Expression, folding, and thermodynamic properties of the bovine oxytocin-neurophysin precursor: relationships to the intermolecular oxytocin-neurophysin complex.

Earlier thermodynamic studies of the intermolecular interactions between mature oxytocin and neurophysin, and of the effects of these interactions on neurophysin folding, raised questions about the intramolecular interactions of oxytocin with neurophysin within their common precursor. To address this issue, the disulfide-rich precursor of oxytocin-associated bovine neurophysin was expressed in Escherichia coli and folded in vitro to yield milligram quantities of purified protein; evidence of significant impediments to yield resulting from damage to Cys residues is presented. The inefficiency associated with the refolding of reduced mature neurophysin in the presence of oxytocin was found not to be alleviated in the precursor. Consistent with this, the effects of pH on the spectroscopic properties of the precursor and on the relative stabilities of the precursor and mature neurophysin to guanidine denaturation indicated that noncovalent intramolecular bonding between oxytocin and neurophysin in the precursor had only a small thermodynamic advantage over the corresponding bonding in the intermolecular complex. Loss of the principal interactions between hormone and protein, and of the enhanced stability of the precursor relative to that of the mature unliganded protein, occurred reversibly upon increasing the pH, with a midpoint at pH 10. Correlation of these results with evidence from NMR studies of structural differences between the precursor and the intermolecular complex, which persist beyond the pH 10 transition, suggests that the covalent attachment of the hormone in the precursor necessitates a conformational change in its neurophysin segment and leads to properties of the system that are distinct from those of either the liganded or unliganded mature protein.

Sequence analysis of a cDNA encoding an isotocin precursor and localization of the corresponding mRNA in the brain of the cartilaginous fish Torpedo marmorata.

The nucleotide sequence of a cDNA encoding an isotocin hormone precursor has been elucidated by analyzing a lambda ZAPII library constructed using poly(A)+ RNA from the brain of the cartilaginous fish Torpedo marmorata. The sequence predicts a precursor of 126 amino acid residues that consists of a signal peptide, the isotocin moiety, and a neurophysin carrier protein. In contrast to other known fish isotocin precursor sequences, the Torpedo neurophysin moiety is not extended at its carboxy-terminus by a copeptin-like sequence. The T. marmorata isotocin precursor exhibits highest amino acid sequence identity (61%) to the toad mesotocin precursor. As demonstrated by in situ hybridization, the isotocin mRNA is present in neurons of the preoptic area of the Torpedo brain.

Amino acid sequence and properties of vasopressin-associated elephant neurophysin.

The primary structure of an elephant neurophysin, homologous to vasopressin-associated neurophysins, is reported. The protein contains a Tyr for Asn substitution at position 75, a position in direct contact with residues 77 and 78 of the monomer-monomer interface. This Tyr residue therefore serves as a potential reporter of the path involved in the long-range linkage between peptide binding and dimerization in this system. NMR studies of the protein in unliganded and liganded states demonstrated normal dimerization properties and the expected increase in dimerization associated with binding peptide. In keeping with an elevated pKa of 11.1 assigned to Tyr-75 by UV spectrophotometric titration, the NMR signals from the 3,5 and 2,6 ring protons of Tyr-75 were shifted 0.3 and 0.2 ppm upfield, respectively, relative to their positions in small peptides, indicating significant shielding and/or hydrogen bonding. The Tyr-75 ring proton signals narrowed slightly, with no discernible change in chemical shift, on conversion from dimer to monomer in the unliganded state. Ring protons of Tyr-49, distant from the monomer-monomer interface, but adjacent to the peptide-binding site, were markedly perturbed by dimerization, in accord with their behavior in bovine neurophysins. The results suggest that the secondary and tertiary structure of the region 75-78 is largely unchanged by dimerization, and argue against an important role for this region in dimerization-mediated conformational changes that alter the binding site in the unliganded state.(ABSTRACT TRUNCATED AT 250 WORDS)

Bony fish neurophysins. Identification of MSEL- and VLDV-neurophysins of the pollack (Pollachius virens).

