The primary structure of human secretogranin II, a widespread tyrosine-sulfated secretory granule protein that exhibits low pH- and calcium-induced aggregation

Secretogranin II (previously also called chromogranin C) is a tyrosine-sulfated secretory protein found in secretory granules in a wide variety of endocrine cells and neurons. Here, we have determined the primary structure of human secretogranin II from a full length cDNA clone and have investigated its properties, predicted from the sequence, by studying the behavior of purified secretogranin II under conditions characteristic of the milieu of secretory granules. Analysis of a 2.35-kilobase cDNA clone isolated from a human pituitary library and identified as secretogranin II by various criteria showed that human presecretogranin II is a 617-residue polypeptide containing an NH2-terminal located signal peptide. Secretogranin II lacks the disulfide-bonded loop structure near the NH2 terminus which is conserved in chromogranin A and chromogranin B (secretogranin I), two other widespread constituents of neuroendocrine secretory granules, but like the latter two proteins contains (i) an -E-N/S-L-X-A/D-X-D/E-X-E-L- motif and (ii) multiple potential dibasic cleavage sites for the generation of smaller, perhaps biologically active peptides. Another structural feature that secretogranin II shares with chromogranin A and chromogranin B (secretogranin I) is the abundance of acidic residues all along the polypeptide chain whose negative charge must somehow be neutralized to allow condensation and packaging of the protein into secretory granules. Experiments with purified secretogranin II showed that in the presence of 10 mM calcium at pH 5.2, conditions characteristic of the milieu of neuroendocrine secretory granules, this protein formed aggregates. Immunoglobulin G, a secretory protein that in vivo is not packaged into secretory granules, did not form aggregates under these in vitro conditions and was excluded from the secretogranin II aggregates. Very little aggregation of secretogranin II was observed in the absence of calcium at pH 5.2 or in the presence of calcium at neutral pH. In vivo, ammonium chloride, which is known to neutralize the pH of acidic intracellular compartments, inhibited the packaging of newly synthesized secretogranin II into secretory granules. Our results suggest that the low pH- and calcium-induced aggregation of secretogranin II may be important for the organization of the secretory granule matrix and raise the possibility that aggregation of secretogranin II may be involved in its sorting to secretory granules.     

Sequence analysis, tissue distribution and regulation by cell depolarization, and second messengers of bovine secretogranin II (chromogranin C) mRNA

Secretogranin II is a very acidic, tyrosine-sulfated protein found in secretory granules of cells belonging to the diffuse neuroendocrine system. It gained more general importance recently as a universal immunohistochemical marker for endocrine neoplasms. Sequence information was obtained from secretogranin II isolated from bovine anterior pituitaries, allowing the isolation of cDNA clones and deduction of its primary structure. Bovine secretogranin II is a 586-amino acid protein of 67,455 Da which is preceded by a signal peptide of 27 residues and contains 9 pairs of basic amino acids in its sequence which are used as potential cleavage sites for generation of physiologically active peptides. Moderately abundant mRNA levels were found in adrenal medulla, pituitary, hippocampus, and caudate. Secretogranin II message was absent from parathyroid gland, adrenal cortex, kidney, liver, and spleen. Depolarization of isolated chromaffin cells by various secretagogues significantly up-regulated secretogranin II mRNA levels by mechanisms distinct from those established for chromogranins and neuropeptides, components maintained along with secretogranin II in neuroendocrine storage vesicles.     

Sustained Dopamine Release Induced by Secretoneurin in the Striatum of the Rat: A Microdialysis Study

Abstract: Secretoneurin (SN) is a neuropeptide derived from secretogranin II that is found in brain and endocrine tissues. The aim of the present study was to determine the influence of this novel peptide on dopamine (DA) release from rat striatum using the microdialysis technique. Rat SN (1–30 µmol/L added to the dialysis buffer) enhanced DA outflow of awake rats in a concentration-dependent way without marked effects on the outflow of 3,4-dihydroxyphenylacetic acid or homovanillic acid. The increase in extracellular DA content caused by the peptide was observed throughout the entire period of administration (up to 4 h). Human SN and its 15-amino-acid C-terminal sequence also increased DA outflow, but the effects were smaller than those of rat SN. Two other peptides derived from secretogranin II were without effect on DA efflux. These results establish that SN has a pronounced effect on DA release under in vivo conditions.     

