60 YEARS OF POMC: Purification and biological characterisation of melanotrophins and corticotrophins.

The remarkable conservation of the primary structures and anatomical location of dogfish α-melanocyte-stimulating hormone (MSH), corticotrophin-like intermediate lobe peptide (CLIP) and adrenocorticotrophic hormone (ACTH) compared with mammals reinforced the tissue-specific processing hypothesis of ACTH peptides in the pituitary gland. The cloning of dogfish pro-opiomelanocortin (POMC) led to the identification of δ-MSH and simultaneously revealed the high conservation of the γ-MSH sequence during evolution. These studies have also shown that ß-MSH is much less conserved during evolution and in some species is not even processed from ß-LPH. Human pro-γ-MSH potentiates the corticosteroidogenic activity of ACTH and peptides generated from its N-terminal, in particular big-γ-MSH, appear to have adrenal mitogenic activity. Human big-γ-MSH (from the zona intermedia) may also cause the adrenache. The review finishes with a cautionary note with regard to the misdiagnosis of the ectopic ACTH syndrome in which partial processing of ACTH can result in large concentrations of α-MSH and CLIP, which can interfere in the performance of two-site immunoassays, and the problem of the correct disulphide bridge arrangement in synthetic N-POMC peptides is also discussed.

Cyclic analogs of alpha-melanocyte-stimulating hormone (alphaMSH) with high agonist potency and selectivity at human melanocortin receptor 1b.

Alpha-melanotropin (alphaMSH), Ac-Ser1-Tyr2-Ser3-Met4-Glu5-His6-Phe7-Arg8-Trp9-Gly10-Lys11-Pro12-Val13-NH2,(1) has been long recognized as an important physiological regulator of skin and hair pigmentation in mammals. Binding of this peptide to the melanocortin receptor 1 (MC1R) leads to activation of tyrosinase, the key enzyme of the melanin biosynthesis pathway. In this study, interactions of the human MC1bR (an isoform of the receptor 1a) with the synthetic cyclic analogs of alphaMSH were studied. These ligands were analogs of MTII, Ac-Nle4-cyclo-(Asp5-His6-D-Phe7-Arg8-Trp9-Lys10)-NH2, a potent pan-agonist at the human melanocortin receptors (hMC1,3-5R). In the structure of MTII, the His6-D-Phe7-Arg8-Trp9 segment has been recognized as "essential" for molecular recognition at the human melanocortin receptors (hMC1,3-5R). Herein, the role of the Trp9 in the ligand interactions with the hMC1b,3-5R has been reevaluated. Analogs with various amino acids in place of Trp9 were synthesized and tested in vitro in receptor affinity binding and cAMP functional assays at human melanocortin receptors 1b, 3, 4 and 5 (hMC1b,3-5R). Several of the new peptides were high potency agonists (partial) at hMC1bR (EC50 from 0.5 to 20 nM) and largely inactive at hMC3-5R. The bulky aromatic side chain in position 9, such as that in Trp, was found not to be essential to agonism (partial) of the studied peptides at hMC1bR.

Immunohistochemical localization and comparison of carboxypeptidases D, E, and Z, alpha-MSH, ACTH, and MIB-1 between human anterior and corticotroph cell "basophil invasion" of the posterior pituitary.

Basophil invasion, i.e., invasion of basophilic corticotrophs from the residual intermediate lobe into the posterior lobe of the human pituitary gland, is believed to be a physiological phenomenon. This study evaluated the distribution of CPE, CPD, CPZ, alpha-MSH, ACTH, and Ki-67 immunoreactivity between human anterior pituitary and basophil invasion of the neurohypophysis. Mild to moderate immunoreactivities for CPE and CPZ were distributed relatively uniformly in the majority of the anterior pituitary cells and basophil invasion. In contrast, only corticotrophs exhibited intense CPD immunoreactivity. Basophil invasion showed similar immunoreactivities for alpha-MSH, ACTH, CPE, and CPZ as corticotrophs in the anterior pituitary, except for CPD, which was detected much less frequently. In the posterior lobe, CPE, CPD, and CPZ were present within the Herring bodies. Although no MIB-1 immunoreactivity was identified in anterior pituitary cells, limited MIB-1 labeling was detected in basophil invasion in five of ten cases. Highly selective expression of CPD in corticotrophs suggests that CPD plays a particularly important role in prohormone (POMC) processing in corticotrophs, with minimal or no significant roles in non-corticotrophs. Evidence that corticotrophs in basophil invasion are undergoing proliferation and are also phenotypically different from their counterpart in the anterior pituitary has further raised the possibility of some neoplastic potential.

