60 YEARS of POMC: Lipotropin and Beta-Endorphin: a Perspective

56:3 D G SMYTH Lipotropin and beta-endorphin 56:4 T13–T25 Thematic Review 60 YEARS OF POMC Lipotropin and beta-endorphin: a perspective Correspondence should be addressed to D G Smyth D G Smyth Email [email protected] Department of Endocrinology, William Harvey Research Institute, London, UK Abstract Many important fields of research had a humble origin. In the distant past, A J P Martin’s Key Words discovery that amino acids could be separated by paper chromatography and Moore f lipotropin and Stein’s use of columns for quantitative amino acid analysis provided the first steps f neurotransmitters towards the determination of structure in complex biologically active molecules. They f beta-endorphin opened the door to reveal the essential relationship that exists between structure and f POMC function. In molecular endocrinology, for example, striking advances have been made by chemists with their expertise in the identification of structure working with biologists who contributed valuable knowledge and experience. Advantage was gained from the convergence of different background, and it is notable that the whole is greater than the sum. In the determination of structure, it may be recalled that four of the world’s great pioneers (Archibald Martin, Rodney Porter, Fred Sanger and Vincent du Vigneaud) were acknowledged for their fundamental contributions when individually they were awarded the Nobel Prize. They foresaw that the identification of structure would prove of outstanding importance in the future. Indeed, study of the structures of β-endorphin Journal of Molecular Endocrinology Journal of Molecular and enkephalin and the different forms of opiate activity they engender has led to a Endocrinology transformation in our understanding of chemical transmission in the brain. (2016) 56, T13–T25 Lipotropin: precursor of β-endorphin It is well known, of course, that most if not all peptide unexpected discoveries: firstly, that a 31-residue fragment hormones are formed as fragments of larger peptides and of lipotropin shown to be present in pituitary and brain lipotropin is no exception. This 91-residue polypeptide, possessed potent opiate activity (Feldberg & Smyth 1976, first isolated by Birk and Li (1964) and sequenced by Bradbury et al. 1976a, Li & Chung 1976); and secondly, Li et al. (1965), possessed lipolytic activity, and it was that lipotropin was itself a fragment of a precursor, which provisionally assigned the title ‘lipotrophic hormone’. proved to be a multifunctional prohormone (Mains et al. The primary structure included a sequence of amino acids 1977, Roberts & Herbert 1977, Nakanishi et al. 1979). It that exhibited a marked similarity to α-melanotropin is of interest to recall the background to these advances (α-MSH), and it was generally assumed that ‘β-MSH’ was which stemmed from the study of prohormone processing the biologically active counterpart to α-MSH in lipotropin. and generation of their biologically active constituents. As a melanotrophic hormone, however, β-MSH was much less active than -MSH and alarm bells began to ring when α Lipotropin: from structure to function it was reported that β-MSH was not present in the human pituitary (Bloomfield et al. 1974), which raised question For several years, the principal interests of my on its physiological significance. This set the stage for two laboratory at the National Institute for Medical Research http://jme.endocrinology-journals.org © 2016 Society for Endocrinology This paper is part of a thematic review section on 60 Years of POMC. DOI: 10.1530/JME-16-0033 Printed in Great Britain The Guest Editors for this section were Adrian Clark and Philip Lowry. Published by Bioscientifica Ltd. Downloaded from Bioscientifica.com at 09/30/2021 01:38:48PM via free access 10.1530/JME-16-0033 Thematic Review D G SMYTH Lipotropin and beta-endorphin 56:4 T14 (Mill Hill, London) were focused on β-endorphin and This implied that the C-terminal peptide rather than its formation from lipotropin. Donald Steiner’s classical β-MSH might be functionally significant, so I arranged studies on insulin precursors had demonstrated that a collaborative study with James Edwardson of Imperial insulin is formed from a prohormone, and it seemed College, London, to investigate its activity. As the likely that this would prove to be a general phenomenon. C-terminal peptide 1, named ‘C-fragment of lipotropin’, As fragments of prohormones might be retained in tissues had been isolated from the pituitary which secretes its where the prohormones are synthesised, our strategy was hormones into the circulation, Edwardson started by to isolate and identify the constituent fragments of a investigating potential peripheral activities such as putative prohormone and then reassemble them to reveal effects on sodium transport in the kidney and influence its linear structure. In 1974, we were successful in isolating on insulin secretion from the pancreas. a series of novel peptides from porcine