Peroxisomal localization of the proopiomelanocortin-derived peptides beta-lipotropin and beta-endorphin.

The peptide hormones ACTH, MSHs, ß-lipotropin (ß-LPH), and ß-endorphin are all derived from the precursor molecule proopiomelanocortin (POMC). Using confocal laser microscopy and immunoelectron microscopy in human pituitary gland, we demonstrate a peroxisomal localization of ß-endorphin and ß-LPH in cells expressing the peroxisomal ATP-binding cassette-transporter adrenoleukodystrophy protein (ALDP). The peroxisomal localization of ß-LPH and ß-endorphin was not restricted to the pituitary gland but was additionally found in other human tissues that express high levels of ALDP, such as dorsal root ganglia, adrenal cortex, distal tubules of kidney, and skin. In contrast to the peptide hormones ß-LPH and ß-endorphin, which are derived from the C terminus of POMC, the N-terminal peptides ACTH, α-MSH, and γ-MSH were never detected in peroxisomes. This novel peroxisomal localization of ß-endorphin and ß-LPH in ALDP-positive cells was confirmed by costaining with ALDP and the peroxisomal marker catalase. Moreover, peroxisomal sorting of ß-LPH could be modeled in HeLa cells by ectopic expression of a POMC variant, modified to allow cleavage and release of ß-LPH within the secretory pathway. Although ß-LPH and ß-endorphin were only associated with peroxisomes in cells that normally express ALDP, the transporter activity of ALDP is not necessary for the peroxisomal localization, as demonstrated in tissues of X-linked adrenoleukodystrophy patients lacking functional ALDP. It remains to be elucidated whether and how the peroxisomal localization of POMC-derived hormones has a role in the endocrine dysfunction of peroxisomal disease.

Molecular cloning and characterization of preproopiomelanocortin (prePOMC) cDNA from the ostrich (Struthio camelus).

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.

[Molecular and genetic study of the role of hormones, receptors, and enzymes in regulation of reproduction, lipid metabolism, and other human physiological functions].

Prevalence of uterine progesterone receptors over estrogen ones, high uterine cAMP level, and low uterine prostaglandin level are necessary conditions of normal pregnancy. In cases of spontaneous and antiprogestin RU486-induced abortions, estrogen receptors prevail over progesterone ones, cAMP level decreases, and prostaglandin concentration in decidual tissue increases. Porcine and bovine beta-lipotropines were the first proteins, whose correct amino acid sequence was first determined in Russia. Several research centers carried out collaborative studies of the nucleotide sequences of human and animal proopiomelanocortin (lipotropin precursor) and prolactin cDNA. Researchers constructed genetic engineering producers of human pre-proinsulin and somatostatin, identified structural genes expressed in pancreatic beta-cells, studied antigenic properties of glutamic acid decarboxylase (GAD), which determine insulin-dependent diabetes, and identified the cholesterase determinant. They revealed mutations in the genes of proopiomelanocortin and melanocortin receptors (MC4-P), which inhibit leptin regulation of appetite and are associated with human obesity.

Identification of two proopiomelanocortin genes in zebrafish (Danio rerio).

Characterization of a newly cloned proopiomelanocortin (POMC) gene in the teleost zebrafish, Danio rerio, is reported. This gene is formed by three exons and two introns, and its complete cDNA codes for a polypeptide of 222 amino acids. Zebrafish proopiomelanocortin (zfPOMC) contains the consensus sequences for ACTH, gamma-LPH, beta-MSH and beta-endorphin (beta-END). RT-PCR expression studies indicate that zfPOMC is selectively expressed in nervous tissue and in the pituitary gland. An homologous sequence to zfPOMC in the zebrafish genome is also presented. It is possible that this sequence represents part of a duplicate POMC, which might have appeared as a result of an extra genome duplication that have taken place in the cyprinidae family or even in all teleosts. Comparisons between the two zebrafish beta-endorphins and among these peptides and its homologues in other species are also presented.

Post-translational modification of bovine pro-opiomelanocortin. Tyrosine sulfation and pyroglutamate formation, a mass spectrometric study.

