CD and (13)C(alpha)-NMR studies of folding equilibria in a two-stranded coiled coil formed by residues 190-254 of alpha-tropomyosin.

Synthesis and CD and (13)C(alpha)-NMR studies in a near-neutral saline buffer are reported for a 65-residue peptide ((190)Tm(254)) comprising residues 190-254 of the alpha-tropomyosin chain. CD on a version disulfide cross-linked via the N-terminal cysteine side chains indicates that this dimer is highly helical and melts near 48 degrees C. The CD is independent of peptide concentration, showing that association of (190)Tm(254) stops at the two-strand level. Similar studies on the reduced version show much lower helix content at low temperature, melting points below room temperature, and the expected concentration dependence. The observed melting temperature of the reduced peptide is far below (by 27 degrees C) that expected from an extant analysis of calorimetry data on parent tropomyosin that designates (190)Tm(254) as an independently melting "cooperative block." This disagreement and the pronounced nonadditivity seen when data for (190)Tm(254) are combined with extant data for other subsequences argue decisively against the concept of specific independently melting blocks within the tropomyosin chain. The data for (190)Tm(254) also serve to test recent ideas on the sequence determinants of structure and stability in coiled coils. Analysis shows that some ideas, such as the stabilizing effect of leucine in the d heptad position, find support, but others--such as the destabilizing effect of alanine in d, the dimer-disfavoring effect of beta-branching in d and its dimer-favoring effect in a, and the dimer-directing effect of asparagine in a--are more questionable in tropomyosin than in the leucine zipper coiled coils. (13)C(alpha)-NMR data at two labeled sites, L228(d) and V246(a), of (190)Tm(254) display well-separated resonances for folded and unfolded forms at each site, indicating that the transition is slow on the NMR time scale and thus demonstrating the possibility of obtaining thermodynamic and kinetic information on the transition at the residue level.

Nerve growth factor alpha subunit: effect of site-directed mutations on catalytic activity and 7S NGF complex formation.

Mouse alpha- and gamma-nerve growth factor (NGF) are glandular kallikreins that form a non-covalent complex (7S NGF) with beta-NGF. gamma-NGF is an active arginine-specific esteropeptidase; the alpha-subunit is catalytically inactive and has a zymogen-like conformation. Site-directed mutagenesis of alpha-NGF to alter the N-terminus and three residues in loop 7, a region that contributes to the catalytic center, restored substantial catalytic activity against N-benzoyl arginine-p-nitroanilide as substrate in two derivatives although they were not as active as recombinant gamma-NGF. Seven of the 15 derivatives that remained more alpha-like were able to substitute for native alpha-NGF in reforming 7S complexes; the other eight derivatives that were more gamma-like showed greatly reduced ability to do so. However, the most gamma-like alpha-NGF derivative could not substitute for native gamma-NGF in 7S complex formation. These findings suggest that the alpha-NGF backbone can be corrected to a functional enzyme by the addition of a normal N-terminal structure and two catalytic site substitutions and that the 7S complex requires one kallikrein subunit in the zymogen form and one in an active conformation.

A soluble gradient of the neuropeptide secretoneurin promotes the transendothelial migration of monocytes in vitro.

Secretoneurin, derived from the chromogranin secretogranin II, triggers the selective migration of human monocytes, eosinophils, fibroblasts, endothelial and smooth muscle cells. More recently, we located specific binding sites on the human monocytic cell line MonoMac-6. Differentiated U937 transendothelial diapedesis was evaluated using an in vitro model of the vascular wall and specific monoclonal antibodies against CD11/CD18 and the alpha-chains of the very late activation antigen (VLA)-4 were used to evaluate involved adhesion molecules. Results showed a significant migratory response to secretoneurin between 10(-8) to 10(-10) M. Migration was comparable to a maximal effect induced by the monocyte chemotactic agent N-formyl-Met-Leu-Phe (fMLP, 10(-8) M). Rabbit anti-secretoneurin antibodies were able to block the neuropeptide effect but not of fMLP and a trypsinized secretoneurin preparation as well as the secretogranin II-fragment EL-17 were ineffective in eliciting migration. Transmigration of U937 across endothelial-layers toward secretoneurin is inhibited by antibodies to CD11/CD18 adhesion molecules. The novel neuropeptide secretoneurin may play a role in regulating migration of monocytes into the subendothelial space, supposing a role in inflammatory responses.