Proteolytic Processing of Chromogranin B and Secretogranin II by Prohormone Convertases

Abstract: Two experimental approaches were used to study the processing of chromogranin B and secretogranin II by prohormone convertases. In GH₃ cells various prohormone convertases were overexpressed together with the substrate chromogranin B by use of a vaccinia virus infection system. PC1 appeared to be by far the most active enzyme and converted chromogranin B to several smaller molecules, including the peptide PE-11. In brain this peptide is cleaved physiologically from chromogranin B. Some processing of chromogranin B and formation of free PE-11 were also observed with PC2 and PACE4. Furin produced larger fragments, whereas PC5-A and PC5-B had negligible effects. As a second model, PC12 cells were stably transfected with PC1 or PC2 to investigate the processing of endogenous chromogranins. Both enzymes effectively cleaved chromogranin B and secretogranin II, liberating the peptides PE-11 and secretoneurin, respectively. However, in transfection experiments the ability to generate the free peptides was more pronounced with PC2 than with PC1. The extent of proprotein processing achieved by prohormone convertases apparently differed depending on the experimental system applied. This suggests that in vivo mechanisms to support and fine-tune the activity of the processing enzymes exist, which might be overlooked by using only one methodological approach.     

Processed forms of neuroendocrine proteins 7B2 and secretogranin II are found in porcine pituitary extracts.

The complete structure of the novel polypeptide 7B2 recently deduced from cDNA clones has been reported to be highly conserved in a variety of species. The deduced amino acid sequence of the mature protein is predicted to be 185 or 186 amino acids long. While its biological role is still unknown, its occurrence in neuroendocrine secretory granules has been largely documented. This report shows: (i) that the protein, isolated from a large quantity of porcine pituitary glands, does not correspond to the full predicted cDNA structure but, on the contrary, to a truncated form; (ii) that the latter could arise from proteolytic cleavage at position 150 following pairs of basic residues; (iii) that it contains an extra residue at position 100 which is absent in the cDNA sequence; and, finally, (iv) that it displays a higher than expected molecular weight on SDS-polyacrylamide gel electrophoresis. In addition, a copurifying peptide was identified as an NH2-terminal related fragment of the secretogranin II molecule. Protein sequencing of the latter demonstrates (i) that the correct amino terminus of mature porcine secretogranin II is an Ala residue and not the previously proposed Gln residue and (ii) that this fragment could also arise from proteolytic cleavage at a pair of basic residues located within the secretogranin II sequence.     

Secretogranin II: Relative Amounts and Processing to Secretoneurin in Various Rat Tissues

Abstract: Secretoneurin is a 33-amino-acid peptide produced in vivo from secretogranin II. An antiserum raised against this peptide recognizes both the free peptide and its precursors. By HPLC and radioimmunoassay we characterized the immunoreactive molecules and determined the levels of immunoreactivity in various rat organs. In adrenal medulla and to a lesser degree in the anterior pituitary processing of secretogranin II to secretoneurin was very limited, whereas in all other organs studied (brain, intestine, endocrine pancreas, thyroid gland, and posterior pituitary) a high degree of processing was apparent. Thus, practically all of the immunoreactivity was present as free secretoneurin. This was also true for serum. When the total amount of secretoneurin immunoreactivity was calculated for the various organs, the largest pools in descending order were in the intestine, CNS, anterior pituitary, pancreas, and adrenal gland. This makes it likely that secretoneurin in serum is mainly derived from the intestine. The high degree of processing of secretogranin II in most organs is consistent with the concept that this protein acts as a precursor of a functional peptide, i.e., secretoneurin.     