The amino-terminal fragment of beta-lipotropin (i.e. beta-lipotropin (1-40)) and joining peptide portions of pro-opiomelanocortin have been purified from extracts of bovine posterior pituitaries. Peptides were purified using a combination of reversed-phase and ion-exchange batch extraction procedures followed by reversed-phase high performance liquid chromatography. beta-Lipotropin (1-40) was found to consist of four major components while joining peptide was found to consist of two major components. Fast atom bombardment-mass spectrometric analysis of the tryptic fragments of both peptides revealed that the observed heterogeneity could be explained in terms of post-translational modifications. beta-Lipotropin (1-40) was found to be sulfated at tyrosine residue 28 to an extent of about 50%. The tyrosine residue in beta-lipotropin (1-40) is situated within an amino acid sequence with a preponderance of glutamate residues. Sulfation of this amino acid residue is entirely compatible with the known primary structure requirements of the sulfotransferase enzyme located in the trans-Golgi fraction. Both beta-lipotropin (1-40) and joining peptide were found to have pyroglutamate at their amino termini to an extent of about 50%. The cDNA sequence for bovine pro-opiomelanocortin predicts the presence of glutamic acid at position 1 of both peptides. Pyroglutamate is normally formed through the cyclization of glutamine. This reaction is thought to be catalyzed by a pyroglutamate forming enzyme located within the secretory granule fraction. Under certain circumstances peptides with glutamate at their amino termini may act as substrates for this enzyme.

[In vitro processing of the artificial analog of beta-lipotropin]. / Issledovanie protsessinga in vitro iskusstvennogo analoga beta-lipotropina.

The processing of the recombinant analogue of beta-lipotropin (beta-LHP) having 11 additional N-terminal amino acid residues and separated from the hormone by the processing signal, was investigated using rat adrenal secretory granule lysate as a test system of processing "in vitro". It was found that incubation of the beta-LPH analogue with secretory granule enzymes leads to its limited specific degradation with a release of native beta-endorphin. It is concluded that the additional N-terminal amino acids induced no qualitative changes in beta-LPH processing.

Posttranslational processing of opioid pro-opiomelanocortin products in piglets.

We have measured levels of beta-lipotropin, beta-endorphin, and N-acetyl-beta-endorphin in the plasma, cerebrospinal fluid (CSF), and caudal medullary brain containing the respiratory-related portion of the nucleus tractus solitarius (NTS) of 2.5 +/- 1.0- (SD) and 38.2 +/- 3.7-day-old naive uninstrumented piglets. Time of day, ambient atmosphere, temperature, handling, sound, light, and nutritional status were kept constant. Experimental procedure included decapitation and rapid collection, processing, and freezing of tissues until analysis by radioimmunoassay. Young, compared with older piglets, have higher measured levels of beta-lipotropin in the plasma and CSF and of N-acetyl-beta-endorphin in all three body compartments. Although measured levels of beta-endorphin-like immunoreactivity are also higher in the plasma and CSF of the young group, the calculated level of beta-endorphin is higher only in the CSF. In the NTS, both the measured and calculated active endorphin appear higher in the older group, but this difference is not significant. Excess beta-endorphin in the CSF of neonates may explain the relative immaturity of their respiratory functions at birth.

Processing reactions in the later stages of hormone activation.

Three tiers of processing have been investigated in the reactions that transform prohormones into their mature end products. Evidence is presented that the proteolytic reactions that convert lipotropin into shortened forms of beta-endorphin take place in individually distinct stages. After these cleavages have occurred, the removal of basic residues by carboxypeptidase H and amidation of the products are effected by independent reactions which do not synergise. Experiments are also described which show that the amidating enzyme can accept certain imino acids as substrates and utilises a mechanism that involves hydroxylation; it is implicit that peptide amidation proceeds by a similar mechanism. These results point to a general concept that pro-hormone processing involves consecutive reactions which take place in a predetermined order.

[Expression in Escherichia coli cells of the nucleotide sequence of DNA coding for the bovine lipotropic hormone]. / Ekspressiia v kletkakh Escherichia coli nukleotidnoi posledovatel'nosti DNK, kodiruiushchei lipotropnyi gormon byka.

A cDNA fragment of bovine proopiomelanocortin coding for beta-lipotropic hormone was joined with a promoter and ribosome binding site of B. amyloliquefaciens and cloned in E. coli in pBR 327 plasmid. The level of beta-lipotropin synthesis in bacterial cells transformed by the obtained plasmid was estimated immunochemically. The level of beta-lipotropin production was shown to be 5 mg per liter of bacterial culture.

Posttranslational processing of proadrenocorticotropin/endorphin-derived peptides during postnatal development in the rat pituitary.

