Dibasic Processing Sites Research Articles

Diversity of Neuropeptide Cell-Cell Signaling Molecules Generated by Proteolytic Processing Revealed by Neuropeptidomics Mass Spectrometry

Neuropeptides are short peptides in the range of 3-40 residues that are secreted for cell-cell communication in neuroendocrine systems. In the nervous system, neuropeptides comprise the largest group of neurotransmitters. In the endocrine system, neuropeptides function as peptide hormones to coordinate intercellular signaling among target physiological systems. The diversity of neuropeptide functions is defined by their distinct primary sequences, peptide lengths, proteolytic processing of pro-neuropeptide precursors, and covalent modifications. Global, untargeted neuropeptidomics mass spectrometry is advantageous for defining the structural features of the thousands to tens of thousands of neuropeptides present in biological systems. Defining neuropeptide structures is the basis for defining the proteolytic processing pathways that convert pro-neuropeptides into active peptides. Neuropeptidomics has revealed that processing of pro-neuropeptides occurs at paired basic residues sites, and at non-basic residue sites. Processing results in neuropeptides with known functions and generates novel peptides representing intervening peptide domains flanked by dibasic residue processing sites, identified by neuropeptidomics. While very short peptide products of 2-4 residues are predicted from pro-neuropeptide dibasic processing sites, such peptides have not been readily identified; therefore, it will be logical to utilize metabolomics to identify very short peptides with neuropeptidomics in future studies. Proteolytic processing is accompanied by covalent post-translational modifications (PTMs) of neuropeptides comprising C-terminal amidation, N-terminal pyroglutamate, disulfide bonds, phosphorylation, sulfation, acetylation, glycosylation, and others. Neuropeptidomics can define PTM features of neuropeptides. In summary, neuropeptidomics for untargeted, global analyses of neuropeptides is essential for elucidation of proteases that generate diverse neuropeptides for cell-cell signaling. Graphical Abstract ᅟ.

Evolution of gastrointestinal hormones: the cholecystokinin/gastrin family

To describe recent advances in our understanding of the evolution of gastrointestinal hormones, with the gastrin/cholecystokinin (CCK) family as a model. The release of 11 genomic sequences in the last year has provided a wealth of additional information on peptide hormone sequences. The alternative approach of reverse genetics has identified a separate class of CCK receptor ligands in the nematode Caenorhabditis elegans. Three classes of ligands, insect sulfakinins, nematode neuropeptide-like proteins and vertebrate gastrins/cholecystokinins, have now been described for the family of CCK receptors. Although all terminate in an amidated phenylalanine, similarity between the three classes is minimal elsewhere in the sequences. The occurrence of separate gastrin and CCK genes in the dogfish Squalus acanthias dates the divergence of gastrin and CCK to at least 528 +/- 56 Myr ago. The presence of a polyglutamate sequence in marsupial gastrins suggests that the ability to bind ferric ions, which is a critical determinant of biological activity for nonamidated gastrins, was acquired at least 173 +/- 12 Myr ago. Comparison of gastrin or CCK sequences between species suggests that, apart from the C-terminal tetrapeptide amide that is required for receptor binding, conservation is largely restricted to the dibasic processing sites and to the C-terminal flanking peptides of gastrin and CCK. The independent conservation of the latter peptide may be either a consequence of a requirement for precise processing, or may indicate a separate function.

Differential Accessibilities of Dibasic Prohormone Processing Sites of Proenkephalin to the Aqueous Environment Revealed by H−D Exchange Mass Spectrometry

Proenkephalin (PE) is a prohormone containing dibasic sites that are cleaved by proteases to generate peptide neurotransmitters and hormones. Little is known about the conformational features of such protease cleavage sites within prohormone substrates. Therefore, the goal of this study was to investigate the relative accessibilities of multiple dibasic processing sites of PE by peptide amide hydrogen-deuterium exchange mass spectrometry (DXMS). DXMS demonstrated differences in the relative accessibilities of the KR, KK, and RR cleavage sites of PE to the aqueous environment. DXMS assesses relative rates of exchange of hydrogens of the polypeptide backbone of PE with deuterium atoms from D(2)O (heavy water) in solvent. Analyses of peptides spanning each of the 12 dibasic PE cleavage sites illustrated differences in H-D exchange rates that reflect relative solvent accessibility. The mid-domain cleavage sites (dibasic sites 4-8) exhibited greater accessibility to the aqueous solvent compared to regions of the NH(2) and COOH domains (dibasic sites 2, 3, and 9-11, respectively). The NH(2)- and COOH-terminal domains both exhibited relatively high H-D exchange rates. The hydrogen exchange rate profile of PE, as well as its circular dichroism (CD) features for secondary structure, was modified in trifluoroethanol, an organic solvent that represents a more hydrophobic environment. These findings suggest that the dibasic protease cleavage sites of the PE prohormone with differences in accessibility to the aqueous environment undergo proteolytic processing to generate active neuropeptides for cell-cell communication in neuroendocrine systems.