The role of pancreatic chromogranins in islet physiology

Chromogranins are acidic secretory glycoproteins with a widespread but specific distribution in neuroendocrine tissues. The chromogranin family is heterogenous, consisting of propeptides such as chromogranin-A, chromogranin-B and secretogranin II, which can either elicit an effect themselves, or serve as precursors to a large number of peptides, which are biologically more active. Chromogranin processing varies in different neuroendocrine tissues. Furthermore, it is more marked in pancreatic islets than in many other tissues. Chromogranin-A and chromogranin-B are expressed in all types of pancreatic islet cells, whereas secretogranin II has not been found in pancreatic tissue. The aim of the present mini review is to focus on chromogranin-A, chromogranin-B and their derived peptides, in the function of pancreatic islets.     

Processing of secretogranin II by prohormone convertases: Importance ofPC1 in generation of secretoneurin

Secretoneurin is a recently characterized neuropeptidepresent in the primary amino acid sequence of secretogranin II. We investigated the proteolytic processing of secretogranin II by prohormone convertases in vivo in a cellular system using the vaccinia virus system. Both PC1 and PC2 can cleave the secretogranin II precursor at sites of pairs of basic amino acids to yield intermediate-sized fragments. Other convertases like PACE4, PC5 and furin were not active. For the formation of the free neuropeptide secretoneurin a different pattern was found. Only PC1 but none of the other convertases tested including PC2 were capable of generating secretoneurin. Our results demonstrate that the prohormone convertases PC1 and PC2 are involved in proteolytic processing of secretogranin II. The neuropeptide secretoneurin can only be generated by PC1 suggesting tissue-specific processing of secretogranin II in neurons expressing different subsets of the prohormone convertases.     

Recently identified a novel neuropeptide manserin colocalize with the TUNEL-positive cells in the top villi of the rat duodenum.

We recently isolated a novel 40 amino acid neuropeptide designated manserin from the rat brain. Manserin is derived from secretogranin II, a member of granin acidic secretory protein family by proteolytic processing, as previously reported secretoneurin and EM66. Manserin peptide are localized in the endocrine cells of the pituitary. In this study, we further investigated the manserin localization in the digestive system by immunohistochemical analysis using antimanserin antibody. In the duodenum, manserin immunostaining was exclusively observed in the nuclei of top villi instead of cytosol as observed in neurons in our previous study. Interestingly, manserin-positive cells in the duodenum are colocalized with terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) positive cells, the cells whose DNA was damaged. Since the top villi of duodenum epithelial cells are known to undergo spontaneous apoptosis during epithelial cell turn over, and since other peptides such as secretoneurin and EM66 derived from SgII have been reported to be cancer-related, these results indicated that manserin peptide may have a role in apoptosis and/or cancer pathogenesis in the digestive organ.     

Chromogranin/secretogranin proteins in murine heart: myocardial production of chromogranin A fragment catestatin (Chga364–384)

In the heart, the secretory granules containing the atrial natriuretic peptides (ANP) and B-type myocardial natriuretic peptide (BNP) provide the basis for the endocrine function of this organ. We sought to determine whether atrial and myocardial secretory granules contain chromogranin/secretogranin proteins including chromogranin A (CHGA/Chga), chromogranin B (CHGB/Chgb) and secretogranin II (SCG2/Scg2). Deconvolution microscopy on immunolabeled proteins revealed the presence of Chga, Chgb, and Scg2 in murine cardiac secretory granules. The presence of low plasma catestatin (CST: mChga_364–384) in older mice indicates diminished processing of Chga to CST with advancement of age, which is comparable to that found in humans. We have previously shown that CST (hCHGA_352–372) exerts potent cardio-suppressive effects on frog and rat heart, but the source of CST for such action has remained elusive. In the present study, we found CST-related peptides in cardiomyocytes and in heart, which establishes an autocrine/paracrine function of CST in cardiac tissue. We conclude that cardiac secretory granules contain Chga, Chgb and Scg2 and that Chga is processed to CST in murine heart.     