The two types of neurophysins known in vertebrate species, namely MSEL-neurophysin (vasopressin-like hormone-associated neurophysin) and VLDV-neurophysin (oxytocin-like hormone-associated neurophysin) have been purified from the pollack (Pollachius virens) pituitary through a combination of molecular sieving and high-pressure liquid chromatography (HPLC). Homogeneity has been checked by gel electrophoresis and return in HPLC. The apparent molecular masses measured by SDS-electrophoresis are near 12 kDa, significantly higher than those found for their mammalian homologues (10 kDa). The two types of neurophysins have been recognized through their N-terminal amino acid sequences. The primary structure of MSEL-neurophysin has been partially determined using automated Edman degradation applied on native and reduced-alkylated protein, as well as peptides derived by trypsin or staphylococcal proteinase hydrolyses. Comparison of pollack MSEL-neurophysin with ox, goose and frog counterparts reveals that particular positions in the polypeptide chain are subjected to substitutions and that the numbers of substitutions do not seem closely related to the paleontological times of divergence between the different vertebrate classes.

Synthesis, turnover, and release of peptides from the neurohypophysis of the Jerboa Jaculus orientalis.

Peptide contents of neural lobes from adult jerboas (Jaculus orientalis) under different states of hydration were determined by radioimmunoassay. The amounts of vasopressin, oxytocin, and their associated neurophysins in animals dehydrated for up to 4 weeks were not significantly different from those of controls. The different neurohypophyseal peptide were separated on two different types of gradient using reverse-phase high-performance liquid chromatography. The shape of the chromatograms suggests that, in contrast to the case of the rat, for which only three types of neurophysins have been shown, there are, in jerboa, many subspecies of neurophysins. This was also shown using two-dimensional electrophoresis. Injection of [35S]cysteine into the supraoptic nucleus followed by HPLC of extracts from the neural lobes from animals under different states of dehydration showed that the labeled material is not released any faster in dehydrated animals than in controls. Labeled vasopressin, oxytocin, and neurophysins could still be detected by HPLC 4 weeks after injection. Neural lobes from animals injected with [35S]cysteine were perfused in vitro and the release of neuropeptides was triggered by bursts of electrical pulses and also by K(+)-induced depolarization. The amplitude of the rate constant for release and the amounts of vasopressin and of radiolabeled material released were similar in animals dehydrated for up to 3 weeks and in controls. Under physiological conditions similar to those that would be expected to occur in their natural habitat, the jerboas appear to have a hypothalamoneurohypophyseal system which is down-regulated.

Modulation of allosteric interactions in neurophysin induced by succinylation of serine-56 or cleavage of residues 1-8.

Neurophysin is an allosteric protein in which peptide binding and self-association are positively linked. Reaction of neurophysin with succinic anhydride led to a large decrease in peptide affinity assignable to succinylation of a serine or threonine hydroxyl group. To identify the residue involved, acetimidated protein was reacted with [14C]succinic anhydride and the active and inactive components were separated by affinity chromatography. Performic acid oxidation and tryptic and Asp-N mapping of the two components, followed by automated Edman degradation, allowed identification of the critical residue as Ser-56. This residue is not a direct participant in peptide binding and is distant from the subunit interface of the dimer, but it is immediately adjacent to the site of one of the known mutations associated with familial diabetes insipidus. Examination in solution of the peptide affinity of neurophysin succinylated at Ser-56 indicated a binding affinity approximately 1/20th that of the native protein or of protein succinylated at other residues, and a loss of the normal dependence of binding affinity on protein concentration. Under the same buffer conditions, loss of the concentration dependence of binding, in addition to the previously demonstrated loss of binding affinity, also accompanied excision of residues 1-8, an effect attributed to the loss of binding site residue Arg-8. However, in contrast to the effects of succinylation on native neurophysin, only minor effects of succinylation on the binding affinity of the des-1-8 protein were observed.(ABSTRACT TRUNCATED AT 250 WORDS)

Membrane translocation and relationship with MHC class I of a human thymic neurophysin-like protein.