Specific binding of a 125I-secretoneurin analogue to a human monocytic cell line.

Secretoneurin (SN) is a novel neuropeptide expressed in the central and peripheral nervous system as well as in various endocrine tissues. SN inhibits growth of aortic pulmonary and endothelial cells and is a potent chemoattractant for endothelial cells, skin fibroblasts and monocytes. We investigated here the presence of specific high affinity binding sites for SN on a target tissue. SN was iodinated with the Bolton-Hunter (BH) reagent and purified by isocratic reversed phase chromatography. Specific binding sites for 125I-BHSN were identified on human Mono Mac 6 cells, a monocytic cell line. Scatchard analysis revealed a single class of binding sites with a Kd value of 7.3 nM and a Bmax of 322 (fmol/mg protein). Competition studies demonstrated that the 15 C-terminal amino acids of SN could displace authentic SN, whereas shorter fragments were inactive. Other sensory neuropeptides like substance P, calcitonin gene-related peptide or galanin as well as the chemokine receptor ligand Rantes or the typical chemoattractant FMLP could not displace SN. Our studies demonstrate specific high affinity binding sites for SN on a monocytic cell line. Since SN exerts a potent chemotactic activity towards monocytes and increases cytosolic calcium in these cells, these binding sites might well represent a putative functional plasma membrane receptor for SN.

Inhibition of proliferation and stimulation of migration of endothelial cells by secretoneurin in vitro.

Vascular cell responses in inflammation are affected by several neuropeptides of perivascular nerve fibers. Secretoneurin is a 33-amino acid peptide that is coreleased from these nerve endings with other proinflammatory neuropeptides, eg, substance P and calcitonin gene-related peptide. Furthermore, secretoneurin has been shown to be chemotactic for human skin fibroblasts and human blood monocytes in vitro and in vivo. An action on cellular components of the vascular wall is not yet reported. We therefore investigated in vitro effects of this novel sensory neuropeptide on endothelial cells. Secretoneurin exerted a potent and reversible inhibitory effect both on endothelial cell growth under low serum conditions (1% fetal calf serum) and endothelial cell growth factor-activated endothelial cell proliferation. We show in the present study that secretoneurin exerts this effect on aortic (rat) and pulmonary artery (bovine) endothelial cells, as well as venous (human umbilical vein) endothelium. Endothelial cell chemotaxis was tested by means of three different migration assays employing nitrocellulose and polycarbonate micropore filters. Secretoneurin consistently exhibited potent chemoattractant activity. The effective concentrations for the observed effects were in the picomolar range. The combination of chemotactic and antiproliferative effects on endothelial cells suggests that secretoneurin may act as a regulatory factor of vascular cell functions.

Molecular cloning and characterization of NESP55, a novel chromogranin-like precursor of a peptide with 5-HT1B receptor antagonist activity.

The chromogranins comprise a class of acidic proteins that are secreted from large dense core vesicles and expressed in neuronal and endocrine tissues. We describe here the molecular characterization of NESP55 (neuroendocrine secretory protein of Mr 55,000), a novel member of the chromogranins. Several NESP55 cDNA clones were isolated from bovine chromaffin cell libraries. The cDNA sequence of NESP55 totals 1499 nucleotides. All of the clones that were isolated contained in their 3'-untranslated mRNA a sequence that was homologous to exon 2 of the G-protein Gsalpha. The open reading frame encodes for an acidic and hydrophilic protein of 241 amino acids with a predicted molecular mass of 27,494 Da. An antiserum directed against the C terminus of NESP55 labeled a band of Mr 55,000 with an acidic pI ranging from 4.4 to 5.2 in one- and two-dimensional immunoblots of secretory proteins from chromaffin granules. NESP55 is localized within the cell to the large dense secretory vesicles and is expressed, apart from the adrenal medulla, in the anterior and posterior pituitary and various regions of the brain. For the physiological function, one interesting factor has emerged. NESP55 is proteolytically processed within the chromaffin granule to smaller peptides that might be physiologically active. One tetrapeptide, Leu-Ser-Ala-Leu (LSAL), present in the NESP55 sequence and flanked by arginine residues suitable for cleavage by prohormone convertases, has been identified recently as an endogenous antagonist of the serotonergic 5-HT1B receptor subtype. Alterations in the serotonergic system are thought to play an important role in mental disorders, especially depression, and might be related to abnormal ethanol consumption. It is tempting to speculate that increased expression of NESP55 or its proteolytically derived peptide LSAL might contribute to the pathophysiology of the serotonergic transmission.