Structural characterization of peptides derived from prosomatostatins I and II isolated from the pancreatic islets of two species of teleostean fish: the daddy sculpin and the flounder

The primary structures of three peptides from extracts from the pancreatic islets of the daddy sculpin (Cottus scorpius) and three analogous peptides from the islets of the flounder (Platichthys flesus), two species of teleostean fish, have been determined by automated Edman degradation. The structures of the flounder peptides were confirmed by fast-atom bombardment mass spectrometry. The peptides show strong homology to residues (49-60), (63-96) and (98-125) of the predicted sequence of preprosomatostatin II from the anglerfish (Lophius americanus). The amino acid sequences of the peptides suggest that, in the sculpin, prosomatostatin II is cleaved at a dibasic amino acid residue processing site (corresponding to Lys61-Arg62 in anglerfish preprosomatostatin II). The resulting fragments are further cleaved at monobasic residue processing sites (corresponding to Arg48 and Arg97 in anglerfish preprosomatostatin II). In the flounder the same dibasic residue processing site is utilised but cleavage at different monobasic sites takes place (corresponding to Arg50 and Arg97 in anglerfish preprosomatostatin II). A peptide identical to mammalian somatostatin-14 was also isolated from the islets of both species and is presumed to represent a cleavage product of prosomatostatin I.     

Secretoneurin: A new peptide in the human enteric nervous system

Secretoneurin is a functional neuropeptide derived from secretogranin II (chromogranin C). This proprotein is processed to varying degrees in neuroendocrine tissues. In the present study we established by gel filtration high performance liquid chromatography that in human intestinal wall and mucosa an antiserum against secretoneurin detects as the major immunoreactive moiety the free peptide secretoneurin. In the mucosa some larger immunoreactive peptides were also present, however, a significant amount of the intact proprotein secretogranin II could not be detected. By immunohistochemistry we studied the distribution of secretoneurin within the gut. Antibodies to protein gene product 9.5 and chromogranin A were used to identify all neurons and endocrine cells, respectively, whilst those to the peptides substance P. CGRP and somatostatin were used for the further characterization of individual secretoneurin-positive structures. Secretoneurin immunoreactivity was found in nerve fibres in all layers of the gut wall. In both myenteric and submucous plexuses, nerve fibres and the majority of ganglion cells were secretoneurin-immunoreactive. In the mucosa, some secretoneurin-positive nerve processes ran parallel to the basal membrane of epithelial cells, occasionally invading the epithelial layer. Secretoneurin immunoreactivity was found in endocrine cells, mostly D cells, in the following regions in descending order of density: stomach/duodenum; rectum; colon; ileum. Thus, secretoneurin is a new major peptide within the human enteric neuroendocrine system. Its presence in abundant myenteric ganglion cells may imply a role in the modulation of gastrointestinal motility. The chemotactic properties of secretoneurin and its possible localization in sensory fibres suggest that this peptide may be involved in the genesis of intestinal inflammation.     

Post-translational processing of the porcine gastrin precursor by phosphorylation of the COOH-terminal fragment

The gene sequence encoding porcine preprogastrin is known; in order to clarify pathways of post-translational processing of the predicted precursor peptide we have characterized material reacting with antibodies to a synthetic peptide corresponding to the expected extreme COOH-terminal portion of the precursor. Radioimmunoassay was used to identify and monitor the purification of peptides in porcine antral mucosa. Two peptides (I and II) were isolated to homogeneity by steps involving gel filtration, ion exchange, and reversed-phase high performance liquid chromatography. The two co-eluted on gel filtration but were separated on anion-exchange chromatography. The more acidic peptide (II) was less hydrophobic on high performance liquid chromatography. Automated gas-phase microsequencing revealed the less acidic peptide (I) to have the sequence of porcine preprogastrin 96-104 (SAEEGDQRP); it would be produced by tryptic-like cleavage of Arg95-Ser96. The second peptide did not yield a phenylthiohydantoin-derivative on the first cycle but thereafter it sequenced as the first peptide (i.e. -AEEGDQRP). Incubation in alkali liberated almost equimolar amounts of phosphate from peptide II but not from I. In addition, alkaline phosphatase liberated phosphate and converted the acidic peptide to the less acidic one. The results suggest that serine in the first position is phosphorylated in peptide II but not I. The tripeptide -Ser(P)-Ala-Glu- also occurs in adrenocorticotropic hormone; this tripeptide is a substrate for physiological casein kinase. Potential phosphorylation sites occur at comparable positions in the precursors of a number of regulatory peptides.     