The anterior pituitary content of pro-ACTH/endorphin-related peptides increased 5-fold from birth to 4 weeks and increased another 3-fold by adulthood. In contrast, the neurointermediate lobe content of pro-ACTH/endorphin-related peptides increased 15-fold from birth to 4 weeks and another 10-fold by adulthood. Despite the dramatic increase in content, posttranslational processing of pro-ACTH/endorphin in the neurointermediate lobe of the neonate closely resembled intermediate lobe processing in the adult; alpha MSH- and beta-endorphin-sized molecules (rather than ACTH and beta-lipotropin) accounted for more than 90% of the immunoreactivity in both neonates and adults. In the neurointermediate pituitary of both the neonate and the adult, the alpha MSH-sized material was largely diacetylated, and the beta-endorphin was both alpha-N-acetylated and C-terminally shortened. However, the extent of C-terminal shortening of beta-endorphin in the neurointermediate lobe of the neonate was not as great as that observed by postnatal day 21 or that in the adult. In the anterior pituitary, distinct differences in processing occurred between birth and adulthood. Proteolytic processing of pro-ACTH/endorphin was not as extensive on day 1 as in the adult, and pro-ACTH/endorphin accounted for 40-50% of the total immunoreactive peptide. The extent of processing of precursor increased around day 21, and a higher percentage of ACTH-(1-39) and beta-endorphin-(1-31)-sized material was found. Neonatal anterior pituitary contained substantial amounts of alpha MSH-sized material, whereas in adult anterior pituitary, less than 1-2% of the ACTH-related material was alpha MSH-sized. Despite these differences in the extent of proteolytic processing, neonatal anterior pituitary corticotropes resembled those of adults, in that they did not alpha-N-acetylate beta-endorphin or alpha MSH. Immunocytochemical studies demonstrated that a subset of the neonatal anterior pituitary corticotropes produced alpha MSH-related molecules.

[Genetic engineering of peptide hormones]. / Geneticheskaia inzheneriia peptidnykh gormonov.

The application of different approaches for preparing DNAs coding for peptide hormones was demonstrated. The libraries of human, bovine and porcine pituitaries cDNA were obtained starting from their total mRNAs. Screening of these libraries revealed clones containing human, bovine and porcine growth hormone sequences, cDNAs for bovine ACTH-beta-lipotropin precursor and for bovine and porcine prolactin. The gene of human calcitonin was created by combination of chemical and enzymatic synthesis. This synthetic gene was further cloned in pBR322. The expression of cloned human growth hormone cDNA under control of different Escherichia coli promoters was studied and physico-chemical and biological properties of the growth hormone produced by E. coli were tested.

Tissue distribution of messenger RNAs coding for opioid peptide precursors and related RNA.

All of the endogenous opioid peptides thus far identified are derived from three types of precursors, i.e. the corticotropin/beta-lipotropin precursor, preproenkephalin A and preproenkephalin B. Poly(A)-containing RNA from various bovine and porcine tissues has been subjected to blot hybridization analysis with the use of cDNA probes specific for the three opioid peptide precursors. Analysis with a corticotropin/beta-lipotropin precursor cDNA probe has revealed, in addition to the pituitary mRNA, a smaller hybridizable RNA species present in bovine extrapituitary tissues, such as the adrenal medulla, thyroid, thymus, duodenum and lung. The hypothalamus contains both these RNA species. DNA complementary to the smaller RNA species from the bovine adrenal medulla has been cloned. Analysis of the cloned cDNA, in conjunction with endonuclease S1 mapping of poly(A)-rich RNA from the adrenal medulla, has indicated that the smaller RNA species represents the 3'-terminal 712-729 nucleotides, excluding the poly(A) tail, of the pituitary corticotropin/beta-lipotropin precursor mRNA, having heterogeneous start sites. Analysis with a preproenkephalin A cDNA probe has shown the presence of hybridizable RNA in the bovine hypothalamus, duodenum and pituitary neurointermediate lobe in addition to the adrenal medulla. The hybridizable RNA species from all these tissues are indistinguishable in size. RNA hybridizable with a preproenkephalin B cDNA probe has been found in the porcine spinal cord and ileum besides the hypothalamus, and these RNA species exhibit an indistinguishable size. The results presented indicate that each opioid peptide precursor is synthesized in different tissues.

Heterogeneity of 3' nontranslated regions in proopiomelanocortin (POMC) precursor mRNA of chum salmon Onchorynchus keta: polymorphism of the gene.

Heterogeneity of salmon pituitary proopiomelanocortin (POMC) mRNA was shown by comparison of the nucleotide sequence of independently isolated cDNA clones encoding POMC, pSSM90, pSSM53 and pSSM17, the last of which was previously characterized. Newly isolated clones pSSM90 and pSSM53 contained inserts of 1228 and 666 base pairs, respectively (excluding poly(A)). Sequence analysis revealed that the former contained sequences coding for the carboxy half of putative corticotropin (ACTH), the whole region of beta-lipotropin (beta-LPH), and the entire 3' nontranslated region, while the latter contained only the 3' nontranslated region. Sequence comparison of the three clones revealed that there are some definite nucleotide changes in the 3' nontranslated regions, i.e., base replacements and base additions at multiple sites, whereas no single change was observed in the coding regions, thus demonstrating heterogeneity and hence polymorphism of the gene in the salmon genome.

