Secondary literature sources for NPP

The following references were automatically generated.

Kobayashi Y et al.
cDNA cloning of proopiomelanocortin (POMC) and mass spectrometric identification of POMC-derived peptides from snake and alligator pituitaries.
Gen Comp Endocrinol. 2007; 152: 73-81
Display abstract
Proopiomelanocortin (POMC) is the precursor of melanocyte-stimulating hormone (MSH) and beta-endorphin, and is suggested to have evolved by the insertion and deletion of ancestral MSH segments. Here, the primary structure of POMC was determined with cDNA cloning of brown tree snakes of Squamata and American alligators of Crocodylia to show an overview of the molecular evolution of POMC in reptiles. Snake and alligator POMCs are composed of alpha-, beta-, and gamma-MSH segments and a single beta-END segment as in other tetrapods; however, the gamma-MSH segment in snake POMC has a mutation in the essential sequence from His-Phe-Arg-Trp to His-(d)-(d)-Arg, in which (d) means deletion. It is conceivable that the ancestry of snake gamma-MSH had weak functional constraint and lacked biological significance during evolution. Phylogenetic analyses using the neighbor-joining method show that snake prePOMC is most diverged, and alligator prePOMC is most conserved in reptilian POMCs while it shows the highest sequence identity with ostrich prePOMC. These relationships are comparable to those observed in mitochondrial DNA. On the other hand, analyses of the pituitary with mass spectrometry revealed several peptides by post-translational processing as predicted by the locations of processing sites consisting of basic amino acid residues in snake and alligator POMCs. Remarkably, the monobasic site at the N-terminal side of the snake beta-MSH is suggested to act as a processing site. Thus, the study shows the divergence of snake POMC such as the critical mutation of gamma-MSH and high conservation of hormone organization of alligator POMC.

Naude R, Oelofsen W, Takahashi A, Amano M, Kawauchi H
Molecular cloning and characterization of preproopiomelanocortin (prePOMC) cDNA from the ostrich (Struthio camelus).
Gen Comp Endocrinol. 2006; 146: 310-7
Display abstract
To date proopiomelanocortin (POMC), the precursor protein for melanotropin (MSH), adrenocorticotropin (ACTH), lipotropins (LPH), and beta-endorphin (beta-END) in the pituitary gland, has been studied extensively over a wide spectrum of vertebrate classes. A paucity of information exists, however, with regard to POMC in the avian class, where to date POMC from only one species, the domestic chicken, appears to have been fully characterized. In the present study, we report the use of three clones of cDNA to provide the complete nucleotide sequence of ostrich prePOMC cDNA, consisting of 1072 bp (excluding the poly(A) tail). The deduced amino acid sequence of 253 amino acid residues includes the N-terminal signal peptide of 17 amino acid residues. The predicted amino acid sequence in the overall arrangement of its domains, conforms to that found in other tetrapods. Sequence domains for gamma-MSH, ACTH, alpha-MSH, gamma-LPH, beta-MSH, and beta-END are located at positions 74-85, 134-172, 134-146, 175-220, 203-220, and 223-253, respectively, in ostrich prePOMC, but some of them may not be released in the ostrich pituitary gland, despite the presence of nine potential processing sites consisting of 2-4 dibasic amino acids each. Substitution of glutamic acid for a dibasic amino acid at position 202 in ostrich prePOMC could prevent release of beta-MSH. To date the release of pro-gamma-MSH, beta-LPH, ACTH, gamma-LPH, and beta-END have been confirmed by direct isolation and characterization from ostrich pituitary extracts. In the present study, we have also identified ACTH, gamma-LPH and beta-END in a single frozen ostrich pituitary slice by means of MALDI-TOF mass spectrometry. When compared to a wide range of vertebrate prePOMC molecules, ostrich prePOMC revealed a high level of amino acid sequence identity (77%) with chicken prePOMC, which is the only other avian sequence available. As with other vertebrate classes, considerable intraclass differences were also evident between chicken and ostrich prePOMCs, which belong to different avian orders. Identity of ostrich prePOMC with non-avian tetrapod counterparts is only moderate (53-56%), whereas lower identities (20-49%) are evident over a range of fish prePOMCs.

Takahashi A, Amano M, Amiya N, Yamanome T, Yamamori K, Kawauchi H
Expression of three proopiomelanocortin subtype genes and mass spectrometric identification of POMC-derived peptides in pars distalis and pars intermedia of barfin flounder pituitary.
Gen Comp Endocrinol. 2006; 145: 280-6
Display abstract
Proopiomelanocortin (POMC) is a common precursor of adrenocorticotropic hormone (ACTH), melanophore-stimulating hormone (MSH), and endorphin (END). In pituitary gland, POMC receives posttranslational processing by which different peptides are generated in the pars distalis (PD) and pars intermedia (PI). Recently, we cloned three subtypes of the POMC gene in pituitary gland of barfin flounder. The present study was undertaken to elucidate whether the three POMC genes are expressed in both the PD and PI of barfin flounder pituitary, and to identify peptides derived from POMCs in these lobes. We amplified the transcripts of POMC-A, -B and -C in both the PD and PI by the reverse transcription-polymerase chain reaction. In situ hybridization also detected signals for these three subtypes in the PD and PI. These results demonstrated that all three POMC genes are expressed in both the PD and PI of barfin flounder pituitary. By mass spectrometric analyses, ACTH-A, Des-acetyl (Ac)-alpha-MSH-A/B (amino acid sequence of alpha-MSH-A is identical to that of alpha-MSH-B), beta-MSH-A, corticotropin-like intermediate lobe peptide (CLIP)-A, and N-terminal peptide (N-POMC)-A were identified in the PD. Moreover, Des-Ac-alpha-MSH-A/B, alpha-MSH-A/B, beta-MSH-A and -B, N-beta-lipotropin-A, CLIP-A, N-Ac-beta-END-A(1-41) (C-terminally truncated form of N-Ac-beta-END-A), and N-POMC-A were identified in the PI. Predominant detection of POMC-A-derived peptides indicates the greatest production of POMC-A and no detection of POMC-C-derived peptides indicates the lowest production of POMC-C in both the PD and PI. ACTH-A is specifically produced in the PD, however, the occurrence of Des-Ac-alpha-MSH-A, CLIP-A, and beta-MSH-A shows that the entire POMC-A is further cleaved into small peptides as in the PI. In the PI, some peptides receive modification or truncation as shown by the occurrence of alpha-MSH-A/B and N-Ac-beta-END-A(1-41). These results show differential posttranslational processing of POMC between the PD and PI in barfin flounder pituitary.