Thymic epithelial and nurse cells (TEC/TNC) synthesize an oxytocin (OT)-like peptide in association with a neurophysin (NP)-related protein in a way similar to in the hypothalamo-neurohypophysial (NHP) system. The central T-cell tolerance of the NHP neuroendocrine functions have been proposed to be mediated through these thymic NHP-related peptides due to their close homology with the NHP neurohormones OT and vasopressin (VP). In order to investigate their putative presentation by proteins of the major histocompatibility complex (MHC), human thymic membranes were purified and passed through an immunoaffinity column using mAb B9.12 directed to the monomorphic determinant of human MHC class I proteins. This methodology provided the following observations: (1) a NP-like protein is translocated in human thymic membranes and is retained by B9.12 on the column; (2) the MW of this NP-like material (50-55 kD) is quite different from the MW of hypothalamic NP proteins (10 kD), and (3) this thymic NP-like protein could be identified on Western blots with mAb B9.12. The precise extent of this relationship between the thymic NP-like protein and the Ig/MHC superfamily is actually investigated through the characterization of the genetic mechanisms responsible for the thymic expression of NHP-related peptides. Given the physiological importance of OT and of its binding to NP for transport along the axonal processes of the NHP tract, we postulate that, somewhat analogously, the thymic NP-/MHC class I-related protein could be involved in the presentation of the OT-like peptide to immature T-cells.

Oriented immobilization of peptide ligands on solid supports.

A synthetic procedure was developed for the direct immobilization on preactivated affinity supports of peptidic ligands requiring free alpha-amino groups to recognize their targets properly. The peptidic ligand is assembled by solid-phase peptide synthesis on an octa-branched heptalysine core through a polyglycine spacer, similar to the method developed for the production of multiple antigenic peptides. After deblocking from the resin, peptide is dialysed, lyophylized and used directly for coupling to preactivated supports. Following immobilization, only a limited number of peptide chains are covalently linked to the solid phase, leaving the remainder facing the mobile phase and sufficiently spaced to interact properly. This procedure was applied successfully to the design, synthesis and oriented immobilization of a multimeric tripeptide ligand (Met-Tyr-Phe) for affinity purification of bovine neurophysin.

Neurophysin-neurohypophyseal hormone interactions: studies using a dansylated vasotocin analogue.

We have synthesized a neurohypophyseal hormone analogue containing an extrinsic fluorescence probe by linking a dansyl (DNS) group to the epsilon-amino group of the lysine at residue 8 of vasotocin. The fluorescence properties of this analogue have been characterized by steady-state and time-resolved spectroscopic methods and compared with those of epsilon-DNS-lysine and the dansylated carboxyl terminal tripeptide Pro-Lys(DNS)-GlyNH2. The binding of this hormone analogue to purified isoforms of bovine neurophysins, the natural carrier proteins of the neurohypophyseal hormones, results in changes in several fluorescence parameters of the dansyl probe. These changes include an increase in intensity and average lifetime, a shift of the emission band to higher energies, and an increase in the emission anisotropy. Anisotropy changes have been used to determine dissociation constants for binding to these neurophysin isoforms. Based on the changes in the fluorescence properties of the dansyl probe, the dansyl group itself interacts with the protein. The degree of the dansyl-neurophysin interaction, however, appears to be different for the full sequence isoform of neurophysin I and the Val89 isoform of neurophysin II.

Binding and fluorescence studies of the relationship between neurophysin-peptide interaction and neurophysin self-association: an allosteric system exhibiting minimal cooperativity.