pituitary (Bradbury et al. 1975, 1976b) and we observed that a group of them Discovery of -endorphin accounted for the sequence of adrenocorticotrophic β hormone (ACTH) together with the sequence of lipotropin. In September 1975, there was an astonishing coincidence. At that time, it was not known that ACTH and lipotropin I was invited to Imperial College to give a talk on the are formed from a common prohormone, although the peptides we had isolated, and I described how we had possibility had been discussed informally at Meetings of identified a series of novel peptides that accounted for the Endocrine Society (see Lowry 2016). the sequence of lipotropin. I explained that we had The peptides we isolated could have been inert reason to believe that one of the peptides, the C-fragment fragments ‘left over’ after activation of a prohormone, of lipotropin, might possess biological activity. To or they might possess an activity that was waiting to be demonstrate this, I showed a diagram illustrating the discovered. To obtain an indication of this, we examined alignment of our peptides in the lipotropin sequence the sensitivity of lipotropin to cleavage by trypsin and (Fig. 1) and I drew attention to a peptide we had also by an enzyme with similar specificity which we had synthesised to model the region of lipotropin where isolated from the pituitary, because it was believed that release of its C-fragment takes place. The model peptide peptide hormones were generated from their precursors contained the sequence Lys–Asp–Lys–Arg–Tyr–Gly, which by enzymes with basic specificity (Smyth 1975). We found on digestion with enzymes with basic specificity released that one of the peptides we identified in the pituitary Tyr–Gly (Bradbury et al. 1976b). could be released from the C-terminus of lipotropin by Journal of Molecular Endocrinology limited digestion with either enzyme (Bradbury et al. C-Fragment of lipotropin: Tyr–Gly–Gly–Phe–Met... 1976c), but surprisingly there was negligible cleavage ...His–Lys–Lys–Gly–Gln on the N-terminal side of the sequence at the centre of Enkephalin 1: Tyr–Gly–Gly–Phe–Met lipotropin necessary for the release of β-MSH, which at the time was believed to be the biologically active region. Enkephalin 2: Tyr–Gly–Gly–Phe–Leu Figure 1 Alignment of peptides isolated from porcine pituitary in the amino acid sequence of lipotropin (LPH). The C-fragment of lipotropin (β-endorphin) corresponds to lipotropin residues 61–91, the Cʹ-fragment to lipotropin residues 61–87, and β-MSH to residues 41–58. Reprinted from Biochemical and Biophysical Research Communications, 69, Bradbury AF, Smyth DG & Snell CR, Lipotropin: Precursor to two biologically active peptides, 950–956. Copyright (1976), with permission from Elsevier. http://jme.endocrinology-journals.org © 2016 Society for Endocrinology Published by Bioscientifica Ltd. DOI: 10.1530/JME-16-0033 Printed in Great Britain Downloaded from Bioscientifica.com at 09/30/2021 01:38:48PM via free access Thematic Review D G SMYTH Lipotropin and beta-endorphin 56:4 T15 In discussion with Howard Morris after my lecture, it Sir Arnold Burgen, the director of the National became apparent that the four residues at the N-terminus of Institute for Medical Research (NIMR), Mill Hill, London, the Leu-enkephalin and Met-enkephalin peptides which had at once arranged a meeting with Kosterlitz and two of been identified corresponded to this N-terminal sequence of his colleagues, John Hughes and Howard Morris, and the C-fragment of lipotropin, pointing to a structural and myself, to take place in his office at the NIMR. A few days possibly functional relationship between these peptides. later, Kosterlitz and Hughes came down from Aberdeen Morris told me that he was involved in an important to London for the meeting, and we discussed the but confidential collaboration which he was not free to background and progress made in their identification of discuss, but the next morning Hans Kosterlitz, the head of the enkephalins and our investigation of the C-fragment a pharmacology group in Aberdeen, Scotland, telephoned of lipotropin. It was clear that there was an unexpected to let me know what the secrecy was about. but highly significant overlap in our research, so it was Journal of Molecular Endocrinology Figure 2 Rate of release of N-terminal tyrosine from β-endorphin and structurally related synthetic peptides. The peptides (0.34 mM) were incubated at 37˚C with aminopeptidase M (lower figure, 0.02 mg/mL) or with a preparation of an aminopeptidase from rat brain (upper figure) in 0.1 M ammonium bicarbonate at pH 7.4. dl-Norleucine was included as an internal standard. At intervals, aliquots of the solutions were added to 1 mL of 0.2 M citrate at pH 2.2 and the liberated tyrosine was determined by amino acid analysis. Reprinted from Biochemical and Biophysical Research Communications, 76, Austin BM & Smyth DG, The NH2-terminus of C-Fragment is resistant to the action of aminopeptidases, 476–482.

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