Artuc M et al.
Human mast cells in the neurohormonal network: expression of POMC, detection of precursor proteases, and evidence for IgE-dependent secretion of alpha-MSH.
J Invest Dermatol. 2006; 126: 1976-81
Display abstract
Human mast cells have been shown to release histamine in response to the neuropeptide alpha-melanocyte-stimulating hormone (alpha-MSH), but it is unknown whether these cells express proopiomelanocortin (POMC) or POMC-derived peptides. We therefore examined highly purified human skin mast cells and a leukemic mast cell line-1 (HMC-1) for their ability to express POMC and members of the prohormone convertase (PC) family known to process POMC. Furthermore, we investigated whether these cells store and secrete alpha-MSH. Reverse transcriptase-PCR (RT-PCR) analysis revealed that both skin mast cells and HMC-1 cells express POMC mRNA and protein. Expression of the POMC gene at the RNA level in HMC-1 cells could be confirmed by Northern blotting. Transcripts for both PC1 and furin convertase were detectable in skin-derived mast cells and HMC-1 cells, as shown by RT-PCR. In contrast, PC2 transcripts were detected only in skin mast cells, whereas transcripts for paired basic amino acid converting enzyme 4 (PACE4) were present only in HMC-1 cells. Radioimmunoassays performed on cell lysates and cell culture supernatants from human skin-derived mast cells disclosed immunoreactive amounts of alpha-MSH in both fractions. Stimulation with an anti-IgE antibody significantly reduced intracellular alpha-MSH and increased extracellular levels, indicating IgE-mediated secretion of this neuropeptide. Our findings show that human mast cells are active players in the cutaneous POMC system. Mast cell-derived alpha-MSH may contribute to cutaneous hyperpigmentation as seen in patients with urticaria pigmentosa. Moreover, IgE-dependent release of alpha-MSH suggests an immunomodulatory role of this neurohormone during inflammatory and allergic reactions of the skin.

Karsi A, Waldbieser GC, Small BC, Liu Z, Wolters WR
Molecular cloning of proopiomelanocortin cDNA and multi-tissue mRNA expression in channel catfish.
Gen Comp Endocrinol. 2004; 137: 312-21
Display abstract
Channel catfish (Ictalurus punctatus) proopiomelanocortin (POMC) cDNA was cloned to investigate its structure, evolution, and expression in different tissues. POMC is an important gene in the hypothalamus-pituitary-adrenal axis, the main mediator of the stress response. POMC gene was isolated from a pituitary cDNA library and nucleotide sequence was determined. POMC cDNA is composed of 1164 nucleotides with a 639 nucleotide open reading frame encoding a protein of 212 amino acids. Catfish POMC protein contains a signal peptide (SP, Met(1)-Ala(28)), N-terminal peptide (Gln(29)-Glu(101)), adrenocorticotropic hormone (ACTH, Ser(104)-Met(142)), alpha-melanocyte stimulating hormone (alpha-MSH, Ser(104)-Val(116)), corticotropin-like intermediate lobe peptide (CLIP, Arg(121)-Met(142)), beta-lipotropin (beta-LPH, Glu(145)-His(212)), gamma-lipotropin (gamma-LPH, Glu(145)- Ser(177)), beta-MSH (Asp(161)-Ser(177)), and beta-endorphin (beta-EP, Tyr(180)-His(212)). Catfish POMC protein does not contain a gamma-MSH region and most of the joining peptide and part of the gamma-LPH are deleted. Protein sequence alignment showed the highest similarity with the carp (Cyprinus carpio) POMC I (66.5%) and POMC II (67%), while the sea lamprey (Petromyzon marinus) POC (17.9%) and POM (18.8%) were the most divergent. The average similarity was 46.95% among the 44 POMC proteins from 36 different species analyzed. Compared to the POMC mRNA levels in the pituitary, the concentration of the POMC mRNA was 0.0594% in the anterior kidney and 0.0012-0.0045% in all the other tissues except in the skin where the lowest expression (0.0005%) was observed. Overall architecture of channel catfish POMC is highly similar to those from other teleosts.

Alrubaian J, Sollars C, Danielson PB, Dores RM
Evaluating the radiation of the POMC gene in teleosts: characterization of American eel POMC.
Gen Comp Endocrinol. 2003; 132: 384-90
Display abstract
A distinctive feature of the pituitary hormone precursor, proopiomelanocortin (POMC), is the presence of multiple melanocortin core sequences (HFRW), and one copy of the opioid, beta-endorphin. In the older lineages of ray-finned fish (i.e., orders Acipenseriformes and Semionotiformes), certain extant lobe-finned fish (Australian lungfish and African lungfish), and the tetrapods there are three melanocortin regions in POMC: ACTH/alphaMSH, beta-MSH, and gamma-MSH. However, among the teleosts, the most recent radiation of the ray-finned fishes, the gamma-MSH sequence is absent from the POMC genes of euteleosts like the carp, tilapia, chum salmon, sockeye salmon, and rainbow trout. The objective of this study was to determine whether the gamma-MSH sequence still may be present in the POMC gene of a more basal lineage of the teleosts such as a representative from subdivision Elopomorpha. To this end, a POMC cDNA was cloned and sequenced from the pituitary of the American eel, Anguilla rostrata (order Anguilliformes, family Anguillidae). The open reading frame of the eel POMC cDNA was 648 nucleotides in length and encoded 216 amino acids. As predicted, eel POMC contained the deduced amino acid sequences for beta-endorphin, ACTH/alpha-MSH, and beta-MSH. These end-products displayed primary sequence features that are common to ray-finned fish. Eel POMC lacks a gamma-MSH sequence and a large portion of the joining peptide region. In this regard, the eel POMC gene thus displays features very similar to the POMC genes that have been sequenced from euteleosts. Although it is conceivable that the gamma-MSH sequence may be present in representatives from the other basal extant lineages of teleosts (i.e., subdivisions Osteoglossomorpha or Clupeomorpha), it is also possible that the deletion that resulted in the loss of the gamma-MSH sequence occurred in the ancestral neopterygian that gave rise to the teleosts. In this case, the gamma-MSH sequence should be absent in all extant teleosts.