alpha-MSH acetylation in the pituitary gland of the sea bream (Sparus aurata L.) in response to different backgrounds, confinement and air exposure.

MSH is a pituitary hormone derived by post-translational processing from POMC and involved in stress and background adaptation. N-terminal acetylation of MSH to monoacetyl alpha-MSH or diacetyl alpha-MSH increases the bioactivity of the peptide. The aim of this study was to characterize alpha-MSH acetylation in the sea bream (Sparus aurata L.) pituitary gland in response to the stressors air exposure and confinement, as well as in fish adapted for 15 days to a white, gray or black background. Pituitary homogenates were purified by reversed-phase HPLC (RP-HPLC). The alpha-MSH content of fractions was measured by RIA. Immunoreactive RP-HPLC fractions were further analyzed by electrospray mass spectrometry and the peptide sequence determined as SYSMEHFRWGKPV-NH2. In the pituitary gland of sea bream, des-, mono- and diacetyl alpha-MSH were identified. Then plasma alpha-MSH levels were measured in sea bream adapted to different backgrounds. Surprisingly, we found the highest plasma alpha-MSH levels in white-adapted as compared with black-adapted sea bream with intermediate values for gray-adapted fish. This observation is in contrast with results that have been obtained in eel, trout or terrestrial vertebrates. Next, des-, mono- and diacetyl alpha-MSH forms were measured in homogenates of the pituitary gland and in plasma of sea bream exposed to air, to confinement, or to different backgrounds. Monoacetyl alpha-MSH was the predominant form in all control and experimental groups. The lowest content of monoacetyl alpha-MSH relative to des- and diacetyl alpha-MSH was found in white-adapted fish. Levels of des- and diacetyl alpha-MSH forms were similar under all conditions. We observed that monoacetyl alpha-MSH is the most abundant isoform in the pituitary gland after background adaptation, confinement and air exposure, in sea bream. These data indicate that the physiologically most potent isoform of alpha-MSH may vary from species to species.

POMC-related products in the intermediate pituitary of the amphibian, Bufo marinus: differential subcellular processing in the Golgi and secretory granules.

In the intermediate pituitary of the anuran amphibian, Bufo marinus, the N-acetylation of ACTH(1-13)-NH2 to yield alpha-MSH occurs as a cosecretory processing event, whereas the N-acetylation of beta-endorphin occurs as a posttranslational processing event. To understand how these two N-acetylation reactions are segregated, B. marinus intermediate pituitary cells were analyzed by immunogold labeling electron microscopy, and by using an ultracentrifugation procedure. The immunogold labeling studies indicated that ACTH(1-13)-NH2-related immunoreactivity was colocalized with N-acetylated beta-endorphin-related immunoreactivity in secretory granules. Furthermore, ACTH(1-13)-NH2-related immunoreactivity was not detected in either the ER or the Golgi. N-Acetylated beta-endorphin-related immunoreactivity, however, was detected in the Golgi. Ultracentrifugation analysis revealed that in an ER/microsomal fraction, beta-LPH-sized and nonacetylated beta-endorphin-sized immunoreactive material were present in a molar ratio of 1:2. No N-acetylated forms of beta-endorphin were detected in the ER/microsomal fraction. In a Golgi/secretory granule fraction, the molar ratio of beta-LPH to beta-endorphin was 1:9 with 58% of the beta-endorphin being N-acetylated. Collectively, these data support the following hypotheses. The proteolytic cleavage of ACTH (1-39) to yield ACTH (1-13)-NH2 is a late processing event occurring in secretory granules. The cleavage of beta-LPH to yield nonacetylated beta-endorphin is an early processing event that may occur in the ER or the Golgi. Because N-acetylated beta-endorphin and nonacetylated ACTH(1-13)-NH2 are colocalized in secretory granules, it appears, therefore, that the N-acetylation of beta-endorphin is completed prior to loading into secretory granules. Thus, there is a spatial and temporal separation of the posttranslational processing events associated with the beta-LPH portion and ACTH portion of the POMC biosynthetic pathway in amphibian intermediate pituitary cells.