Alternative Processing of Proproteins in AspergillikexBGene Disruptants under Hyperosmotic Conditions

Disruption of the kexB gene encoding a subtilisin-like processing protease in Aspergillus oryzae and Aspergillus nidulans led to remarkable morphological defects, and these phenotypes were suppressed under hyperosmotic conditions. In this study, we investigated to determine whether non-KexB proteases might complement the in vivo function of KexB in the two Aspergillus kexB disruptants. Neither overexpression of opsA or opsB encoding A. oryzae aspartyl proteases homologous to yeast yapsins (YPS1/2) suppressed the kexB mutation, although yapsins are multicopy suppressors for the yeast kex2 mutation. A. nidulans and A. oryzae kexB disruptants grown under hyperosmotic conditions processed a recombinant fusion protein carrying a synthetic dibasic processing site (Lys-Arg) although the disruptants grown under normal growth conditions did not cleave the site. These results suggest that the two Aspergilli have other potential processing proteases that are induced and/or activated under hyperosmotic conditions and consequently complement, at least in part, the in vivo function of KexB.

Prohormone convertases 1/3 and 2 together orchestrate the site-specific cleavages of progastrin to release gastrin-34 and gastrin-17

Cellular synthesis of peptide hormones requires PCs (prohormone convertases) for the endoproteolysis of prohormones. Antral G-cells synthesize the most gastrin and express PC1/3, 2 and 5/6 in the rat and human. But the cleavage sites in progastrin for each PC have not been determined. Therefore, in the present study, we measured the concentrations of progastrin, processing intermediates and alpha-amidated gastrins in antral extracts from PC1/3-null mice and compared the results with those in mice lacking PC2 and wild-type controls. The expression of PCs was examined by immunocytochemistry and in situ hybridization of mouse G-cells. Finally, the in vitro effect of recombinant PC5/6 on progastrin and progastrin fragments containing the relevant dibasic cleavage sites was also examined. The results showed that mouse G-cells express PC1/3, 2 and 5/6. The concentration of progastrin in PC1/3-null mice was elevated 3-fold. Chromatography showed that cleavage of the Arg(36)Arg(37) and Arg(73)Arg(74) sites were grossly decreased. Accordingly, the concentrations of progastrin products were markedly reduced, alpha-amidated gastrins (-34 and -17) being 25% of normal. Lack of PC1/3 was without effect on the third dibasic site (Lys(53)Lys(54)), which is the only processing site for PC2. Recombinant PC5/6 did not cleave any of the dibasic processing sites in progastrin and fragments containing the relevant dibasic processing sites. The complementary cleavages of PC1/3 and 2, however, suffice to explain most of the normal endoproteolysis of progastrin. Moreover, the results show that PCs react differently to the same dibasic sequences, suggesting that additional structural factors modulate the substrate specificity.

Pattern analysis for the prediction of fungal pro-peptide cleavage sites

Support vector machines (SVMs) have many applications in investigating biological data from gene expression arrays to understanding EEG signals of sleep stages. In this paper, we have developed an application that will support the prediction of the pro-peptide cleavage site of fungal extracellular proteins which display mostly a monobasic or dibasic processing site. Many of the secretory proteins and peptides are synthesized as inactive precursors and they become active after post-translational processing. A collection of fungal pro-protein sequences are used as a training data set. A specifically designed kernel is expressed as an application of the well-known Gaussian kernel via feature spaces defined for our problem. Rather than fixing the kernel parameters with cross validation or other methods, we introduce a novel approach that simultaneously performs model selection together with the test of accuracy and testing confidence levels. This leads us to higher accuracy at significantly reduced training times. The results of the server ProP1.0 which predicts pro-peptide cleavage sites are compared with the results of this study. A similar mathematical approach may be adapted to pro-peptide cleavage prediction in other eukaryotes.