Lu J, Swinnen E, Proost P, De Vreese B, Vankelecom H, Denef C
Isolation and structure-bioactivity characterization of glycosylated N-pro-opiomelanocortin isoforms.
J Neuroendocrinol. 2002; 14: 869-79
Display abstract
The N-terminal fragment of mouse pro-opiomelanocortin (N-POMC) was isolated from AtT-20 cell-conditioned medium on the basis of immunoreactivity to an anti-POMC1-50 monoclonal antibody by a concentration step, a cation exchange step, reversed phase high-performance liquid chromatography (HPLC) and size exclusion HPLC. Two groups of N-POMC isoforms with a molecular weight (MW) of approximately 11 kDa and 13 kDa, respectively, were identified by mass spectrometry and N-terminal amino acid sequencing. C-terminal sequencing indicated that 11 kDa isoforms correspond to POMC1-74 and 13 kDa isoforms to POMC1-95. Isoforms from both groups enhanced the prolactin mRNA content (measured by means of TaqMan real-time reverse transcription-polymerase chain reaction) in cultured rat pituitary cell aggregates in a dose-dependent manner, but not all of them showed this activity. POMC1-74 compounds were significantly more potent than POMC1-95 isoforms. The observed effects were abolished by coincubation with the monoclonal anti-POMC1-50 antibody, showing the specificity of this biological action. Incorporation of bromodeoxyuridine into DNA of immunostained lactotrophs was enhanced by only a minor part of the isoforms. Some of these had no effect on prolactin mRNA expression. The N-POMC isoforms appeared to be N- and at least in part O-glycosylated. After enzymatic N-deglycosylation of selected N-POMC isoforms, the stimulatory effect on the prolactin mRNA level was depressed (in case of the POMC1-95 isoforms) or totally abolished (in case of the POMC1-74 isoforms). The present findings show that N-POMC is a mixture of differentially glycosylated isoforms, that the isoforms of POMC1-74 are the biologically more effective forms and that different isoforms induce different biological responses in the same cell population. The data also show the essential role of N-glycosylation in the biological response.

Stefano GB, Salzet-Raveillon B, Salzet M
Mytilus edulis hemolymph contains pro-opiomelanocortin: LPS and morphine stimulate differential processing.
Brain Res Mol Brain Res. 1999; 63: 340-50
Display abstract
Mytilus edulis hemolymph contains mammalian-like proopiomelanocortin (POMC). The 20 kDa protein was purified by high pressure gel permeation chromatography, anti-adrenocorticotropin (ACTH)-affinity column and reverse-phase HPLC. The amino acid sequence determination was by Edman degradation, enzymatic treatments and Western blot analysis. Of the six peptides found in this opioid precursor, methionine-enkephalin, gamma-melanocyte stimulating hormone (MSH), alpha-MSH and ACTH exhibited 100, 80, 85 and 74% sequence identity, respectively, with the mammalian counterparts. beta-Endorphin and gamma-LPH exhibited only 25 and 10% sequence identity. Dibasic amino acid residues were found at the C-terminus of MSH and ACTH, indicating cleavage sites. The alpha-MSH is flanked at the C-terminus by Gly-Lys-Lys, representing an amidation signal. ACTH and CLIP (80% sequence identity) are also C-terminally flanked by dibasic amino acid residues. Furthermore, morphine, in a dose-dependent manner, increased the hemolymph levels of alpha-MSH and ACTH (1-39) in a naloxone and phosphoramidon antagonizable manner, indicating a neutral endopeptidase (24.11; NEP) mediated cleavage. Lipopolysaccharide (10 microg/animal) stimulated the processing of ACTH (1-39) yielding ACTH (1-24) in a cleavage that is independent of NEP, but dependent on aspartyl proteases, demonstrating differential enzymatic cleavage of ACTH (1-39). Taken together, POMC is present in invertebrates and its processing can be altered depending on the signal.

Bicknell AB, Savva D, Lowry PJ
Pro-opiomelanocortin and adrenal function.
Endocr Res. 1996; 22: 385-93
Display abstract
Pro-opiomelanocortin (POMC) is a polyhormone precursor produced predominantly in the pars distalis and pars intermedia of the pituitary gland where it undergoes tissue specific processing to produce a whole array of peptides. We have shown previously that peptides derived from the N-terminal region of POMC are involved in adrenal growth in rats. Using specific two site immunoradiometric assays we have found that the plasma of 17 week old fetal sheep contain a 50 fold excess of pro-gamma-MSH over ACTH. As term approached, the levels of pro-gamma-MSH fell and ACTH rose with evidence of fragmentation of pro-gamma-MSH, suggesting that these peptides act in concert in the development of the fetal adrenal cortex and also provide the necessary drive to bring about parturition. In an attempt to explore the pathophysiology of adrenal function we have cloned human POMC cDNA which led to the discovery of a 9bp addition/deletion mutation in the C-terminus of gamma 3-MSH between positions 67-73. Chinese hamster ovary cells (CHO) cells stably transfected with constructs containing the variant POMC cDNAs have shown a degree of partial processing. Work is currently underway to further investigate the effects of these mutations on the processing by the prohormone converting enzymes PC1 and PC2.