High-performance liquid chromatographic assay for Melanotan-1 ([Nle4-DPhe7]alpha-melanocyte-stimulating hormone) in biological matrices.

The overall objective of this research was to develop a sensitive, specific, and stability-indicating HPLC assay for the determination of the [Nle4-DPhe7]alpha-melanocyte-stimulating hormone analog known as Melanotan-1 (MT-1) in biological matrices, i.e., cell culture transport media and human plasma. Separation was accomplished isocratically within 8.0 min using a C8 reversed-phase column. The mobile phase consisted of 0.1 M phosphate buffer-acetonitrile (80:20, v/v) with 18 microliters/l triethylamine at pH 2.50. The flow-rate was 1 ml/min with detection at 214 nm. Standard curves (n = 5) were linear over the concentration range 100-1000 ng/ml. The precision, accuracy, intra- and inter-day variations were good with C.V.s typically within 8.7% for concentrations greater than 100 ng/ml. This method was applied to a study of the transport of MT-1 in the Caco-2 cell monolayer model.

alpha-Melanocyte-stimulating hormone during human perinatal life.

To obtain information on human pituitary intermediate lobe activity throughout the perinatal period, plasma alpha MSH immunoreactivity (IR) was measured in 106 newborns at delivery and during the first week of postnatal life. Subjects were divided into groups according to gestational age at birth, mode of parturition, and antenatal state of health. Plasma alpha MSH IR decreased progressively from severe preterm to fullterm neonates born by vaginal delivery (VD; P < 0.001) or cesarean section (CS) with and without prenatal distress (P < or = 0.001 in both cases). alpha MSH IR was due, in all studied conditions, to three major forms: desacetyl alpha MSH, alpha MSH, and diacetyl alpha MSH. Desacetyl alpha MSH was always the most represented form, but it decreased from 75-80% of the total in severe premature to 40-45% in mature infants. In term neonates, total alpha MSH IR values were higher in subjects born by normal VD than by elective CS (P < or = 0.05), in complicated than in normal VD (P < or = 0.01), and in CS performed because of fetal distress than in elective CS (P < or = 0.01). No significant difference was detectable in mature subjects in the percentages of the three alpha MSH forms in relation to the mode of delivery and fetal state during antenatal life or at parturition. Twelve hours after birth, total alpha MSH IR significantly decreased in all groups of term newborns, reaching a plateau of 0.8-1.4 pmol/L. In premature infants, similar concentrations were detectable by the fourth postnatal day. We conclude that 1) alpha MSH IR intermediate lobe secretion progressively decreases throughout the third trimester of pregnancy; 2) stress, including that pertinent to parturition, stimulates alpha MSH IR release; and 3) pituitary intermediate lobe activity declines shortly after birth independently of the maturity reached by the fetus, the mode of parturition, and the presence of antenatal chronic distress, although the process is slightly retarded in premature newborns.

Partial characterization of IR-alpha-MSH peptides found in melanoma tumors.