Unique Form and Osmoregulatory Function of a Neurohypophysial Hormone in a Urochordate

The cyclic nonapeptides, oxytocin and vasopressin, are neurohypophysial hormones that regulate many significant physiological processes related especially to reproduction and osmoregulation. In this study, we characterized an oxytocin-related peptide cDNA from a urochordate, Styela plicata, thought to be a sister group to vertebrates. Sequence analysis of the deduced precursor polypeptide revealed that the precursor is composed of three segments: a signal peptide, an oxytocin-like sequence flanked by a Gly C-terminal amidation signal and a Lys-Arg dibasic processing site, and a neurophysin domain, similar to other oxytocin/vasopressin family precursors. However, unlike other members of this family, the tunicate oxytocin-like peptide (CYISDCPNSRFWST-NH2) is a tetradecapeptide. We termed this peptide Styela oxytocin-related peptide (SOP). Furthermore, analyses of mass spectrometry, in situ hybridization, and immunohistochemistry demonstrated production of mature SOP in the cerebral ganglion. To elucidate the physiological action of SOP, we kept the tunicate for 2 d under the three different concentrations of seawater, 60, 100, and 130%, and measured the expression levels of SOP mRNA in the cerebral ganglion. The greatest expression of SOP mRNA was observed in the 60% seawater. In 60% seawater, but not in 100 or 130%, the tunicate mostly closed the atrial and branchial siphons. Therefore, we investigated the contractile effects of SOP on the siphons in vitro. SOP caused contractions in both siphons in a dose-dependent manner. Taken together, these results suggest that SOP acts to prevent the influx of a low concentration of seawater into the body and thus play an important role in osmoregulation.

Identification of putative crustacean neuropeptides using in silico analyses of publicly accessible expressed sequence tags

The development of expressed sequence tags (ESTs) for crustacean cDNA libraries and their deposition in publicly accessible databases has generated a rich resource for peptide discovery in this commercially and ecologically important arthropod subphylum. Here, we have conducted in silico searches of these databases for unannotated ESTs encoding putative neuropeptide precursors using the BLAST program tblastn, and have predicted the mature forms of the peptides encoded by them. The primary strategy used was to query the database with known decapod prepro-hormone sequences or, in some instances, insect precursor protein sequences. For neuropeptides for which no prepro-hormones are known, the peptides themselves were used as queries. For those peptides expected to originate from a common precursor, the individual sequences were combined, with each peptide flanked by a dibasic processing site and, if amidated, a glycine residue. Using these approaches, 13 unannotated ESTs encoding putative neuropeptide precursors were found. For example, using the first strategy, putative Marsupenaeus japonicus prepro-hormones encoding B-type allatostatins, neuropeptide F (NPF), and orcokinins were identified. Similarly, several Homarus americanus ESTs encoding putative orcokinin precursors were found. In addition to the decapod prepro-hormones, ESTs putatively encoding a NPF isoform and a red pigment concentrating hormone-like peptide were identified from the cladoceran Daphnia magna, as was one EST putatively encoding multiple tachykinin-related peptides from the isopod Eurydice pulchra. Using the second strategy, we identified a Carcinus maenas EST encoding HIGSLYRamide, a peptide recently discovered via mass spectrometry from Cancer productus. Using mass spectral methods we confirmed that this peptide is also present in Carcinus maenas. Collectively over 50 novel crustacean peptides were predicted from the identified ESTs, providing a strong foundation for future investigations of the evolution, regulation and function of these and related molecules in this arthropod taxon.