The mechanism of peptide-enhanced neurophysin self-association was investigated to address questions raised by the crystal structure of a neurophysin-dipeptide complex. The dependence on protein concentration of the binding of a broad range of peptides to the principal hormone-binding site confirmed that occupancy of this site alone, and not a site that bridges the monomer-monomer interface, is the trigger for enhanced dimerization. For the binding of most peptides to the principal hormone-binding site on bovine neurophysin I, the affinity of each dimer site was at least 10 times that of monomer under the conditions used. No interactions between the two sites of the dimer were evident. Fluorescence polarization studies of pressure-induced dimer dissociation indicated that the volume change for this reaction was almost 4 times greater in the liganded than in the unliganded state, pointing to a significant alteration of the monomer-monomer interface upon peptide binding. Novel conformational changes in the vicinity of the single neurophysin tyrosine, Tyr-49, induced by pressures lower than required for subunit dissociation, were also observed. The bovine neurophysin I dimer therefore appears to represent an allosteric system in which there is thermodynamic and functional communication between each binding site and the monomer-monomer interface, but no communication across the interface to the binding site of the other subunit. A model for the peptide-enhanced dimerization is proposed in which intersubunit contacts between monomers reduce the large unfavorable free energy associated with binding-induced intrasubunit conformational change. Structural origins of the lack of communication across the interface are suggested on the basis of the low volume change associated with dimerization in the unliganded state and monomer-monomer contacts in the crystal structure. Potential roles for the peptide alpha-amino group and position 2 phenyl ring in triggering conformational change are discussed.

Complete amino acid sequence of goose VLDV-neurophysin. Traces of a putative gene conversion between promesotocin and provasotocin genes.

Goose VLDV-neurophysin (mesotocin-associated neurophysin) has been purified from posterior pituitary glands through molecular sieving on Sephadex G-75 and high-pressure reverse-phase liquid chromatography on Nucleosil C-18 columns. Despite apparent molecular mass of unreduced VLDV-neurophysin measured by polyacrylamide gel electrophoresis with sodium dodecylsulfate appeared near 17 kDa, this value fell to 11 kDa after reduction with mercaptoethanol, suggesting the existence of a homodimer. Complete amino acid sequence (93 residues) of goose VLDV-neurophysin has been determined. N- and C-terminal sequences of the protein have been established by Edman degradation (microsequencing) and use of carboxypeptidase Y, respectively. Peptides derived from oxidized or carboxamidomethylated neurophysin by trypsin or staphylococcal proteinase hydrolyses have been isolated by high-pressure liquid chromatography and microsequenced, allowing determination of the complete sequence. Comparison within the vertebrate VLDV-neurophysin lineage, namely goose VLDV-neurophysin to mammalian VLDV-neurophysins and to deduced toad VLDV-neurophysin, reveals a residue insertion between positions 66 and 67 in the nonmammalian VLDV-neurophysins. When goose MSEL-neurophysin (vasotocin-associated neurophysin) and goose VLDV-neurophysin are compared to their bovine counterparts, identical substitutions are found in positions 17 (Asn in both goose neurophysins instead of Gly in both ox neurophysins), 18 (Arg instead of Lys), 35 (Tyr instead of Phe), and 41 (Thr instead of Ala). Identity of the sequences 10-74 in both ox neurophysins has been explained by partial gene conversion between oxytocin and vasopressin genes, and identical substitutions in both goose neurophysins might reveal a similar gene conversion between mesotocin and vasopressin genes in birds.

Non-mammalian "big" neurophysins--complete amino acid sequence of a two-domain MSEL-neurophysin from goose.

Vasotocin-associated neurophysin (MSEL-neurophysin) has been purified from goose neurohypophysis through molecular sieving and high-pressure reverse-phase liquid chromatography (HPLC). The protein has a molecular mass (measured by SDS-polyacrylamide gel electrophoresis) of 17 kDa in contrast to 10 kDa found for the mammalian MSEL-neurophysins. Complete amino acid sequence (131 residues) has been determined mainly through tryptic or staphylococcal proteinase peptides derived from carboxyamidomethylated neurophysin, isolated by HPLC and microsequenced. N- and C-terminal sequences have been established by Edman degradation or action of carboxypeptidase Y, respectively, applied on the native protein. Goose MSEL-neurophysin is homologous to the two-domain "big" MSEL-neurophysin previously identified in the frog. It appears that in non-mammalian tetrapods, namely birds and amphibians, the proteolytic processing of the pro-vasotocin involves only one cleavage, releasing the hormone moiety and a "big" neurophysin with two domains homologous to mammalian MSEL-neurophysin and copeptin, respectively. Comparison of the avian protein with its mammalian and amphibian counterparts reveals that the first half of the polypeptide chain is evolutionarily much less variable than the second and that the goose protein resembles the frog protein much more than the mammalian one.