Our previous work indicated that IR-alpha-MSH (immunoreactive alpha-melanocyte-stimulating hormone) plasma levels are three times as high in melanoma patients with progressing disease than in disease-free patients, and that the melanoma tumor itself may be the source of IR-alpha-MSH. Further identification of the material in tumor extracts has been carried out in this study, and the results presented here show that the immunoreactivity is associated with a major fraction of about 16 kDa and another of 5-9 kDa. Significant amounts of the immunoreactive material were also found in human melanoma cells but not in culture supernatants. The presence of this material may be related to the melanogenic status of the tumor cells. We have estimated the intracellular IR-alpha-MSH to be within a 0.4 to 2.3 nM range in melanoma tumor cells. We have investigated the melanogenic effect of the IR-alpha-MSH material and its relationship to alpha-MSH. Purified extracts both from metastases and cultured cells were found to promote frog skin darkening as well as tyrosinase activity in Cloudman S91 melanoma cells. The IR material could also displace labeled alpha-MSH from its binding sites in human melanoma cells. Our data clearly indicate that melanoma cells engage in an autocrine production of alpha-MSH-like bioactive peptides by melanoma cells, of larger mol.wt., which are able to bind to MSH receptors. These peptides may be involved in the regulation of melanogenesis and possibly in the growth and proliferation of melanoma cells by an autocrine/paracrine mechanism.

Isolation and structural characterization of peptides related to alpha- and gamma-melanocyte-stimulating hormone (MSH) from the frog brain.

Peptides that are derived from the processing of proopiomelanocortin were isolated in pure form from the brain of the frog Rana ridibunda. The primary structure of the most abundant of those peptides was established as: Ser-Tyr-Ser-Met-Glu-His-Phe-Arg-Trp-Gly-Lys-Pro-Val. This amino acid sequence is identical to that of mammalian and frog pituitary alpha-melanocyte-stimulating hormone (MSH) and the peptide co-eluted with synthetic desacetyl alpha-MSH, indicating that it is COOH-terminally alpha-amidated. A second component, which exhibited a shorter retention time, co-eluted with the glycine-extended form of desacetyl alpha-MSH [ACTH(1-14)]. The primary structure of the third peptide isolated in pure form from the brain extract was established as: Lys-Tyr-Val-Met-Ser-His-Phe-Arg-Trp-Asn-Lys-Phe-NH2. This sequence corresponds to Lys-gamma 1-MSH as predicted from the nucleotide sequence of frog proopiomelanocortin. The presence of substantial amounts of desacetyl alpha-MSH and Lys-gamma 1-MSH in the frog brain supports the concept that, in amphibia, melanotropins may act as neurotransmitters and/or neuromodulators as well as hormonal peptides.

Detection and partial characterization of proopiomelanocortin-related end-products from the pars intermedia of the toad, Bombina orientalis.

Steady-state analyses were performed on the proopiomelanocortin (POMC)-related end-products present in acid extracts of the pars intermedia of the anuran amphibian, Bombina orientalis. Sephadex G-75 gel filtration chromatography indicated that immunoreactive alpha-MSH-sized material and N-acetylated beta-endorphin-related material are the major POMC-related products present in this tissue. The alpha-MSH-sized immunoreactivity was further fractionated by reversed phase HPLC. The major peak of immunoreactivity isolated by this procedure eluted with the same retention time as synthetic ACTH(1-13)amide. Cation exchange chromatography supported the conclusion that the major storage form of alpha-MSH in the pars intermedia of Bombina is ACTH(1-13)amide. Analysis of Bombina pars intermedia in culture indicated that mono-acetylated and di-acetylated alpha-MSH were the major forms of alpha-MSH secreted into the medium. The major peak of N-acetylated beta-endorphin-related material was further analyzed by cation exchange chromatography and Sephadex G-25 gel filtration column chromatography. The major storage form of beta-endorphin in this tissue is N-acetylated, has a net positive charge at pH 2.75 of +1, and has an apparent molecular weight of 1.2K. The beta-endorphin present in the pars intermedia of this tissue does not undergo further N-acetylation at the time of secretion. These results indicate that in the pars intermedia of the archaeobatrachian, Bombina orientalis, the N-acetylation of alpha-MSH is a cosecretory processing event, whereas N-acetylation of beta-endorphin is a post-translational processing event. These results are compared to other archaeobatrachian and neobatrachian pituitary POMC systems that have been analyzed.