Leviev I, Grimmelikhuijzen CJ
Molecular cloning of a preprohormone from sea anemones containing numerous copies of a metamorphosis-inducing neuropeptide: a likely role for dipeptidyl aminopeptidase in neuropeptide precursor processing.
Proc Natl Acad Sci U S A. 1995; 92: 11647-51
Display abstract
Neuropeptides are an important group of hormones mediating or modulating neuronal communication. Neuropeptides are especially abundant in evolutionarily "old" nervous systems, such as those of cnidarians, the lowest animal group having a nervous system. Cnidarians often have a life cycle including a polyp, a medusa, and a planula larva stage. Recently, a neuropeptide, < Glu-Gln-Pro-Gly-Leu-Trp-NH2, has been isolated from sea anemones that induces metamorphosis in a hydroid planula larva to become a hydropolyp [Leitz, T., Morand, K. & Mann, M. (1994) Dev. Biol. 163, 440-446]. Here, we have cloned the precursor protein for this metamorphosis-inducing neuropeptide from sea anemones. The precursor protein is 514-amino acid residues long and contains 10 copies of the immature, authentic neuropeptide (Gln-Gln-Pro-Gly-Leu-Trp-Gly). All neuropeptide copies are preceded by Xaa-Pro or Xaa-Ala sequences, suggesting a role for dipeptidyl aminopeptidase in neuropeptide precursor processing. In addition to these neuropeptide copies, there are 14 copies of another, closely related neuropeptide sequence (Gln-Asn-Pro-Gly-Leu-Trp-Gly). These copies are flanked by basic cleavage sites and, therefore, are likely to be released from the precursor protein. Furthermore, there are 13 other, related neuropeptide sequences having only small sequence variations (the most frequent sequence: Gln-Pro-Gly-Leu-Trp-Gly, eight copies). These variants are preceded by Lys-Arg, Xaa-Ala, or Xaa-Pro sequences, and are followed by basic cleavage sites, and therefore, are also likely to be produced from the precursor. Thus, there are at least 37 closely related neuropeptides localized on the precursor protein, making this precursor one of the most productive preprohormones known so far. This report also shows that unusual processing sites are common in cnidarian preprohormones.

van Strien FJ, Devreese B, Van Beeumen J, Roubos EW, Jenks BG
Biosynthesis and processing of the N-terminal part of proopiomelanocortin in Xenopus laevis: characterization of gamma-MSH peptides.
J Neuroendocrinol. 1995; 7: 807-15
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The aim of this study was to determine the terminal products of processing of the N-terminal part of proopiomelanocortin (POMC) in pituitary melanotrope cells of Xenopus laevis. Biosynthetic in vitro labelling studies showed that POMC is rapidly processed to form N-terminal peptides with an estimated molecular mass of 18 kDa, 9 kDa and 4 kDa. All peptides were released into the medium, indicating that they are processing end products. An antiserum was raised against the synthetic N-terminal eight amino acids of the putative Xenopus gamma-MSH which is present in the N-terminal part of POMC. With immunocytochemistry we demonstrated that gamma-MSH-immunoreactive material in the pituitary gland is restricted to the pars intermedia. A radioimmunoassay in combination with reversed-phase HPLC revealed the presence of at least two gamma-MSH-like peptides. Complete purification followed by electrospray ionization mass spectrometry and amino acid sequence determination showed that these peptides are gamma 1-MSH and glycosylated gamma 3-MSH. The amounts of these gamma-MSH peptides were low compared to the other POMC-derived peptides, alpha-MSH and beta-endorphin. Only 10% of POMC is processed into gamma-MSH peptides and the 4 kDa peptide, leaving the 18 kDa and 9 kDa peptides as the major end products.

van der Meer JR, Rollema HS, Siezen RJ, Beerthuyzen MM, Kuipers OP, de Vos WM
Influence of amino acid substitutions in the nisin leader peptide on biosynthesis and secretion of nisin by Lactococcus lactis.
J Biol Chem. 1994; 269: 3555-62
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Structural genes for small lanthionine-containing antimicrobial peptides, known as lantibiotics, encode N-terminal leader sequences which are not present in the mature peptide, but are cleaved off at some stage in the maturation process. Leader sequences of the different lantibiotics share a number of identical amino acid residues, but they are clearly different from sec-dependent protein export signal sequences. We studied the role of the leader sequence of the lantibiotic nisin, which is produced and secreted by Lactococcus lactis, by creating site-directed mutations at various positions in the leader peptide sequence. Mutations at Arg-1 and Ala-4, but not at the conserved Pro-2, strongly affected the processing of the leader sequence and resulted in the extracellular accumulation of a biologically inactive precursor peptide. Amino acid analysis and 1H NMR studies indicated that the precursor peptide with an Ala-4-->Asp mutation contained a modified nisin structural part with the (mutated) unmodified leader sequence still attached to it. The Ala-4-->Asp precursor peptide could be activated in vitro by enzymatic cleavage with trypsin, liberating nisin. These results confirmed that cleavage of the leader peptide is the last step in nisin maturation and is necessary to generate a biologically active peptide. Several mutations, i.e. Pro-2-->Gly,Pro-2-->Val, Asp-7-->Ala,Lys-9-->Leu,Ser-10-->Ala/Ser-12-->Ala and Val-11-->Asp/Val-13-->Glu in the leader peptide did not have any detectable effect on nisin production and secretion, although some of them affected highly conserved residues. When mutations were created in the -18 to -15 region of the nisin leader peptide (i.e. Phe-18-->Leu,Leu-16-->Lys,Asp-15-->Ala), no secretion or intracellular accumulation could be detected of nisin or its precursors. This suggested that these conserved residues are involved in the maturation process and may interact with lantibiotic-specific modifying enzymes.

Tam WW, Andreasson KI, Loh YP
The amino-terminal sequence of pro-opiomelanocortin directs intracellular targeting to the regulated secretory pathway.
Eur J Cell Biol. 1993; 62: 294-306
Display abstract
The molecular signal which targets the pro-opiomelanocortin (POMC) prohormone into the regulated secretory pathway was investigated. DNA sequences encoding the first 10, 26, 50, and 101 N-terminal amino acids of mouse POMC were fused in frame to the chloramphenicol acetyltransferase (CAT) gene and expressed in AtT-20 cells. Immunofluorescence microscopy using antibody directed against CAT indicated that fusion proteins carrying 26, 50 and 101 amino acids of N-POMC were directed to secretory granules. This finding was confirmed by secretion studies in which 1 microM forskolin stimulated the release of fusion proteins carrying 26 and 101 amino acids of N-POMC, whereas no regulated secretion was observed with the shortest fusion protein. Subcellular fractionation studies also indicated the presence of the fusion proteins with 26 and 101 amino acids of N-POMC in secretory granules. These results provide evidence that the signal directing POMC to secretory granules is contained within the N-terminus of the prohormone, with the first 26 amino acids being sufficient for targeting. Binding studies showed that N-POMC1-76 bound to secretory granule membranes specifically on the luminal side and in a pH-sensitive manner. Only N-POMC1-76 bound optimally to secretory granule membranes at pH 5 to 6.5, but not the ACTH1-39 (mid), corticotropin-like intermediate lobe peptide (CLIP) and beta-lipotropin (C-terminal) domains of POMC. Such binding may be involved in the mechanism of sorting POMC to the regulated secretory pathway.