Expression of human α1-proteinase inhibitor in Aspergillus niger

BackgroundHuman α1-proteinase inhibitor (α1-PI), also known as antitrypsin, is the most abundant serine protease inhibitor (serpin) in plasma. Its deficiency is associated with development of progressive, ultimately fatal emphysema. Currently in the United States, α1-PI is available for replacement therapy as an FDA licensed plasma-derived (pd) product. However, the plasma source itself is limited; moreover, even with efficient viral inactivation steps used in manufacture of plasma products, the risk of contamination from emerging viruses may still exist. Therefore, recombinant α1-PI (r-α1-PI) could provide an attractive alternative. Although r-α1-PI has been produced in several hosts, protein stability in vitro and rapid clearance from the circulation have been major issues, primarily due to absent or altered glycosylation.ResultsWe have explored the possibility of expressing the gene for human α1-PI in the filamentous fungus Aspergillus niger (A. niger), a system reported to be capable of providing more "mammalian-like" glycosylation patterns to secretable proteins than commonly used yeast hosts. Our expression strategy was based on fusion of α1-PI with a strongly expressed, secreted leader protein (glucoamylase G2), separated by dibasic processing site (N-V-I-S-K-R) that provides in vivo cleavage. SDS-PAGE, Western blot, ELISA, and α1-PI activity assays enabled us to select the transformant(s) secreting a biologically active glycosylated r-α1-PI with yields of up to 12 mg/L. Matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) analysis further confirmed that molecular mass of the r-α1-PI was similar to that of the pd-α1-PI. In vitro stability of the r-α1-PI from A. niger was tested in comparison with pd-α1-PI reference and non-glycosylated human r-α1-PI from E. coli.ConclusionWe examined the suitability of the filamentous fungus A. niger for the expression of the human gene for α1-PI, a medium size glycoprotein of high therapeutic value. The heterologous expression of the human gene for α1-PI in A. niger was successfully achieved to produce the secreted mature human r-α1-PI in A. niger as a biologically active glycosylated protein with improved stability and with yields of up to 12 mg/L in shake-flask growth.

Processing of multiple forms of preprosodefrin in the abdominal gland of the red-bellied newt Cynops pyrrhogaster: regional and individual differences in preprosodefrin gene expression

Peptides derived from the post-translational processing of preprosodefrin were isolated from an extract of the abdominal glands of male red-bellied newts Cynops pyrrhogaster obtained 5 months prior to the onset of the breeding season. Structural characterization of the peptides showed that the pheromone sodefrin (SIPSKDALLK) is stored in a biologically inactive COOH-terminally extended form (SIPSKDALLKISA). It follows, therefore, that the activation of a protease that cleaves at a Lys–Ile bond to generate the active pheromone must occur by the time of onset of reproductive behavior. Additional peptides (representing preprosodefrin-(146–175)-peptide and preprosodefrin-(159–173)-peptide), that are derived from the precursor by cleavage at monobasic and dibasic processing sites, were also purified from the extract. The isolation of paralogs of these peptides, including an inactive COOH-terminally extended form of [Asn 10]sodefrin, provides evidence for the expression of multiple genes encoding preprosodefrin. PCR products derived from total RNAs from the abdominal gland of individual newts collected from three different regions of Japan were analyzed. The data confirm the existence of multiple genes encoding sodefrin and its variants whose expression varied according to the individuals and the regions. However, genes encoding sodefrin were found to be expressed in all the specimens sampled.

Progastrin Is Directed to the Regulated Secretory Pathway by Synergistically Acting Basic and Acidic Motifs

Bioactivation of prohormones occurs in the granules of the regulated secretory pathway of endocrine cells, which release hormones in response to external stimulation. How secretory granules are formed and how the cargo is selected is still unclear, but it has been shown for several prohormones and processing enzymes that domains within the prohormone structure can act as "sorting signals" for this pathway. The domains mediate interactions with other proteins or with the membrane or facilitate aggregation of the (pro)peptides. We have now searched for domains in progastrin that are active in sorting the prohormone into secretory granules. Truncation studies showed that the N-terminal 30 residues of progastrin are dispensable, whereas the last 49 residues are sufficient for correct biosynthesis of bioactive gastrin. Thus, further N-terminal truncation abolished gastrin expression. C-terminal truncation of 8 residues resulted in an increase in basal secretion as did point mutations in the dibasic processing sites of progastrin. These mutants, however, still responded to secretagogues, suggesting a residual sorting capacity to the regulated pathway. Amino acid substitutions in an acidic, polyglutamate motif within gastrin-17, the main bioactive, cellular gastrin form, did not alter secretion per se, but when these residues were substituted in C-terminally truncated mutants, double mutants increased in basal secretion and did not respond to secretagogue stimulation. This implies that the mutants are constitutively secreted. Our data suggest that the dibasic processing sites constitute the most important sorting domain of progastrin, and these sites act in synergy with the acidic domain.

Molecular characterization and cell-specific expression of a Manduca sexta FLRFamide gene.