Complete structure of the porcine pro-opiomelanocortin mRNA derived from the nucleotide sequence of cloned cDNA.

Polyadenylated RNA isolated from porcine pituitary neurointermediate lobes was used to construct a cDNA library. The library was screened with a rat genomic DNA fragment specific for pro-opiomelanocortin sequences. Two positive clones, pJA-19 and pJA-20, containing respectively 850 bp and 550 bp were characterized. Sequence analysis of the cDNA inserts revealed the complete structure of the porcine pro-opiomelanocortin mRNA. This mRNA would include 129 5'-untranslated nucleotides, 801 nucleotides coding for the 267 amino acids precursor and 162 3'-untranslated nucleotides. Comparison with pro-opiomelanocortin mRNA sequences from other species shows regions of high homology not only in the coding sequences but also in the 5'untranslated region where the first 50 nucleotides are over 80% purines.

Complete nucleotide sequence of the human corticotropin-beta-lipotropin precursor gene.

The nucleotide sequence of an 8658-base-pair human genomic DNA segment containing the entire corticotropin-beta-lipotropin precursor gene has been determined, and some sequence features of the gene and its flanking regions have been analysed. The gene is composed of 7665 base pairs including two introns of 3708 and 2886 base pairs. Comparison of the 5'-flanking sequences of the human, bovine and mouse corticotropin-beta-lipotropin precursor genes reveals the presence of a highly conserved region, which contains sequences of 14-15 base pairs homologous with sequences located upstream of the mRNA start site of other glucocorticoid-regulated genes.

Sequence requirement for transcription in vitro of the human corticotropin/beta-lipotropin precursor gene.

Using HeLa whole cell extracts, we have demonstrated that transcription in vitro of the cloned human and bovine corticotropin/beta-lipotropin precursor genes is initiated accurately and efficiently. DNA sequences required for promoter function have been assessed by using a series of 5'-deletion mutants of a fusion gene that contains the 5'-flanking sequence and capping site of the human corticotropin/beta-lipotropin precursor gene and the structural sequence of the herpes simplex virus thymidine kinase gene. The results obtained have shown that the region between 22 base pairs and 35 base pairs upstream from the capping site is essential for the correct and efficient transcriptional initiation in vitro. Thus, the 'TATA box' present in this region seems to be the main promoter element for transcription of the human corticotropin/beta-lipotropin precursor gene in the HeLa cell-free system. We have also developed a transcription system in vitro from the corticotropin-producing mouse pituitary tumor cell line AtT-20 in culture. Deletion mapping of the fusion gene promoter has indicated that the 'TATA box' region is required for the accurate and efficient transcriptional initiation in this system as well. Characteristic of this system is that the deletion of the sequence lying between 53 base pairs and 59 base pairs upstream from the capping site increases the transcriptional efficiency. Because this effect is observed in the AtT-20 cell-free system, but hardly in the HeLa cell-free system, it seems reasonable to assume that the interaction of this upstream sequence with some factor(s) in the AtT-20 cell extract is responsible for the modulation of transcription of the human corticotropin/beta-lipotropin precursor gene.

Structural analysis of repetitive DNA sequences in the bovine corticotropin-beta-lipotropin precursor gene region.

Repetitive DNA sequences in the bovine corticotropin-beta-lipotropin precursor gene region have been mapped and subjected to nucleotide sequence analysis. Two of the four repetitive DNA segments found are located in the 5'-flanking region, and one each within the intervening sequences. Each repetitive DNA segment contains one to three highly homologous unit sequences with an approximate length of 120 base pairs. All the unit sequences are flanked on the 3' side by tandem repeats. There are about 10(5) copies of the repetitive DNA in the bovine genome. Comparison of the bovine repetitive sequences with those of other mammalian species reveals the presence of a homologous segment of approximately 40 base pairs. This segment and the region preceding it in the bovine repetitive DNA exhibit sequence homology with the region encompassing the origin of DNA replication in papovaviruses.

Repetitive DNA sequences in the human corticotropin-beta-lipotrophin precursor gene region: Alu family members.

Repetitive DNA sequences in the human corticotropin-beta-lipotropin precursor gene region have been studied by blot hybridization analysis and DNA sequencing. Six repetitive sequences are present in this gene region; five of them are Alu family members with an approximate length of 300 base pairs, and the other consists of a portion of an Alu family sequence. Two of these Alu family members are located in the 5'-flanking region of the gene, and the remaining four within the intervening sequences. These Alu family sequences constitute inverted repeats in the intervening sequences as well as in the 5'-flanking region of the gene.