Azaryan AV et al.
Purification and characterization of a paired basic residue-specific yeast aspartic protease encoded by the YAP3 gene. Similarity to the mammalian pro-opiomelanocortin-converting enzyme.
J Biol Chem. 1993; 268: 11968-75
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Yeast cells express an alternate enzyme encoded by the YAP3 gene which can process pro-alpha-mating factor when this pheromone is overexpressed in KEX2-deficient mutants. The YAP3 gene product is an aspartic protease (YAP3) that cleaves at paired basic residues (Egel-Mittani, M., Flygenring, H.P., and Hansen, M. T. (1990) Yeast 6, 127-137). In this study, the YAP3 gene was overexpressed in the BJ 3501 strain of Saccharomyces cerevisiae. YAP3 was purified to apparent homogeneity using concanavalin A and pepstatin A affinity chromatography. The enzyme was characterized as an M(r) 68,000 glycoprotein with a pH optimum of 4.0-4.5. It was inhibited by pepstatin A and activated by 5 mM Ca2+. YAP3 cleaved at paired basic residues of mouse pro-opiomelanocortin (POMC) to yield adrenocorticotropin (ACTH) and beta-lipotropin (LPH); human beta-LPH to yield beta-endorphin-(1-31), beta-endorphin-(1-29), beta-endorphin-(1-28), gamma-LPH, and beta-melanocyte-stimulating hormone; and bovine N-POMC1-77 to yield gamma 3-melanocyte-stimulating hormone. It also cleaved the tetrabasic residues of ACTH1-39 to yield primarily ACTH1-15 and Lys-Arg-corticotropin-like intermediate lobe peptide. The physical properties, pH optimum, and specificity of YAP3 indicate that it is a homologue of the mammalian POMC-converting enzyme (EC 3.4.23.17), a paired basic residue-specific aspartic protease from bovine pituitary intermediate lobe secretory granules (Loh, Y. P., Parish, D.C., and Tuteja, R. (1985) J. Biol. Chem. 260, 7194-7205).

Levy B, Chauvet MT, Chauvet J, Acher R
Ontogeny of bovine neurohypophysial hormone precursors. II. Foetal copeptin, the third domain of the vasopressin precursor.
Int J Pept Protein Res. 1986; 27: 320-4
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Vasopressin, MSEL-neurophysin and a glycopeptide, here referred to as copeptin, are three fragments of a common protein precursor processed during axonal transport from hypothalamus to neurohypophysis. Neurohormones and neurophysins purified from 7-9-month-old bovine foetuses have previously been shown to be identical with those found in the adult. Copeptin has now been isolated from 7-9-month and 3-month-old bovine foetuses and chemically characterized. It can be concluded from the nature of the three precursors that the same vasopressin gene is expressed in the adult and the 7-9-month-old foetus.

Chretien M, Benjannet S, Lazure C, Seidah NG
Biosynthesis of hormonal and neural peptides.
Trans Am Clin Climatol Assoc. 1984; 95: 19-25

Lee TH, Lee MS
Isolation of corticotropin-beta-lipotropin common precursor from ovine pituitary glands.
Biochim Biophys Acta. 1984; 800: 188-93
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Ovine corticotropin-beta-lipotropin common precursor was purified to homogeneity from commercial frozen ovine pituitary glands. A crude preparation was obtained following a procedure published elsewhere (Lee, T.H. and Lee, M.S. (1977) Biochemistry 16, 2824-2829) and was purified by gel filtration on Sephadex G-200 in the presence of 0.5% SDS and 0.1% 2-mercaptoethanol, and under an atmosphere of nitrogen. The gel filtration was repeated once. The partially purified preparation obtained from the second Sephadex G-200 gel filtration was further fractionated by preparative SDS-acrylamide gel electrophoresis, using immunoprecipitated and electrophoretically purified [125I]corticotropin-beta-lipotropin common precursor as a marker. The preparation was judged homogeneous by the appearance of a single protein band in analytical SDS-acrylamide gel electrophoresis, which exhibited both corticotropin and beta-lipotropin immunoreactivities, and a single symmetrical peak in high-pressure liquid chromatography on a reverse phase C18 column. The isolated ovine corticotropin-beta-lipotropin common precursor possessed specific activities of 116 micrograms of immunoreactive corticotropin and 210 micrograms of immunoreactive beta-lipotropin per mg of protein, equivalent to 89 and 62% of theoretical values, respectively. The amino acid composition of the homogeneous preparation was determined.

Marx JL
Synthesizing the opioid peptides.
Science. 1983; 220: 395-7

Randle JC, Moor BC, Kraicer J
Differential control of the release of pro-opiomelanocortin-derived peptides from the pars intermedia of the rat pituitary. Response to serotonin.
Neuroendocrinology. 1983; 37: 131-40

Hoshina H, Hortin G, Boime I
Rat pro-opiomelanocortin contains sulfate.
Science. 1982; 217: 63-4
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Intermediate lobes isolated from rat pituitary glands incorporated [35S]sulfate into pro-opiomelanocortin and other adrenocorticotropic hormone-containing peptides. Incubation of intermediate lobes in medium containing the arginine analog canavanine inhibited the cleavage of pro-opiomelanocortin into smaller products. Pro-opiomelanocortin that accumulated in the presence of canavanine was also sulfated.

Akil H, Shiomi H, Thompson R, Lax S, Coy D, Watson S
The signal peptide of pro-opiomelancortin: validation of a specific radioimmunoassay.
Life Sci. 1982; 31: 2271-3
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The N-terminus portion of the POMC leader sequence (signal peptide) was synthesized, and an antiserum was raised against it. A radioimmunoassay was developed which is effective at a dilution of 1:500,000, and sensitive at less than 1 fmole/tube. Since leader sequences often exhibit structural homologies, and since synthetic peptides are not readily available, we resorted to an unusual procedure to establish specificity. This involved extraction of pituitary RNA, cell-free translation to produce the pre-prohormones, and purification by B-END and signal antibody affinity columns. The eluates were then tested by SDS gel electrophoresis and by multiple immunoprecipitations. All results showed that the signal antibody captured a single molecular species, approximately 30,000 in MW, which was also captured by the B-END column, and was immunoprecipitable by B-END and ACTH antisera. It therefore appears that this antibody selectively measures the POMC leader sequence and should be valuable in measuring the newly synthesized pre-prohormone.