FMRFamide-related peptides (FaRPs) are a large group of neuropeptides containing a common RFamide C-terminus; they have been identified in vertebrates and invertebrates. We have isolated the cDNA that encodes three FaRPs in the tobacco hornworm, Manduca sexta, including the amidated decapeptide F10. The larger FaRPs are the partially processed precursors of F10, a neuropeptide belonging to the myosuppressin family of peptides. The presence of all three FaRPs in different tissues suggests differential utilization of typical dibasic processing sites and atypical processing sites C-terminal to leucine residues. F10 mRNA was detected in the brain, nerve cord, and midgut, and the mRNA levels in the nervous system are dynamically regulated during development. In situ hybridization analysis localized the F10 mRNA to a variety of cell types within the central nervous system (CNS), a peripheral neurosecretory cell (L1), and midgut endocrine cells, which suggests diverse functions. Distribution of the F10-containing neurons within the central nervous system is segment-specific, and the developmental profile suggests that the F10 gene products may have stage-specific functions. Molecular characterization of the F10 gene has provided insights into its regulation and cell-specific distribution that will enhance our understanding of how these FaRPs modulate different physiological systems and ultimately behavior.

Diversity of Arabidopsis Genes Encoding Precursors for Phytosulfokine, a Peptide Growth Factor

Phytosulfokine-alpha (PSK-alpha), a unique plant peptide growth factor, was originally isolated from conditioned medium of asparagus (Asparagus officinalis) mesophyll cell cultures. PSK-alpha has several biological activities including promoting plant cell proliferation. Four genes that encode precursors of PSK-alpha have been identified from Arabidopsis. Analysis of cDNAs for two of these, AtPSK2 and AtPSK3, shows that both of these genes consist of two exons and one intron. The predicted precursors have N-terminal signal peptides and only a single PSK-alpha sequence located close to their carboxyl termini. Both precursors contain dibasic processing sites flanking PSK, analogous to animal and yeast prohormones. Although the PSK domain including the sequence of PSK-alpha and three amino acids preceding it are perfectly conserved, the precursors bear very limited similarity among Arabidopsis and rice (Oryza sativa), suggesting a new level of diversity among polypeptides that are processed into the same signaling molecule in plants, a scenario not found in animals and yeast. Unnatural [serine-4]PSK-beta was found to be secreted by transgenic Arabidopsis cells expressing a mutant of either AtPSK2 or AtPSK3 cDNAs, suggesting that both AtPSK2 and AtPSK3 encode PSK-alpha precursors. AtPSK2 and AtPSK3 were expressed demonstrably not only in cultured cells but also in intact plants, suggesting that PSK-alpha may be essential for plant cell proliferation in vivo as well as in vitro. Overexpression of either precursor gene allowed the transgenic calli to grow twice as large as the controls. However, the transgenic cells expressing either antisense cDNA did not dramatically decrease mitogenic activity, suggesting that these two genes may act redundantly.

Mutational screening of the CART gene in obese children: identifying a mutation (Leu34Phe) associated with reduced resting energy expenditure and cosegregating with obesity phenotype in a large family.

Cocaine- and amphetamine-regulated transcript (CART) inhibits feeding and induces the expression of c-Fos in hypothalamic areas implicated in appetite regulation. Furthermore, the CART peptide is found in neurons regulating sympathetic outflow, which in turn play an integral role in regulating body temperature and energy expenditure. The CART gene was screened by single-strand conformation polymorphism and automatic sequencing in 130 (72 girls) unrelated obese Italian children and adolescents. Their Z-scores (mean +/- SD) of relative to BMI percentiles was 3.9 +/- 1.8, and the average age at obesity onset was 4.7 +/- 2.6 years. Two previously described silent polymorphisms were found in the 3' untranslated region: an adenine deletion at position 1457 in 9 patients (allele frequency 0.035) and an A/G substitution at position 1475 in 11 patients (allele frequency 0.042). We found no difference between the obese patients heterozygous for one of these polymorphisms and those patients homozygous for the wild allele with respect to their age of obesity onset, BMI Z-scores, and leptin levels. A missense mutation of G729C resulting in the substitution of Leu with Phe at codon 34, within the NH2-terminal CART region, has been detected in the heterozygous state in a 10-year-old obese boy who has been obese since the age of 2 years. The patient belongs to a large family of obese subjects. The mutation cosegregated with the severe obesity phenotype over three generations and was not found in the control population. Resting metabolic rates were lower than expected in the propositus (-14%) and his mother (-16%), who carried the mutation. Leucine at codon 34, conserved in this position in the human and in the rat sequences, immediately precedes a couple of lysine residues that may well represent a dibasic processing site. The Leu34Phe mutation might alter the susceptibility to proteolysis of this potential processing site, likely altering the CART effect on thermogenesis and energy expenditure.