Atrial granules contain an amino-terminal processing enzyme of atrial natriuretic factor

At least three enzymes have been identified in atrial tissue homogenates that are capable of processing pro-atrial natriuretic factor to active atrial peptides. The atrial peptides possess potent natriuretic, diuretic, vasorelaxant, and hemodynamic properties, and their existence has implicated the mammalian heart as an endocrine organ. We have purified and characterized a serine proteinase (Mr approximately equal to 70,000) associated with atrial granules that preferentially hydrolyzes the Arg-Ser bond in the synthetic substrates Gly-Pro-Arg-Ser-Leu-Arg, benzoyl-Gly-Pro-Arg-Ser-Leu-Arg, and benzoyl-Gly-Pro-Arg-Ser-Leu-Arg-Arg-2-naphthylamide, the Arg-2-naphthylamide bond in the substrate benzoyl-Gly-Pro-Arg-2-naphthylamide, and the Arg-Ser bond in a 31-residue substrate (Gly96-Tyr126 peptide) corresponding to residues Arg98-Ser99 in pro-atrial natriuretic factor. The Gly96-Tyr126 peptide contains the putative processing site in pro-atrial natriuretic factor and the sequence for the bioactive peptides. Our results indicate that the minimum processing site sequence is -Gly-Pro-Arg-Ser-Leu-Arg-Arg- and that the Ser99-Tyr126 natriuretic peptide is the predominant hydrolytic product. After prolonged incubation or at high enzyme concentrations, the Ser103-Tyr126 natriuretic peptide may also be formed. The Ser103-Arg125 natriuretic peptide was only a very minor product. The doublet of basic amino acids is not the primary processing site in pro-atrial natriuretic factor, but their presence may influence cleavage at the single Arg residue "upstream." Our findings are consistent with the idea that the pro-protein and the processing enzymes are packaged into the secretory granule and in response to the proper stimulus, the pro-protein is processed to the active peptides, probably during the process of secretion. The processing pathway of pro-atrial natriuretic factor is discussed.     

Purification and sequence of a non-opioid peptide derived from ovine proenkephalin: implications for possible species specific processing

A non-enkephalin containing pentadeca peptide derived from ovine adrenal proenkephalin has been purified and sequenced. The sequence of the peptide is: Phe-Ala-Glu-Pro-Leu-Pro-Ser-Glu-Glu-Glu-Gly-Glu-Ser-Tyr-Ser (preproenkephalin 237-251) representing the amino portion of peptide B (preproenkephalin 237-268). The sequence is identical to bovine preproenkephalin 237-251, differing from the corresponding human sequence at positions 240 and 244. This peptide can be generated by a processing event common to other opioid peptides and is present in chromaffin granules in significant amounts. The presence of this peptide in substantial quantities suggests a possible difference in proenkephalin processing between the bovine and ovine adrenal medulla.     

The organisation of the mouse secretogranin II gene.

We have characterized the gene which encodes mouse secretogranin II (previously also referred to as chromogranin C), a tyrosine-sulfated secretory protein belonging to the granin (chromogranin/secretogranin) family which is found in secretory granules of most endocrine cells and neurons. The secretogranin II gene was found to contain 2 exons. In contrast to chromogranin A and chromogranin B, the two previously characterized granin genes, the entire secretogranin II protein is encoded by a single exon, exon 2, with exon 1 containing only a 5'-untranslated sequence. Consistent with previous data on the expression of secretogranin II, the putative promoter region was found to contain a cAMP-responsive element and a potential AP-1 binding site.     