Boksa P, Seidah NG, Chretien M, Livett BG
Effects of pro-opiomelanocortin fragments on release of catecholamines from adrenal chromaffin cells.
Neurosci Lett. 1982; 28: 199-204
Display abstract
The effects of pro-opiomelanocortin (POMC) peptide fragments on the basal and agonist-induced release of catecholamines (CAs) from monolayer cultures of purified bovine adrenal chromaffin cells were tested. None of the 5 peptides tested, i.e. beta-MSH, ACTH1-39, gamma-MSH1-13, gamma 3-MSH and N-terminal POMC fragment, had any effect on basal CA release. However, beta-MSH (10(-5) M), gamma-MSH1-13 (10(-6)-10(-5) M), gamma 3-MSH (10(-5) M) and N-terminal POMC fragment (10(-5) M) inhibited the nicotine-induced release of CAs from the chromaffin cells. The possible physiological significance of this inhibitory neuromodulation is discussed.

Crine P, Dufour L
Effects of monensin on the processing of pro-opiomelanocortin in the intermediate lobe of the rat pituitary.
Biochem Biophys Res Commun. 1982; 109: 500-6

Chretien M et al.
An aldosterone stimulating agent: a novel human pituitary hormone from pro-opiomelanocortin.
Trans Assoc Am Physicians. 1981; 94: 225-35

Seidah NG, Rochemont J, Hamelin J, Benjannet S, Chretien M
The missing fragment of the pro-sequence of human pro-opiomelanocortin: sequence and evidence for C-terminal amidation.
Biochem Biophys Res Commun. 1981; 102: 710-6

Maloy WL, Nathenson SG, Coligan JE
Primary structure of murine major histocompatibility complex alloantigens. Amino acid sequence of the NH2-terminal ninety-eight residues of the H-2Db glycoprotein.
J Biol Chem. 1981; 256: 2863-72
Display abstract
The NH2-terminal 98 amino acid residues of the murine histocompatibility antigen H-2Db have been assigned using radiochemical methodology. This represents the first extensive, continuous sequence information for a histocompatibility antigen encoded by the H-2D locus and allows comparison with the recently determined amino acid sequence of the H-2Kb molecule. The amino acid sequence was obtained from the sequences of three CNBr peptides, CN-E, CN-D, and CN-B, which comprise residues 1-5, 6-52, and 53-98, respectively. The amino acid sequence of CN-E was determined directly while the sequences of CN-D and CN-B were determined by NH2-terminal sequence analyses and sequence determinations of peptides produced by thrombin, staphylococcal V8 protease, and trypsin cleavage. Alignment of the CNBr peptides was accomplished by NH2-terminal sequence analysis of the H-2Db papain fragment (CN-E to CN-D) and by analyzing peptides from a tryptic digest of the intact H-2Db molecule. Positive identification was possible for all amino acids except Asp and Asn-86 which were indirectly assigned (in italics). The sequence obtained was Gly-Pro-His-Ser-Met-Arg-Tyr-Phe-Glu-Thr-Ala-Val-Ser-Arg-Pro-Gly-Leu-Glu-Glu-Pro -Arg-Tyr-Ile-Ser-Val-Gly-Tyr-Val-Asp-Asn-Lys-Glu-Phe-Val-Arg-Phe-Asp-Ser-Asp-Ala- Glu-Asn-Pro-Arg-Tyr-Glu-Pro-Arg-Ala-Pro-Trp-Met-Glu-Gln-Glu-Gly-Pro-Glu-Tyr-T rp-Glu-Arg-Glu-Thr-Gln-Lys-Ala-Lys-Gly-Gln-Glu-Gln-Trp-Phe-Arg-Val-Ser-Leu-Arg-As n-Leu-Leu-Gly-Tyr-Tyr-Asn-Gln-Ser-Ala-Gly-Gly-Ser-His-Thr-Leu-Gln-Gln-Met.

Lis M et al.
Effect of N-terminal portion of pro-opiomelanocortin on aldosterone release by human adrenal adenoma in vitro.
J Clin Endocrinol Metab. 1981; 52: 1053-6
Display abstract
An adrenal cortex adenoma, surgically removed from a female patient with primary aldosteronism, was used to examine the effect of ACTH, angiotensin II, gamma 3-MSH, and the N-terminal fragment of pro-opiomelanocortin purified from porcine anterior pituitaries on aldosterone release in vitro. Primary cultures of tumor cells were incubated as a monolayer in a 96-well microtitration plate and the aldosterone release was measured in the incubation medium after 2 h of incubation in the presence of absence of different concentrations of the peptides. On a molar basis, the N-terminal portion of pro-opiomelanocortin seems to have the highest activity of all of the peptides assayed.

Seidah NG, Rochemont J, Hamelin J, Lis M, Chretien M
Primary structure of the major human pituitary pro-opiomelanocortin NH2-terminal glycopeptide. Evidence for an aldosterone-stimulating activity.
J Biol Chem. 1981; 256: 7977-84
Display abstract
The isolation and complete purification of a human glycopeptide representing the major immunoreactive form of the pituitary NH2-terminal segment of pro-opiomelanocortin is presented. The complete sequence of this peptide was determined following CNBr fragmentation and it is shown to be 76 amino acids long. It bears an O-glycosylation site at Thr 45 and an N-glycosidic linkage at Asn 65. Compared to the reported genomic DNA sequence (Chang, A. C. Y., Cochet, M., and Cohen, S. N. (1980) Proc. Natl. Acad. Sci. U. S. A. 77, 4890-4894), one variation exists, namely Arg 22 replacing Gly 22. Two disulfide bridges linking Cys 2 to 8 and Cys 20 to 24 have been determined. Based on the sequence and disulfide bridge localization, a large degree of homology exists between the NH2-terminal sequence of the human peptide and all calcitonins, especially porcine calcitonin. The human NH2-terminal peptide is shown to stimulate the release of aldosterone from isolated cells of a human adrenal tumor, in at least equipotency to adrenocorticotropic hormone and the porcine NH2-terminal analogue, which is 100 times more potent than angiotensin II. Finally, this reported sequence completes that of the human DNA which was lacking the first 19 amino acids due to the presence of a 2-kilobase intron.