Diphtheria toxin-related alpha-melanocyte-stimulating hormone fusion toxin. Internal in-frame deletion from Thr387 to His485 results in the formation of a highly potent fusion toxin which is resistant to proteolytic degradation.

We have previously reported the genetic construction and properties of a fusion protein which was composed of the enzymatically active and membrane translocation domains of the diphtheria toxin and the receptor-specific ligand alpha-melanocyte-stimulating hormone (alpha-MSH) (Murphy, J.R., Bishai, W., Borowski, M., Miyanohara, A., Boyd, J., and Nagle, S. (1986) Proc. Natl. Acad. Sci. U.S.A. 83, 8258-8262). While this fusion toxin was found to be selectively toxic for MSH receptor-bearing cells in vitro, it was subject to profound proteolytic degradation in recombinant Escherichia coli making purification difficult. We now report that the deletion of diphtheria toxin fragment B sequences between Thr387 and His485 results in a protease-resistant form of the fusion toxin, DAB389-alpha-MSH. We show that DAB389-alpha-MSH is expressed in high yield in recombinant Escherichia coli, that it is readily purified from crude bacterial lysates by immunoaffinity and high performance liquid chromatography, and its cytotoxic activity toward both human and murine malignant melanoma cell lines is mediated through the MSH receptor.

Regulation of differential release of alpha-melanocyte-stimulating hormone forms from the pituitary of a teleost fish, Oreochromis mossambicus.

Using high-performance liquid chromatography (HPLC) in combination with radioimmunoassay, three forms of alpha-MSH (des-acetyl, mono-acetyl and di-acetyl alpha-MSH) were separated and identified in tilapia neurointermediate lobes and plasma, and in medium from lobes superfused in vitro. The presence of acetylated forms in lobe extracts indicated that the peptides are acetylated intracellularly. Di-acetyl alpha-MSH was, especially in comparison with monoacetyl alpha-MSH, relatively more abundant in lobe extracts than in plasma. This suggests that the three forms of alpha-MSH are not released according to their relative intracellular abundances. The possibility of regulation of this differential release by dopamine and TRH was investigated, using a microsuperfusion system. Dopamine was a potent inhibitor of alpha-MSH release, but did not modulate the relative abundance of the different forms of alpha-MSH released from the MSH cells. TRH was a potent stimulator of alpha-MSH release. It enhanced in vitro the release of di-acetyl alpha-MSH more than the release of mono-acetyl alpha-MSH. Thus tilapia may be able to modulate not only the quantitative but also the qualitative signal from the MSH cells. This might enhance the flexibility of the animals to respond to environmental challenges.

Biologically active monoiodinated alpha-MSH derivatives for receptor binding studies using human melanoma cells.

Three different monoiodinated radioligands of alpha-MSH (alpha-melanocyte-stimulating hormone) were compared in a binding assay with human D10 melanoma cells: [Tyr(125I)2]-alpha-MSH, [Tyr(125I)2,NIe4]-alpha-MSH, and [Tyr(125I)2,NIe4,D-Phe7]-alpha-MSH. They were prepared either by the classical chloramine T method or by the Enzymobead method. A simple and rapid purification scheme was developed consisting of a primary separation on reversed-phase C18 silica cartridges immediately after the iodination, followed by HPLC purification before each binding experiment. Biological testing of the three radioligands showed that they all retained high melanotropic activity in the B16 melanin assay and the Anolis melanophore assay. However, in human D10 melanoma cells, [Tyr(125I)2,NIe4]-alpha-MSH led to a high degree of non-specific binding to the cells which could not be displaced by excess alpha-MSH and only partially by [NIe4]-alpha-MSH. The [Tyr(125I)2,NIe4,D-Phe7]-alpha-MSH tracer gave similar results but with a much lower proportion of non-specific binding. On the other hand, [Tyr(125I)2]-alpha-MSH proved to be an excellent radioligand whose non-specific binding to the D10 cells was not higher than 20% of the total binding.

Alpha-melanocyte-stimulating hormone immunoreactivity in human melanoma metastases extracts.