Identification of two types of neurophysins in Xenopus laevis neurointermediate pituitary homologous to mammalian MSEL- and VLDV-neurophysins.

UNLABELLED: Xenopus laevis neurophysins have been purified from neurointermediate pituitaries through high-pressure reverse-phase liquid chromatography and their N-terminal amino acid sequences have been determined by microsequencing. Two types of neurophysins, corresponding to mammalian MSEL- and VLDV-neurophysins, have been distinguished. A strong homology exists between neurophysins of Xenopus (Pipidae), frog (Ranidae) and toad (Bufonidae). Xenopus MSEL-neurophysin, as frog MSEL-neurophysin, has a high molecular mass suggesting that the C-terminal domain of the vasotocin precursor is not processed in contrast to the two-step processing observed for mammalian vasopressin precursor. ABBREVIATIONS: Mammalian neurophysins are termed MSEL- and VLDV-neurophysins according to the nature of residues in positions 2, 3, 6 and 7 (one-letter symbols for amino acids).

Ostrich MSEL-neurophysin belongs to the class of two-domain "big" neurophysin as indicated by complete amino acid sequence of the neurophysin/copeptin.

Mammalian neurohypophyseal hormones, oxytocin and vasopressin, are known to be synthesized as part of two larger precursors containing, respectively, a VLDV-neurophysin and a MSEL-neurophysin together with its associated glycopeptide. Starting from ostrich neurohypophyses, a "big" neurophysin was isolated and chemically characterized. Following sequence determination of the CNBr-derived fragments and of peptides obtained from trypsin and V8-protease digestion of the oxidized protein, this "big" neurophysin was found to contain an MSEL-neurophysin moiety (94 residues) still covalently associated with the COOH-terminal glycopeptide (38 residues, copeptin). This study demonstrates that the ostrich MSEL-neurophysin sequence closely resembles all known MSEL-neurophysin sequences and that, furthermore, it does not contain the single amino acid insertion shown previously in the ostrich VLDV-neurophysin. It is also shown that the stretch of amino acids, linking the MSEL-neurophysin and the copeptin, is clearly different from its mammalian homologues and lacks the Arg residue normally recognized by the cleaving enzyme. This study also demonstrates that the ostrich copeptin is more closely related to the amphibian copeptin sequence than to its mammalian homologue, leading to the hypothesis that two families of copeptin molecules might exist. Thus, the ostrich MSEL-neurophysin-copeptin molecule is the first "big" neurophysin reported in birds and, together with the guinea pig and amphibian homologues, represents the third example of partial or no neurophysin-copeptin cleavage.

Isolation of neurosecretory granules containing vasopressin and MSEL-neurophysin from guinea pig neurointermediate pituitary.

Neurosecretory granules have been isolated from rat and guinea pig neurointermediate pituitaries and their contents have been analyzed by reverse-phase high-pressure liquid chromatography and polyacrylamide gel electrophoresis. Granule components have been compared with synthetic neurohypophysial hormones and chemically characterized neurophysins. In rat granules, oxytocin, arginine vasopressin and MSEL- and VLDV-neurophysins have been identified. In isolated guinea pig granules, only arginine vasopressin and mature MSEL-neurophysin have been found. From these results it can be concluded that both the "dibasic" cleavage between vasopressin and MSEL-neurophysin and the "monobasic" cleavage between MSEL-neurophysin and copeptin occur within the granule compartment. Previous isolation from frozen guinea pig glands of a partially processed precursor encompassing MSEL-neurophysin and copeptin suggests a two-step processing of the three-domain vasopressin precursor, each involving a distinct enzymic system.

Molecular diagnostics using analytical immuno high performance liquid affinity chromatography.