Herbert E, Phillips M, Budarf M
Glycosylation steps involved in processing of pro-corticotropin-endorphin in mouse pituitary tumor cells.
Methods Cell Biol. 1981; 23: 101-18

McLean C, Hope J, Salacinski P, Estivariz F, Lowry PJ
Purification and characterization of amino-terminal pro-opiocortin peptides from human pituitary glands.
Biosci Rep. 1981; 1: 843-9
Display abstract
Two cystine-containing peptides isolated from human pituitaries were partially sequenced. The peptides are fragments derived from the amino-terminal of pro-opiocortin (NPOC) and have been chemically characterized as NPOC 1-28 (which lacks gamma-MSH) and NPOC 2-59. Their availability enables us to investigate new putative biological roles for the amino-terminal conserved sequence of pro-opiocortin.

Naude RJ, Chung D, Li CH, Oelofsen W
beta-Lipotropin: primary structure of the hormone from the ostrich pituitary gland.
Int J Pept Protein Res. 1981; 18: 138-47
Display abstract
The amino acid sequence of beta-lipotropin from the ostrich pituitary has been determined. It consists of 79 amino acids. The amino acid sequence has been determined as follows: H-(1)AlA-Leu-Pro-Pro-Ala-Ala-Met-Leu-Pro-(10)Ala-Ala-Ala-Glu-Glu-Glu-Glu-Gly-Gl u-Glu-(20)Glu-Glu-Glu-Gly-Glu-Ala-Glu-Lys-Glu-Asp-(30)Gly-Gly-Ser-Tyr-Arg-Met-A rg-His-Phe-Arg-(40)Trp-Gln-Ala-Pro-Leu-Lys-Asp-Lys-Arg-Tyr-(50)Gly-Gly-Phe-Met- Ser-Ser-Glu-Arg-Gly-Arg-(60)Ala-Pro-Leu-Val-Thr-Leu-Phe-Lys-Asn-Ala-(70)Ile-Val -Lys-Ser-Ala-Tyr-Lys-Lys-Gly-(79)Gln-OH. When compared with the primary structures of other known beta-lipotropins, the sequence at the NH2-terminal, beta-melanotropin and beta-endorphin portions of the molecule exhibit considerable variability.

Hakanson R, Ekman R, Sundler F, Nilsson R
A novel fragment of the corticotropin-beta-lipotropin precursor.
Nature. 1980; 283: 789-92

Steiner DF, Quinn PS, Chan SJ, Marsh J, Tager HS
Processing mechanisms in the biosynthesis of proteins.
Ann N Y Acad Sci. 1980; 343: 1-16
Display abstract
Limited proteolysis is a widely occurring mechanism in protein biosynthesis. Protein precursors can be classified according to their functions, localization within cell compartments, and enzymic cleavage mechanisms. The presecretory proteins represent an important class of very rapidly turning over precursors which play an early role in the sequestration of secretory products and whose cleavage appears to be intimately associated with structures formed at the ribosome-membrane junction during protein synthesis. A model is proposed which predicts that the prepeptide forms a beta-pleated sheet structure with other components of the membrane which results in the transfer of a loop of peptide across the microsomal membrane. Proinsulin is representative of the general class of proproteins that are processed post-translationally within their secretory cells either during the formation and maturation of secretory granules (peptides hormones and neurotransmitters, serum albumins) or during the assembly of macromolecular structures (virus capsules, membrane-associated enzyme complexes). The former group are cleaved by Golgi-associated proteases having tryptic and carboxypeptidase B-like specificity. Some precursors are secreted as such and processed extracellularly either in the circulation or at special sites (procollagens, zymogens, provenoms, vitellogenins).

Nakanishi S et al.
The protein-coding sequence of the bovine ACTH-beta-LPH precursor gene is split near the signal peptide region.
Nature. 1980; 287: 752-5
Display abstract
The pituitary hormones corticotropin (ACTH) and beta-lipotropin (beta-LPH) are formed from a large common precursor. Recently, we have elucidated the whole primary structure of the bovine ACTH-beta-LPH precursor (designated alternatively as preproopiocortin) by determining the nucleotide sequence of cloned DNA complementary to the mRNA coding for the precursor protein. The amino acid sequence assigned has disclosed a characteristic repetitive structure of the ACTH-beta-LPH precursor. The repetitive units of the precursor protein each contain a melanotropin (MSH) sequence (alpha-, beta- or gamma-MSH) as well as other peptide components such as beta-endorphin and corticotropin-like intermediate lobe peptide (CLIP). The repetitive units as well as their peptide components are each bounded by paired basic amino acid residues, which apparently represent the sites of proteolytic processing. Several studies have confirmed the translational initiation site and protein structure assigned (see also ref. 11 and refs therein). In view of the recent knowledge about the organization of eukaryotic genes (see refs 12, 13 for reviews), it would be of particular interest to investigate the relationship between the repetitive structure of the ACTH-beta-LPH precursor containing different functional components and the arrangement of the protein-coding sequence in its gene. We have now isolated and characterized bovine genomic DNA fragments encoding this precursor protein and have demonstrated that the protein sequence is encoded by two non-consecutive DNA segments. An intron (intervening sequence) of approximately 2.2 kilobase pairs separates the smaller exon (mRNA-coding sequence), which contains the gene sequence encoding the signal peptide, from the larger exon, which contains the gene sequence for most of the protein structure, including the known biologically active component peptides.

