4465 The Journal of Experimental Biology 210, 4465-4470 Published by The Company of Biologists 2007 doi:10.1242/jeb.012088 Metabolic inactivation of the circadian transmitter, pigment dispersing factor (PDF), by neprilysin-like peptidases in Drosophila R. Elwyn Isaac1,*, Erik C. Johnson2, Neil Audsley3 and Alan D. Shirras4 1Faculty of Biological Sciences, University of Leeds, Leeds, LS2 9JT, UK, 2Department of Biology, Wake Forest University, NC, USA, 3Central Science Laboratory, Sand Hutton, York, YO41 1LZ, UK and 4Department of Biological Sciences, University of Lancaster, LA1 4YQ, UK *Author for correspondence (e-mail: [email protected]) Accepted 4 October 2007 Summary Recent studies have firmly established pigment confirming that such cleavage results in PDF inactivation. dispersing factor (PDF), a C-terminally amidated The Ser7–Leu8 peptide bond was also the principal octodecapeptide, as a key neurotransmitter regulating cleavage site when PDF was incubated with membranes rhythmic circadian locomotory behaviours in adult prepared from heads of adult Drosophila. This Drosophila melanogaster. The mechanisms by which PDF endopeptidase activity was inhibited by the neprilysin –1 functions as a circadian peptide transmitter are not fully inhibitors phosphoramidon (IC50, 0.15·␮mol·l ) and –1 understood, however; in particular, nothing is known thiorphan (IC50, 1.2·␮mol·l ). We propose that cleavage by about the role of extracellular peptidases in terminating a member of the Drosophila neprilysin family of PDF signalling at synapses. In this study we show that PDF endopeptidases is the most likely mechanism for is susceptible to hydrolysis by neprilysin, an endopeptidase inactivating synaptic PDF and that neprilysin might have that is enriched in synaptic membranes of mammals and an important role in regulating PDF signals within insects. Neprilysin cleaves PDF at the internal Ser7–Leu8 circadian neural circuits. peptide bond to generate PDF1-7 and PDF8-18. Neither of these fragments were able to increase intracellular cAMP levels in HEK293 cells cotransfected with the Drosophila Key words: pigment dispersing factor, neprilysin, peptidase, PDF receptor cDNA and a firefly luciferase reporter gene, neuropeptide, circadian rhythm. Introduction afternoon, respectively (Helfrich-Forster, 2005). Normal flies Drosophila melanogaster pigment dispersing factor (PDF; also show anticipation of morning and evening times, as NSELINSLLSLPKNMNDAamide), a member of the arthropod illustrated by initiation of locomotory activity before the lights pigment dispersing hormone/factor (PDH/PDF) family of are switched on and off. This circadian rhythm is maintained peptides, is the solely identified chemical output of clock when flies are transferred from a 12·h:12·h light:dark cycle to a neurons in Drosophila (Helfrich-Forster, 2005; Taghert and constant dark environment. The locomotory activity of pdf 01 Shafer, 2006). The importance of PDF as a neurotransmitter of null mutants is rhythmic under entraining schedules, but differs the circadian clock was suggested by experiments employing from wild-type behaviour in the loss of the anticipation of lights- immunocytochemistry, which showed that PDF is co-expressed on and in the advancement of the evening peak of activity (Renn with clock markers, specifically the period gene (Helfrich- et al., 1999). In constant darkness, pdf 01 flies have reduced Forster et al., 1998). Subsequent mosaic analysis has locomotory response to lights-on, and after 2 days become demonstrated that the LNv (ventral lateral neurons) subset of strongly arrhythmic (Renn et al., 1999). Further studies using pacemaker neurons, which express PDF, are required for the pdf 01mutants showed that PDF is required for synchronising rhythmic circadian locomotory behaviours in adult Drosophila a network of pacemaker cells in adult Drosophila (Lin et al., (Helfrich-Forster et al., 1998). Confirmation that the PDF 2004; Peng et al., 2003). The discovery of the PDF-receptor peptide is a circadian neurotransmitter came from studies of a gene (PdfR) and the behavioural analysis of PdfR mutants Drosophila mutant line (pdf 01) lacking PDF (Peng et al., 2003; confirmed the critical role of PDF signalling in maintaining Renn et al., 1999) and flies misexpressing pdf (Helfrich-Forster robust locomotory circadian rhythms (Hyun et al., 2005; Lear et al., 2000). Wild-type Drosophila entrained to a 12·h:12·h et al., 2005; Mertens et al., 2005). light:dark cycle display robust rhythmic locomotory behaviour There is much interest in understanding how PDF signals in comprising crepuscular (dawn and evening) peaks of activity a rhythmic manner and at what level this is regulated. Clock with periods of ‘sleep’ and ‘rest’ at night time and in the genes are known to control PDF expression, but this occurs at THE JOURNAL OF EXPERIMENTAL BIOLOGY 4466 R. E. Isaac and others a post-translational level, as neither PDF mRNA nor protein Materials and methods levels in the LNv cell bodies cycle (Park and Hall, 1998; Park Materials et al., 