Identification and expression of gastrin and cholecystokinin mRNAs from the turtle, Pseudemys scripta: evidence of tissue-specific tyrosyl sulfation

Gastrin and cholecystokinin (CCK) are related peptide hormones expressed in the brain and gut of vertebrates. In this study, complementary DNAs have been characterised from the red-eared slider turtle, Pseudemys scripta. The encoded preproCCK contains mono and dibasic endoproteolytic processing sites for formation of the previously identified CCK-70, CCK-40 and CCK-8 products, whereas preprogastrin contains two dibasic processing sites for the generation of gastrin-52. Alignment of the predicted preprohormone structures with those of other species, showed that preproCCK has been well conserved among all vertebrates, whereas progastrin is less conserved. Both gastrin and CCK mRNA display expression patterns similar to their mammalian counterparts, with CCK being expressed in the brain, duodenum and small intestine, and gastrin in the antrum. Heterologous expression of turtle preprogastrin in a mammalian endocrine cell line led to production of carboxyamidated gastrin-52 as observed in turtle antrum. However, in contrast to the non-sulfated endogenous peptide, the heterologously expressed gastrin was completely Tyr sulfated. Consequently, it appears that either gastrin producing cells in the turtle gut do not express tyrosylprotein sulfotransferases or the enzyme(s) present in turtle antrum is unable to sulfate turtle gastrin.

Dynorphin A processing enzyme: tissue distribution, isolation, and characterization.

Limited proteolysis of the dynorphin precursor (prodynorphin) at dibasic and monobasic processing sites results in the generation of bioactive dynorphins. In the brain and neurointermediate lobe of the pituitary, prodynorphin is processed to produce alpha and beta neo endorphins, dynorphins (Dyn) A-17 and Dyn A-8, Dyn B-13, and leucine-enkephalin. The formation of Dyn A-8 from Dyn A-17 requires a monobasic cleavage between Ile and Arg. We have identified an enzymatic activity capable of processing at this monobasic site in the rat brain and neurointermediate lobe of the bovine pituitary; this enzyme is designated "dynorphin A-17 processing enzyme." In the rat brain and neurointermediate lobe, a majority of the Dyn A processing enzyme activity is membrane-associated and can be released by treatment with 1% Triton X-100. This enzyme has been purified to apparent homogeneity from the membrane extract of the neurointermediate lobe using preparative iso-electrofocussing in a granulated gel pH 3.5 to 10, FPLC using anion exchange chromatography, and non-denaturing electrophoresis. The Dyn A processing enzyme exhibits a pI of about 5.8 and a molecular mass of about 65 kDa under reducing conditions. The Dyn A processing enzyme is a metalloprotease and has a neutral pH optimum. It exhibits substantial sensitivity to metal chelating agents and thiol agents suggesting that this enzyme is a thiol-sensitive metalloprotease. Specific inhibitors of other metallopeptidases such as enkephalinase [EC 3.4.24.11], the enkephalin generating neutral endopeptidase [EC 3.4.24.15], or NRD convertase do not inhibit the Dyn A processing enzyme activity. In contrast, specific inhibitors of angiotensin converting enzyme inhibit the activity. The purified enzyme is able to process a number of neuropeptides at both monobasic and dibasic sites. These characteristics are consistent with a role for the Dyn A processing enzyme in the processing of Dyn A-17 and other neuropeptides in the brain.

Evidence for Conservation of the Vasopressin/Oxytocin Superfamily in Annelida

Annetocin is a structurally and functionally oxytocin-related peptide isolated from the earthworm Eisenia foetida. We present the characterization of the annetocin cDNA. Sequence analyses of the deduced precursor polypeptide revealed that the annetocin precursor is composed of three segments: a signal peptide, an annetocin sequence flanked by a Gly C-terminal amidation signal and a Lys-Arg dibasic processing site, and a neurophysin domain, similar to other oxytocin family precursors. The proannetocin showed 37.4-45.8% amino acid homology to other prohormones. In the neurophysin domain, 14 cysteines and amino acid residues essential for association of a neurophysin with a vasopressin/oxytocin superfamily peptide were conserved, suggesting that the Eisenia neurophysin can bind to annetocin. Furthermore, in situ hybridization experiments demonstrated that the annetocin gene is expressed exclusively in neurons of the central nervous system predicted to be involved in regulation of reproductive behavior. These findings confirm that annetocin is a member of the vasopressin/oxytocin superfamily. This is the first identification of the cDNA encoding the precursor of an invertebrate oxytocin-related peptide and also the first report of the identification of an annelid vasopressin/oxytocin-related precursor.