Rat brain: distribution of immunoreactivity of PE-11, a peptide derived from chromogranin B.

An antiserum was raised against the peptide PE-11 whose sequence is present in the chromogranin B molecule. The antiserum reacts only with the free C-terminal end of this peptide. PE-11 immunoreactivity in brain was characterized by molecular size exclusion high performance liquid chromatography. Only the free peptide and a N-terminally elongated peptide were detected, indicating that proteolytic processing of chromagranin B in brain is quite extensive. In immunohistochemistry PE-11 immunoreactivity was found in varicosities, fibres and perikarya throughout the brain. Strong staining was detected in the shell sector of the nucleus accumbens, in the lateral septum, in subregions of the extended amygdala, in some areas of the hippocampus and of the hypothalamus, in the locus coeruleus, in the Purkinje cells of the cerebellum and in the dorsal horn of the spinal cord. Our results, which demonstrate significant processing of chromogranin B in brain and its widespread distribution, can be taken as an indication that chromogranin B represents a precursor of peptides with functional relevance for this organ.

Vasostatins, N-terminal products of chromogranin A, are released from the stimulated calf spleen in vitro.

Vasostatins are the N-terminal chromogranin A peptides 7 approximately 22 kDa. They have been shown to be present in several endocrine tissues and exhibit vasoinhibitory activity in vitro. In a first series of experiments, we investigated the presence and subcellular localization of vasostatins in the bovine splenic nerve. Experimental results, obtained using gradient centrifugation, showed that noradrenaline was enriched 25-fold in the large dense core vesicle fraction, compared with the original homogenate. In the latter fraction, the 7 and 18 kDa peptides were observed following immunodetection with antiserum to chromogranin A1-40 and laser densitometric scanning revealed these two fragments as the major N-terminal fragments. Subsequently, we examined the release of the 7 and 18 kDa peptides from perfused calf spleen during veratridine (20 microM) or 1,1-dimethyl-4-phenylpiperazinium iodide (20 microM) stimulation. In the prestimulation samples, we were not able to detect these peptides, however, following stimulation, the 7 and 18 kDa chromogranin A fragments became apparent. The vasostatin-immunoreactivity, in both bovine chromaffin granule lysate and calf spleen perfusate, elutes at the same retention time on reversed-phase high performance liquid chromatography. The present study demonstrated that vasostatins are present in the large dense core vesicles of sympathetic axons and are released from the nerve terminals in response to stimulation. The release of vasostatins from sympathetic nerves in the spleen suggest an in vivo function for N-terminal chromogranin A products of neuronal origin.

Large variations in the proteolytic formation of a chromogranin A-derived peptide (GE-25) in neuroendocrine tissues.

We have established a radioimmunoassay for GE-25, a peptide present in the C-terminal end of the primary amino acid sequence of chromogranin A where it is flanked by typical proteolytic cleavage sites. Gel-filtration HPLC was used to characterize the molecular sizes of the immunoreactive molecules. The antiserum recognized not only the free peptide but also larger precursors including the proprotein chromogranin A. The tissues with the highest levels of GE-25 immunoreactivity were in decreasing order: the adrenal medulla, the three lobes of the pituitary gland, intestinal mucosa, pancreas and various brain regions. In adrenal medulla and parathyroid gland most of the immunoreactivity was found to be present as intact chromogranin A and some intermediate-sized peptides, without significant amounts of the free peptide. In anterior pituitary, and even more so in intestine, a shift to smaller peptides was seen. In the posterior and intermediate pituitary and in pancreas the predominant immunoreactive material was apparently represented by the free peptide GE-25. In reverse-phase chromatography this peptide eluted exactly like the synthetic standard, which allows a tentative identification as GE-25. In brain tissue the processing of chromogranin A was intermediate, with significant amounts of immunoreactivity corresponding to GE-25 as well as precursor proteins being present. We suggest that in those organs (endocrine pancreas, intermediate and posterior pituitary) where the major hormones are proteolytically processed there is also a concomitant proteolysis of further susceptible peptides. Since GE-25 is apparently formed in vivo and is well conserved between species it seems a good candidate for having specific physiological functions.