Urotensin II-related peptide, the endogenous ligand for the urotensin II receptor in the rat brain

Urotensin II (UII) is a piscine neuropeptide originally isolated from the teleost urophysis. The existence of UII in mammals has been demonstrated by cloning of the mammalian orthologs of UII precursor protein genes. While rat and mouse orthologs have been reported, only the tentative structures of UII peptides of these animals have been demonstrated, since prepro-UII proteins lack the typical processing sites in the amino-terminal region of the mature peptides. A novel peptide, UII-related peptide (URP), was discovered by monitoring UII-immunoreactivity in the rat brain, and its amino acid sequence was determined to be ACFWKYCV. cDNAs encoding rat, mouse, and human precursor proteins for URP were cloned and showed that the sequences of mouse and human URP peptides are identical to that for rat URP. URP was found to bind and activate the human or rat urotensin II receptors [GPR14, UT receptor (UTR)] and showed a hypotensive effect when administrated to anesthetized rats. The prepro-URP gene is expressed in several rat tissues, although with lower levels than the prepro-UII gene and, in the human, is expressed comparably to prepro-UII in several tissues except the spinal cord. These results suggest that URP is the endogenous and functional ligand for urotensin II receptor in the rat and mouse, and possibly in the human.     

An examination of the binding behavior of histidine-containing peptides with immobilized metal complexes derived from the macrocyclic ligand, 1,4,7-triazacyclononane

In this study, two different experimental approaches have been employed to examine the binding behavior of histidine-containing peptides with metal ion complexes derived from the macrocyclic ligand 1,4,7-triazacyclononane (tacn). Firstly, a molecular modeling approach has been employed to derive the strain energies for test peptide sequences that have a predicted propensity to readily adopt an α-helical conformation. To this end, binuclear metal complexes were examined with peptides containing two histidine residues in different locations in a pair of peptides of the same composition but different sequence. These modeling results indicate that there are no energetic constraints for two-point binding to occur with dicopper(II) binuclear complexes when two histidine residues are appropriately placed in an α-helical conformation. Secondly, binding experiments were carried out to establish the effect of one or more histidine residues within a peptide sequence on the affinity of a peptide for these Cu(II)–tacn derived binuclear complexes when immobilized onto a chromatographic support material. The results confirm that for all chelating systems, higher affinity is achieved as the histidine number in the peptide structure increases, although the relative location of the histidine residues in these small peptides did not introduce a significant constraint to the conformation on interacting with the immobilized Cu(II) binuclear complexes.     

Greater binding affinity of trivalent antimony to a CCCH zinc finger domain compared to a CCHC domain of kinetoplastid proteins

It has been reported recently that Sb(III) competes with Zn(II) for its binding to the CCHC zinc finger domain of the HIV-1 NCp7 protein, suggesting that zinc finger proteins may be molecular targets for antimony-based drugs. Here, the interaction of Sb(III) with a CCCH zinc finger domain, which is considered to play a crucial role in the biology of kinetoplastid protozoa, has been characterized for the first time. The binding characteristics of Sb(III) were compared between a CCCH-type peptide derived from a kinetoplastid protein and two different CCHC-type zinc finger peptides. The formation of 1 : 1 Zn-peptide and Sb-peptide complexes from the different peptides was demonstrated using circular dichroism, UV absorption, fluorescence spectroscopies and ESI-MS. Titration of the Zn-peptide complexes with SbCl(3) was performed at pH 6 and 7, exploiting the intrinsic fluorescence of the peptides. The differential spectral characteristics of the peptides allowed for competition experiments between the different peptides for binding of Zn(II). The present study establishes that Sb(III) more effectively displaces Zn(II) from the CCCH peptide than CCHC ones, as a result of both the greater stability of the Sb-CCCH complex (compared to Sb-CCHC complexes) and the lower stability of the Zn-CCCH complex (compared to Zn-CCHC complexes). Comparison of the binding characteristics of Sb(III) or Zn(II) between the CCHC-type peptides with different amino acid sequences supports the model that not only the conserved zinc finger motif, but also the sequence of non-conserved amino acids determines the binding affinity of Sb(III) and Zn(II). These data suggest that the interaction of Sb(III) with CCCH-type zinc finger proteins may modulate, or even mediate, the pharmacological action of antimonial drugs.