Finally, in the holocephalian Pacific ratfish, oxytocin, the typical peptide of placental mammals, has been discovered. The duplication of the oxytocin-like hormone gene found in dogfishes has been observed only in some Australian and American marsupials. Cartilaginous fishes have developed an original urea-based osmoregulation involving a glutamine-dependent urea synthesis and blood urea retention through renal urea transporters. Furthermore, marine species use a rectal salt gland for sodium chloride excretion. Although vasopressin, in mammals, and vasotocin, in nonmammalian tetrapods, are clearly implied in water and salt homeostasis, the hormones involved in the blood osmotic pressure regulation of elasmobranchs are still largely unknown. It is suggested that the great diversity of oxytocin-like hormones in elasmobranchs expresses a release from an evolutionary receptor-binding constraint, so that amino-acid substitutions reflect neutral evolution. In contrast, the preservation of vasotocin suggests a selective pressure, which may be related to the regulation of renal urea transporter-recruitment mechanisms, as it has been shown for vasopressin in mammals. J. Exp. Zool. 284:475-484, 1999.

Two multigene families for marsupial neurohypophysial hormones? Identification of oxytocin, mesotocin, lysipressin and arginine vasopressin in the North American opossum (Didelphis virginiana)

Oxytocin, mesotocin ([Ile8]-oxytocin), lysipressin ([Lys8]-vasopressin) and arginine vasopressin have been identified in the North American opossum (Didelphis virginiana) by amino acid composition and high pressure liquid chromatography. The same peptides with the exception of mesotocin have previously been found in two South American opossums (Didelphis marsupialis and Philander opossum). Although a dual heterozygocity could also explain the simultaneous presence of oxytocin/mesotocin on one hand, lysipressin/arginine vasopressin on the other, it is assumed, from the results obtained with individual glands of Australian and South American marsupials, that distinct genes encode for the four peptides.