Biosynthesis of secretogranin II in Xenopus intermediate pituitary

Secretogranin II (SgII) is a sulphated secretory protein found in a broad variety of neuroendocrine cells. We have raised an antiserum against SgII to monitor its fate in Xenopus intermediate pituitary. Pulse-chase incubations in combination with immunoprecipitation analysis showed that SgII was synthesised as an 84-kDa precursor protein which was processed to fragments of 69, 54, 34, 21 and 15 kDa. Secretion of these cleavage products was sensitive to the dopamine D 2 receptor agonist apomorphine, and thus occurred via the regulated secretory pathway. When cells were treated with the fungal metabolite brefeldin A or with the specific vacuolar H +-ATPase inhibitor bafilomycin A1, the processing of SgII and the release of its cleavage products were strongly inhibited, indicating that its processing commenced in the later compartments of the secretory pathway. Pulse-chase and immunoblot analysis showed that the 21-kDa fragment was the major SgII-derived cleavage and release product, and carried secretoneurin, a highly conserved peptide flanked by potential dibasic processing sites. Hence, SgII is cleaved to a variety of products that are released via the regulated secretory pathway, while secretoneurin does not seem to represent a major end-product of SgII processing in Xenopus intermediate pituitary.

PR-39, a proline-rich peptide antibiotic from pig, and FALL-39, a tentative human counterpart

The peptide antibiotic PR-39 was originally isolated from the upper part of pig intestine. It has antibacterial activity against Gram negative bacteria at concentrations comparable with tetracycline. Studies of the mechanism of action showed that PR-39 inhibits both DNA and protein synthesis. Recently, PR-39 was found in wound fluid and was shown to have inductive activity on matrix components as part of the wound repair process. We have now sequenced the complete gene and possible mediators of its expression will be discussed. Our attempts to characterize the human counterpart of PR-39 by probing for the well conserved prepro-part led to a different peptide antibiotic. A clone containing the coding information for this new peptide was isolated from a human bone marrow cDNA library. The putative human peptide antibiotic was designated FALL-39 after the first four residues and the total number of residues. All human peptide antibiotics previously isolated (or predicted) belong to the defensin family with three disulfide bridges, while FALL-39 lacks cysteine. The clone for the prepro-FALL-39 encodes a cathelin-like precursor protein with 170 amino acid residues. We have postulated a dibasic processing site for the mature FALL-39 and chemically synthesized the peptide. In the presence of the basal medium E, synthetic FALL-39 was highly active against Escherichia coli D21 and Bacillus megaterium Bm11. Residues 13–34 in FALL-39 can be predicted to form a perfect amphipatic helix and CD spectra showed that medium E induced 30% helix formation in FALL-39. By Northern blot analyses the transcript was located in bone marrow and testis. The structure of the gene and the chromosomal location is under investigation.

Conformational studies on synthetic peptides reproducing the dibasic processing site of pro-ocytocin-neurophysin.

Synthetic peptides reproducing the proteolytic processing site of pro-ocytocin were studied by different spectroscopic techniques, including circular dichroism, Fourier transform infrared absorption, and mono and bidimensional nuclear magnetic resonance, in order to ascertain the possible role of three-dimensional structure in the recognition process by maturation enzymes. Experimental results were compared with energy minimization calculations and suggest that: (i) the region situated on the N-terminus of the Lys-Arg doublet may form a beta-turn; (ii) the sequential organization of the residues participating in the beta-turn determines the privileged relative orientation of the basic amino acid sidechains and the subtype of turn; and (iii) the peptide segment situated on the C-terminal side of the dibasic doublet may assume a helix arrangement. These findings, in spite of the limitations connected to the flexibility of linear peptides, seem to substantiate the hypothesis that structural motifs around the cleavage site could be important for recognition and processing. however, a straightforward correlation between details of the secondary structure and the in vitro reactivity toward a putative convertase is not yet possible.