Pedersen RC, Brownie AC
Adrenocortical response to corticotropin is potentiated by part of the amino-terminal region of pro-corticotropin/endorphin.
Proc Natl Acad Sci U S A. 1980; 77: 2239-43
Display abstract
Five peptides derived from pro-corticotropin/endorphin (pro-ACTH/endorphin), the pituitary corticotroph cell prohormone, were bioassayed with isolated rat adrenocortical cells: alpha- and beta-melanotropin, beta-lipotropin, beta-endorphin, and the amino-terminal region of pro-ACTH/endorphin known as "16k fragment." The effect of each on steroidogenesis was measured at potentially physiological concentrations (0.01-1 nM) in both the absence and presence of varying concentrations of ACTH-(1-24). Of the peptides tested, only 16k fragment, the amino-terminal region of pro-ACTH/endorphin, has a slight but significant potentiating effect on ACTH-(1-24) action. Prior treatment of 16k fragment with trypsin for 30 sec dramatically increases this dose-dependent synergism. Experiments performed in vivo with hypophysectomized female rats indicate that the trypsin digest of 16k fragment stimulates cholesterol ester hydrolase (cholesterol esterase; sterol-ester acylhydrolase, EC 3.1.1.13) activity in the adrenal cortex but fails to activate cholesterol side-chain cleavage. The effect of the trypsinized material can therefore be qualitatively distinguished from that of ACTH-(1-24). When both ACTH-(1-24) and the digest are administered together, a synergistic increase in serum corticosterone concentration results. We propose that a portion of 16k fragment molecule may play a hormonal role in the control of adrenocortical steroidogenesis.

Pedersen RC, Brownie AC, Ling N
Pro-adrenocorticotropin/endorphin-derived peptides: coordinate action on adrenal steroidogenesis.
Science. 1980; 208: 1044-6
Display abstract
A synthetic peptide, representing a portion of the 16K (16,000 dalton)-fragment sequence within the pro-adrenocorticotropin/endorphin precursor molecule, potentiates the steroidogenic action of the 1 to 24 portion of adrenocorticotropin [ACTH(1-24)] on the rat adrenal cortex. The peptide has 27 amino acid residues and consists of gamma-melanotropin with a carboxyl terminal extension. It affects both the inner and outer adrenocortical zones of hypophysectomized animals, as evidenced by a synergistic augmentation of corticosterone and aldosterone production, respectively. The peptide can be distinguished from adrenocorticotropin by its activation of cholesterol ester hydrolase and its failure to stimulate cholesterol side-chain cleavage.

Seidah NG, Gossard F, Crine P, Gianoulakis C, Routhier R, Chretien M
Chemical characterization of rat alpha MSH/beta-endorphin precursor from pars intermedia.
Ann N Y Acad Sci. 1980; 343: 443-6

Loh YP, Gainer H
Evidence that glycosylation of pro-opiocortin and ACTH influences their proteolysis by trypsin and blood proteases.
Mol Cell Endocrinol. 1980; 20: 35-44
Display abstract
The role of the carbohydrate in the stabilizaion and protection of the glycoprotein, pro-opiocortin, from non-specific proteolysis by trypsin and blood proteases was studied in vitro. [3H]Arginine-labeled, glycosylated and non-glycosylated forms of pro-opiocortin were isolated from frog neurointermediate lobes and subjected to proteolysis by trypsin. The non-glycosylated form was degraded by trypsin more rapidly than the glycosylated form. Analysis of the tryptic products after trypsin treatment, showed that the non-glycosylated pro-opiocortin was cleaved to unidentified peptides within 1 min, whereas the glycosylated prohormone yielded 2 products, mol. wt. 23 000 ACTH and mol. wt. 21 000 ACTH, synthesized by the intact neurointermediate lobe. These data provide direct evidence in support of the hypothesis, derived from studies on the intact lobe (Loh and Gainer, 1978, 1979) that the glycosylation of pro-opiocortin is important: (1) to protect it against non-specific proteolysis in situ, and (2) to direct processing by limiting proteolysis. In addition, we demonstrate that glycosylated forms of ACTH are much more stable in blood than non-glycosylated forms.

Herbert E et al.
Presence of a pre-sequence (signal sequence) in the common precursor to ACTH and endorphin and the role of glycosylation in processing of the precursor and secretion of ACTH and endorphin.
Ann N Y Acad Sci. 1980; 343: 79-93

Mains RE, Eipper BA
Synthesis and secretion of corticotropins, melanotropins, and endorphins by rat intermediate pituitary cells.
J Biol Chem. 1979; 254: 7885-94
Display abstract
The synthesis and secretion of various intermediate pituitary proteins was studied by using dispersed intermediate pituitary cell suspensions. Control studies indicated that the isolated cells were obtained in good yield and that after more than 24 h in culture the isolated cells continued to synthesize a collection of proteins similar to those found in freshly extracted intermediate pituitary tissue. Rat intermediate pituitary cells synthesized a molecule (Mr = 30,000; called 30K) that contained antigenic determinants for beta-endorphin, gamma-lipotropin, corticotropin (ACTH), and 16K fragment (the NH2-terminal region of mouse tumor cell pro-ACTH/endorphin). This 30K molecule, two high molecular weight forms of ACTH(13K and 20K), and 16K fragment were all shown to be glycoproteins. Continuous labeling and pulse-chase incubations were used to define the intracellular biosynthetic processing of the 30K molecule. After a 15-min pulse incubation the 30K molecule was the only labeled protein containing antigenic determinants for beta-endorphin, gamma-lipotropin, ACTH, or 16K fragment. A beta-lipotropin-like molecule served as a biosynthetic intermediate in the production of proteins similar to beta-endorphin and gamma-lipotropin. Methionine-enkephalin and alpha-endorphin were not major products in the intermediate lobe cells. Molecules similar to alpha-melanocyte-stimulating hormone and corticotropin-like intermediate lobe peptide (ACTH(18-39)) were also derived from the same 30K molecule; 20K ACTH served as a biosynthetic intermediate in this conversion. In rat intermediate pituitary cells ACTH(1-39) was not a major final product of the intracellular biosynthetic processing of the 30K molecule. The 30K molecule also served as a precursor to a protein similar to mouse tumor cell 16K fragment and related smaller proteins. With rat intermediate pituitary cells, pulse-chase experiments utilizing [35S]methionine demonstrated almost quantitative conversion of the 30K precursor into labeled proteins similar to beta-endorphin and alpha-melanocyte-stimulating hormone. In the absence of added secretagogues, small amounts of all of the smaller proteins derived from the 30K precursor were secreted coordinately into the culture medium.

Iudaev NA, Pankov IuA
[N-terminal (1-47) sequence of amino acid residues in a molecule of swine beta-lipotropin].
Probl Endokrinol (Mosk). 1970; 16: 49-52