Molecular characterization of immunoreactivities of peptides derived from chromogranin A (GE-25) and from secretogranin II (secretoneurin) in human and bovine cerebrospinal fluid.

Chromogranin A and secretogranin II are members of the so-called chromogranins, the acidic proteins stored in neuroendocrine large dense-core vesicles. We characterized chromogranin A and secretogranin II immunoreactivities in cerebrospinal fluid by radioimmunoassays using synthetic peptides derived from these components (GE-25 for chromogranin A and secretoneurin for secretogranin II). In lumbar cerebrospinal fluid, high levels (more than 1000 fmol/ml) of these two components were found, whereas in ventricular cerebrospinal fluid the secretoneurin levels were relatively low. The cerebrospinal fluid/serum ratio for secretoneurin was close to 170. High-performance liquid chromatography revealed that in both cerebrospinal fluid and extracts from human brain secretoneurin was the predominant immunoreactive component. In cerebrospinal fluid chromogranin A immunoreactivity was present as intermediate-sized peptides with little intact chromogranin A and free GE-25 peptide. In human brain samples smaller peptides including GE-25 were more predominant. Analogous findings for secretoneurin and chromogranin A were obtained for bovine brain samples. We can conclude that chromogranins are present in cerebrospinal fluid in concentrations much higher than those of classical neuropeptides also stored in large dense-core vesicles. Therefore, their degree of proteolytic processing can be analysed with small samples of cerebrospinal fluid. A possible disturbance of proteolytic processing in large dense-core vesicles in various pathological conditions can now be discovered.

Chromogranin A peptide-specific antisera and high-performance size exclusion chromatography demonstrate amino-terminal and carboxy-terminal fragments of the native molecule in human cell lines.

We have studied the pattern of amino-terminal and carboxy-terminal protein processing of chromogranin A (CgA) in five human cell lines, four derived from lung cancers: NCI-H478, NCI-H1011, NCI-H727, and BEN; and one derived from human medullary thyroid carcinoma: TT. This was accomplished by fractionation of cell extracts by high-performance size exclusion chromatography (HPSEC) and measurement of CgA in the fractions by radioimmunoassay (RIA). Three RIA's were used, one specific for the amino-terminus of CgA, one specific for the carboxy-terminal region, and a monoclonal antibody-based assay that recognizes only the native CgA molecule. We demonstrated the presence of different amino- and carboxy-terminal immunoreactive species of CgA in the different cell lines. The amino-terminal assay demonstrated distinct low-molecular-size species in the NCI-H478 and NCI-H1011 cell lines, and a similar peak in the TT cells. The amino-terminal assay did not recognize any distinct species in BEN and NCI-H727 cell lines. The carboxy-regional assay demonstrated distinct low-molecular-size species in the NCI-H478 and NCI-H101 cell lines and high-molecular-size species in the NCI-H727 and BEN cells. Our studies demonstrate with region-specific RIA's the presence of both amino- and carboxy- forms of CgA in human cells that secrete this protein. These results provide direct evidence that CgA-producing cells produce, probably through endoproteolytic processing of the native molecule, amino- and carboxy-terminal forms of the protein.

Bovine posterior pituitary: presence of p65 (synaptotagmin), PC1, PC2 and secretoneurin in large dense core vesicles.