Analytical immuno high performance liquid affinity chromatography (analytical immuno HPLAC) was evaluated as a molecular diagnostic tool. Antibodies raised in rabbits against bovine neurophysin II were immobilized through Protein A crosslinking onto coated silica. Interaction of immobilized antibody with mobile antigen was characterized by zonal and frontal elutions of 14C-labeled bovine neurophysin II under isocratic, nondenaturing conditions. The chromatographic behavior shows that analytical immuno HPLAC with immobilized antibodies can be used to detect the number and functional nature of matrix-interacting antigens in mixtures, thus providing a quantitative chromatographic technology for "antigen mapping."

Analytical high-performance affinity chromatography: evaluation by studies of neurophysin self-association and neurophysin-peptide hormone interaction using glass matrices.

Bovine neurophysin II (BNP II) was covalently immobilized on both nonporous and porous (200-nm pore diameter) glass beads and incorporated in a high-performance liquid chromatograph to evaluate analytical high-performance affinity chromatography as a microscale method for characterizing biomolecular interactions. By extension of the theoretical treatment of analytical affinity chromatography, both the self-association of neurophysin and its binding of the peptide hormone vasopressin were characterized by using a single chromatographic column containing immobilized neurophysin predominantly in the monomer form. Both [3H] [Arg8]vasopressin (AVP) and 125I-BNP II were rapidly eluted (less than 25 min). The relatively symmetrical elution peaks obtained allowed calculation of both equilibrium dissociation constants and kinetic dissociation rate constants. The dissociation constant measured chromatographically for the AVP-immobilized neurophysin complex, KM/L = 11 microM with porous glass beads and 75 microM with nonporous glass (NPG) beads, was in reasonable agreement with those previously obtained by curve fitting of Scatchard plots (16-20 microM) and from binding to [BNP II]Sepharose (50 microM). The values obtained are larger than that for dissociation of AVP from BNP II dimer, by a factor consistent with the intended nature of immobilized BNP II as monomers. Chromatography of BNP II on the [BNP II]NPG gave a dimer dissociation constant of 166 microM, a value in excellent agreement with that derived from equilibrium sedimentation studies (172 microM). In contrast to the agreement of chromatographic equilibrium binding constants with those measured in solution, the dissociation rate, k-3, determined from the variance of the affinity chromatographic elution profile with nonporous beads, was several orders of magnitude smaller than the solution counterpart.(ABSTRACT TRUNCATED AT 250 WORDS)

Precursors of mesotocin and vasotocin in birds: identification of VLDV- and MSEL- neurophysins in chicken, goose, and ostrich.

Precursors of neurohypophysial hormones are small proteins processed into nonapeptide hormones and neurophysins during axonal transport to the neurohypophysis. In mammals, oxytocin is associated with VLDV-neurophysin and vasopressin with MSEL-neurophysin. In birds, mesotocin and vasotocin are found instead of mammalian oxytocin and vasopressin. From goose, chicken and ostrich posterior pituitary glands, two types of neurophysins related to mammalian VLDV- and MSEL-neurophysins, respectively, have been identified by their N-terminal sequences. It is assumed that, as in mammals, hormonal peptide and the first 9 residues of the corresponding neurophysin are encoded by a common exon and that mesotocin and vasotocin, evolutionary predecessors of oxytocin and vasopressin, are associated in the precursors with VLDV-neurophysin and MSEL-neurophysin, respectively.

The neuroendocrine thymus: coexistence of oxytocin and neurophysin in the human thymus.

Immunoreactive oxytocin and neurophysin were identified and measured by radioimmunoassay in human thymus extracts. Serial dilutions of extracts paralleled the appropriate standard curves. Thymus-extracted oxytocin and neurophysin eluted in the same positions as reference preparations on Sephadex G-75. Authenticity of oxytocin was confirmed by biological assay and high-performance liquid chromatography analysis. In most instances, thymus contents of oxytocin and neurophysin were far greater than those expected from known circulating concentrations and declined with increasing age. The molar ratio of oxytocin to neurophysin in thymus was similar to that found in the hypothalamo-neurohypophyseal system, which strongly suggested with the other data a local synthesis of oxytocin. These findings indicate the presence of neurohypophyseal peptides in the human thymus and further support the concept of a neuroendocrine function integrated in an immune structure.