Seven human melanoma metastases were extracted in order to check the possible presence of any alpha-melanocyte stimulating hormone (MSH) immunoreactivity. The aim of that study was to provide some explanation for, mainly, two observations that we have already made and reported: 1) increased plasma alpha-MSH levels in melanoma of tumour-bearing patients as compared with tumour-free patients; 2) the presence of specific alpha-MSH receptors on human melanoma cells in culture. We could measure large amounts of immunoreactive alpha-MSH in all tumours ranging from 0.31 to 4.27 pmoles per g of wet tissue. Further identification of the extracted material by high-performance liquid chromatography revealed compounds of higher molecular weight and more hydrophobic than synthetic alpha-MSH. In addition, purified extracts could also displace 125I-labelled alpha-MSH from its cellular binding sites in an alpha-MSH specific radio-receptor binding assay. Our findings would suggest a possible presence of some hormone precursor(s) inside the melanoma tumours.

Melanin concentrating hormone. V. Isolation and characterization of alpha-melanocyte-stimulating hormone from frog pituitary glands.

The structure of alpha-melanocyte-stimulating hormone (alpha-MSH) has been determined in the pars intermedia of the frog Rana ridibunda. Pulse-chase labeling of frog neurointermediate lobes with selective amino acids revealed that the composition of frog alpha-MSH is similar to that of alpha-MSH from all mammalian species yet studied. Tryptic mapping of nexly synthetized alpha-MSH generated two fragments with the following amino acid composition: (T1) Trp, Pro, Lys, Gly, Val and (T2) Tyr, Arg, Phe, His, Ser, Glu. Concurrently, alpha-MSH was purified from 100 neurointermediate lobes to apparent homogeneity by reverse-phase HPLC. The sequence of the peptide determined by automated Edman degradation was Ser-Tyr-Ser-Met-Glu-His-Phe-Arg-Trp-Gly-Lys-Pro-Val. The structure of frog alpha-MSH is thus identical to mammalian des-N alpha-acetyl alpha-MSH and differs from the sequence of toad (Xenopus laevis) alpha-MSH only by the first residue (Ser instead of Ala). These results confirm that the sequence of alpha-MSH has been highly preserved during evolution.

Expression of diphtheria toxin fragment A and hormone-toxin fusion proteins in toxin-resistant yeast mutants.

Mutants of the eukaryote Saccharomyces cerevisiae, previously selected for resistance to diphtheria toxin, were investigated for their suitability as hosts for the expression of tox-related proteins. The structural gene for the toxin, encoding the fragment A catalytic domain, was modified for efficient intracellular expression in eukaryotes and placed downstream of the yeast GAL1 promoter element in a plasmid. Transformed mutant yeast grown in galactose, which induces that promoter, were viable and contained active fragment A. In contrast, sensitive, wild-type cells harboring this plasmid grew normally under repressing conditions but were killed when the GAL1 promoter was induced. Additional constructions were also prepared that included sequences encoding either the lymphocyte growth factor interleukin 2 or alpha-melanocyte-stimulating hormone along with the lipid-associating domains of fragment B and the leader peptide of the Kluyveromyces lactis killer toxin. Resistant mutant strains transformed with these plasmids efficiently expressed and secreted the expected chimeric toxins.

High-performance displacement chromatography of melanotropins and their derivatives.

Mixtures containing derivatives of the biological active peptide alpha-melanocyte stimulating hormone (alpha-MSH) or beta-melanocyte stimulating hormone (beta-MSH) were purified by using reversed-phase chromatography in the displacement mode. On a 250 x 4.6 mm octadecyl silica column and with instrumentation used in analytical high-performance liquid chromatography about 30 mg of the peptide mixture were separated by using an aqueous solution of benzyldimethyldodecylamonium bromide as the displacer in a single chromatographic run. The results demonstrate the advantages of displacement over elution in preparative chromatography of peptides on the scale of tens of milligrams that is typical for physiological tissue extracts and in solid-phase peptide synthesis.