The subcellular distribution of p65 (synaptotagmin), of the endoproteases PC1 and PC2 and of secretoneurin was studied in bovine posterior pituitary by differential and density gradient centrifugation. All these peptides were found to be present in the neurosecretory granules (large dense core vesicles). p65 can therefore participate not only in exocytosis from small synaptic vesicles but also from large dense core vesicles. Secretoneurin is a peptide derived from secretogranin II. Processing of the propeptide apparently occurs during axonal transport of the large dense core vesicles and is complete in the posterior pituitary. Thus, stimulation of the hypothalamic magnocellular neurons can lead to the release of this newly characterized, functional neuropeptide.

Attraction of human monocytes by the neuropeptide secretoneurin.

Secretoneurin is a newly discovered 33-amino-acid peptide derived from secretogranin II (chromogranin C) that is found in sensory afferent C-fibers. We show here that secretoneurin triggers the selective migration of human monocytes in vitro and in vivo. Combinations of secretoneurin with the sensory neuropeptides, substance P or somatostatin, synergistically stimulate such migration. The attraction of monocytes represents the first established function of secretoneurin as a sensory neuropeptide.

Intracellular and extracellular processing of chromogranin A. Determination of cleavage sites.

Chromogranins are a family of acidic soluble proteins which exhibit widespread distribution in endocrine cells and neurons. Chromogranin A (CGA), the major soluble component of the secretory granules in chromaffin cells of the adrenal medulla, is a single polypeptide chain of 431 residues with an apparent molecular mass of 70-75 kDa and a pI of 4.5-5. In mature bovine chromaffin granules about 50% of the CGA has been processed. In the present paper, the structural features of the proteolytic degradation mechanism have been characterized with regard to the possible function of CGA as a prohormone, as suggested by recent studies. CGA-derived components present in chromaffin granules were subjected to either two-dimensional gel electrophoresis or HPLC and the N-terminal of each fragment was sequenced. Immunoblotting with antisera to specific sequences within the CGA molecule were used to characterize these fragments further at their C-terminal. In addition, a similar approach was performed to characterize CGA-derived fragments released into the extracellular space from directly depolarized bovine cultured chromaffin cells. Our results identified several proteolytic cleavage sites involved in CGA degradation. Intragranular processing occurs at 12 cleavage sites along the peptide chain located in both N- and C-terminal moieties of the protein; a preferential proteolytic attack in the C-terminal part was noted. We found that CGA processing also occurs in the extracellular space after release, generating new shorter fragments. The proteolytic cleavage sites identified in this study were compared with the cleavage points which are thought to be involved in generating CGA fragments with specific biological activity: pancreastatin, chromostatin and N-terminal vasostatin fragments. In addition, a new 12-amino-acid CGA-derived peptide corresponding to the sequence 65-76 was identified in the soluble core of purified chromaffin granules. This short peptide was released, together with catecholamines, after stimulation of cultured chromaffin cells suggesting its presence within the storage complex of chromaffin granules. The specific biological activity of this CGA-derived fragment remains to be determined.

Glycoprotein III (clusterin, sulfated glycoprotein 2) in endocrine, nervous, and other tissues: immunochemical characterization, subcellular localization, and regulation of biosynthesis.

Specific antisera were raised against the A and B chains of glycoprotein III. Immunoblotting revealed that in adrenal medulla both chains migrate very closely together in two-dimensional electrophoresis. Both chains with slightly differing molecular sizes are found in several endocrine tissues and in brain, kidney, liver, and serum. The mRNA has an analogous widespread distribution. In primary cultures of chromaffin cells the level of message becomes significantly increased by treatment with histamine or 12-O-tetradecanoylphorbol 13-acetate/forskolin. However, the increase is small when compared with that of secretogranin II. The subcellular localization of glycoprotein III in endocrine organs and in the posterior pituitary was investigated by subcellular fractionation and immunoelectron microscopy. Glycoprotein III was found to be confined to the large dense-core vesicles of these organs. For a discussion of the function of glycoprotein III, its localization in these organelles has to be taken into account.

Different degrees of processing of secretogranin II in large dense core vesicles of bovine adrenal medulla and sympathetic axons correlate with their content of soluble PC1 and PC2.