2000). Nevertheless, cycling of the mature PDF peptide PDF (NSELINSLLSLPKNMNDAamide) and recombinant has been observed in the terminals of the small LNv cells of human neprilysin were generous gifts from Professor D. Nässel, wild-type flies, suggesting that transport to and release from cell Department of Zoology, University of Stockholm, Stockholm, termini might provide a mechanism for the rhythmic action of Sweden and Professor A. J. Turner, Faculty of Biological PDF (Park et al., 2000). At present the importance of these Sciences, University of Leeds, Leeds, UK. Insect peptides were observations is not entirely clear since it has been shown that either purchased from Bachem (UK) Ltd, St Helens, UK, or some transgenic fly lines display wild-type-like locomotory were custom-synthesised by Pepceuticals, Nottingham, UK. rhythms, even in the absence of any cycling of PDF immuno- PDF1-7 and PDF8-18 were generated enzymatically by reactivity at small LNv cell termini (Kula et al., 2006). incubating PDF with recombinant human neprilysin. The two Therefore questions remain as to the biological mechanism fragments were purified and quantified by high-performance of how PDF functions as a circadian peptide transmitter in adult liquid chromatography (HPLC) and their purity checked by Drosophila. One aspect that has not received attention is the MALDI-MS. Other chemicals, including thiorphan and potential role of neuropeptidases in this mechanism, despite phosphoramidon, were purchased from Sigma Chemical Co., recent reports that several candidate peptidases either have been Poole, Dorset, UK. HPLC solvents were from Rathburns, shown to cycle or are transcriptionally regulated by components Walkerburn, Scotland, UK and the Jupiter 5·␮ column (C18, of the circadian clock (McDonald and Rosbash, 2001). Such 250·mmϫ4.5·mm i.d.) was purchased from Phenomenex, peptidases, typically with preferences for peptides of up to Macclesfield, UK. 4·kDa in size, are known to be important for terminating the signalling activity of neuropeptides at synapses. The best known Preparation of adult D. melanogaster head membranes of these is neprilysin (EC 3.4.24.11), a neutral zinc Drosophila melanogaster (w1118 strain) were cultured on a metallopeptidase with a broad tissue distribution in mammals standard oatmeal/molasses/agar diet at 25°C in a 12·h:12·h (Turner, 2004; Turner et al., 2001). It was first described as a light:dark cycle. Heads (10·mg) were collected from adult major protein of the brush border membrane of the kidney, but w1118 flies that had been frozen in liquid N2 and were was re-discovered as the brain enzyme responsible for the homogenised in 100·mmol·l–1 Hepes buffer, 100·mmol·l–1 NaCl degradation of enkephalin, hence its alternative name: (pH·7). The homogenate was centrifuged at 1000·g for 2·min enkephalinase (Matsas et al., 1985a). As a type II integral and the pellet rehomogenised in 100·mmol·l–1 Hepes buffer, membrane protein, neprilysin has its active site facing the 100·mmol·l–1 NaCl (pH·7) and subjected to a second low-speed extracellular milieu whilst anchored by an N-terminal centrifugation. The previously saved supernatants were cytoplasmic peptide and a transmembrane domain (Turner et al., centrifuged at 30·000·g for 1·h in an OptimaTM MAX Beckman 2001). Enzymatic activity is optimal at neutral pH, and is Coulter using a TLA110 rotor (Beckman Coulter, High inhibited by chelators of bivalent metal ions and by the Wycombe, Bucks, UK). The resulting membrane pellet was Actinomycetes product, phosphoramidon, and additionally by rehomogenised in a high-salt buffer (100·mmol·l–1 Hepes synthetic inhibitors such as thiorphan (Turner, 2004). Although buffer, 0.5·mol·l–1 NaCl, pH·7) and membranes recovered by a neprilysin cleaves a wide range of peptides, it shows specificity second 30·000·g centrifugation step. The final pellet was towards peptide bonds that use the amino group of an amino resuspended in 100·mmol·l–1 Hepes buffer, 100·mmol·l–1 NaCl, acid with a bulky hydrophobic side chain (Matsas et al., 1984; pH·7, using a glass homogeniser to give a protein concentration Turner, 2004). Neprilysin-like enzymes appear early in the of 350·␮g·ml–1. evolution of peptidergic signalling in animal nervous systems, which is consistent with a key role in regulating neuropeptide Hydrolysis of insect peptides activity (Turner et al., 2001). In insects, phosphoramidon- Peptide hydrolysis was performed by incubating sensitive neprilysin activity is enriched in neuropil regions of 100·␮mol·l–1 peptide with either human neprilysin (1.25·ng of the insect brain (Schistocerca gregaria, Locusta migratoria and protein) or D. melanogaster head membranes (1·␮g of protein) Leucophea maderae) and in neural membranes (Drosophila in a final volume of 20·␮l of 100·mmol·l–1 Hepes buffer, melanogaster, Musca domestica and Lymantria dispar) (Isaac, 100·mmol·l–1 NaCl, pH·7 at 35°C. Enzyme activity was stopped 1988; Isaac et al., 2002; Lamango and Isaac, 1993; Masler et by the addition of 5·␮l of 8% (v/v) trifluoroacetic acid and the al., 1996).