We investigated the processing of secretogranin II in large dense core vesicles of adrenal medulla and sympathetic nerve. Despite the fact that both types of vesicles have a very similar biochemical composition, the degree of processing of secretogranin II in vesicles from splenic nerve was significantly higher. The endoproteases PC1 and PC2, two likely candidates for secretogranin II cleavage, are found in both types of vesicles, however, relative to secretogranin II the nerve vesicles have a much higher content of these enzymes. This probably explains the fast and more extensive processing of secretogranin II in these vesicles.

Distribution of secretoneurin, a peptide derived from secretogranin II, in rat brain: an immunocytochemical and radioimmunological study.

The distribution of secretoneurin, a peptide derived from its precursor secretogranin II by proteolytic processing, was studied in the central nervous system of the rat by immunocytochemistry and radioimmunoassay and compared to the distribution of secretogranin II messenger RNA by using in situ hybridization. With a specific antiserum a distinct staining of fibers and to a lesser extent also of perikarya was observed throughout the central nervous system. A high density of immunoreactive fibers and terminals was found in several brain areas, i.e. the lateral septum, the medial parts of the amygdala, some medial thalamic nuclei, the hypothalamus, habenula, nucleus interpeduncularis, locus coeruleus, nucleus tractus solitarii, the substantiae gelatinosae of the caudal trigeminal nucleus and of the spinal cord. The quantitative distribution as measured by a radioimmunoassay agreed well with the varying densities of immunoreactivity found by immunocytochemistry. The highest concentrations of this peptide were present in the hypothalamus, in particular, in the median eminence and are comparable to those of the most highly concentrated neuropeptides. The distribution of immunopositive perikarya corresponded well with that of secretogranin II messenger RNA obtained by in situ hybridization. The pattern of secretoneurin expression in rat brain was widespread and unique, partially overlapping with established chemical transmitters and neuropeptides. The functional significance of this new brain peptide remains to be established.

Secretoneurin releases dopamine from rat striatal slices: a biological effect of a peptide derived from secretogranin II (chromogranin C).

Proteolytic processing of secretogranin II (chromogranin C) in brain leads to the formation of a 33-amino acid peptide which we have named secretoneurin. All the properties of secretoneurin are consistent with the concept that this peptide represents a neuropeptide. However, a biological function has not yet been demonstrated. Therefore, we have now investigated whether secretoneurin could alter transmitter release in brain. Slices of rat caudate-putamen were superfused in an in vitro system and dopamine was measured in the superfusate. Secretoneurin dose-dependently increased the outflow of dopamine. This response was abolished in Ca(2+)-free medium. The secretoneurin-response could also be blocked by preincubation of the peptide with a specific antiserum and was subject to rapid specific and reversible desensitization. This effect on dopamine release constitutes the first discovered biological effect found for a peptide derived from secretogranin II. Thus, secretoneurin can be added to the ever-growing number of neuropeptides.

Secretoneurin--a neuropeptide generated in brain, adrenal medulla and other endocrine tissues by proteolytic processing of secretogranin II (chromogranin C).

Secretogranin II (chromogranin C), originally described as tyrosine sulfated protein of the anterior pituitary, is present in large dense core vesicles of several endocrine cells and neurons. We raised antisera in rabbits to conjugates of two synthetic peptides (bovine secretogranin 133-151 and rat secretogranin 154-186) flanked in the primary structure of secretogranin II by pairs of basic residues and used them to investigate the proteolytic processing of this protein by immunoblotting and a newly developed radioimmunoassay. The sensitivity of this assay was 30 fmol for secretogranin 154-186 and 60 fmol for secretogranin 133-151. The highest degree of processing of secretogranin II (> 90%) occurs in brain. One of the peptides (secretogranin 133-151) is not generated to any significant extent. The other peptide, secretogranin 154-186, however, is formed in vivo, and in brain the free peptide apparently represents the predominant form. The highest concentrations of secretogranin 154-186 are found in the hypothalamus, two- to six-fold lower levels are present in the hippocampus, caudate nucleus, thalamus and brainstem. These concentrations are comparable to those of established neuropeptides. In order to indicate the special relevance of secretogranin II and of this peptide for brain we have named this peptide secretoneurin. The newly developed radioimmunoassay for this peptide will be a useful tool to establish its physiologic role in brain.