Ductus ejaculatorius peptide 99B (DUP99B), a novel Drosophila melanogaster sex-peptide pheromone Philippe Saudan 1 , Klaus Hauck 1 , Matthias Soller 1, *, Yves Choffat 1 , Michael Ottiger 1 , Michael Spo¨ rri 1 , Zhaobing Ding 1 , Daniel Hess 2, †, Peter M. Gehrig 2 , Stefan Klauser 2 , Peter Hunziker 2 and Eric Kubli 1 1 Zoologisches Institut Universita ¨ tZu ¨ rich-Irchel, Zu ¨ rich, Switzerland; 2 Biochemisches Institut der Universita ¨ tZu ¨ rich-Irchel, Zu ¨ rich, Switzerland We have characterized a g lycosylated, 31 amino-acid pep- tide of 4932 Da isolated from Drosophila melanogaster males. The mature p eptide contains a sugar moiety of 1184 Da a t a ND T c onsensus g lycosylation site and a disulfide bond. It is synthesized in the male ejaculatory duct via a 54 a mino-acid precursor containing an N-terminal signal peptide and Arg-Lys at the C-terminus which is cleaved off during maturation. The gene contains an intron of 53 bp and is localized in the cytological region 99B of the D. melanogaster genome. The peptide is therefore named DUP99B (for ductus ejaculatorius peptide, cytological localization 99B). The C-terminal parts of mature DUP99B and D. melanogaster sex-peptide (ACP70A) are highly homologous. Injected into virgin females, DUP99B elicits the same postmating responses as sex-peptide (increased oviposition, reduced receptivity). These effects are also induced by de-gl ycosylated native peptide or synthetic DUP99B lacking the sugar moiety. Presence of the glycosyl group, however, decreases the amount needed to elicit the postmating responses. Homologies in the coding regions of the two exons of DUP99B and sex-peptide, respectively, suggest that the two genes have evolved by gene duplication. Thus, we consider these two genes to be members of the new sex-peptide gene family. Keywords: Drosophila melanogaster; ductus ejaculatorius; oviposition; receptivity; sex-peptides. In many insects the reproductive behaviour of females is influenced by peptides and other substances synthesized in the male genital tract. During mating they are transferred into the female with the seminal fluid (reviewed in [1–4]). Drosophila melanogaster sex-peptide (SP; ACP70A) is one of the well characterized peptides affecting female repro- ductive behaviour [5,6]. Sex-peptide is 36 amino acids in length and is synthesized in the male accessory glands [5]. It causes an increase in oviposition and reduction of receptivity (readiness to mate, [7,8]), two postmating responses observed in females of many insects [1–3]. As SP is synth esized in the m ale and acts in the female, it can be considered as a sex-pheromone as defined by Karlson and Lu ¨ scher [9]. Recently, juvenile hormone was found to induce increased e gg production comparable to SP [10,11]. In accord with this finding, SP stimulates juvenile hormone synthesis in corpora allata/corpora c ardiaca complexes isolated from sexually mature virgins [12,13]. Thus, the corpus allatum might represent one target of SP in vivo. However, other targets must exist, as application of the juvenile hormone analogue methoprene neither elicits oviposition nor reduces receptivity [11]. Indeed, Ottiger et al. [ 14] have identified high affinity binding sites for SP in the central and peripheral n ervous system and also in t he genital tract. Microcautery of the pars intercerebralis, a neuroendocrine centre of the insect brain rich in peptides, inhibits oviposition of mated females, suggesting the involvement of o ther peptides in inducing oviposition [15,16]. Although SP is sufficient to elicit the t wo postmating responses, it is not known whether it is also necessary. Therefore, we have initiated a search for peptides that can also induce the two postmating responses and that may act in parallel with or downstream of SP. In this paper, we report on the isolation and characteri- zation of a closely related peptide which also elicits the two postmating responses when injected into the hemolymph of virgin females. We have isolated and sequenced parts of the peptide and the corresponding cDNA, together with the complete gene. T he expression of the gene w as studied in both sexes by Northern blot analysis and who le mount in situ hybridization. The gene was named Dup99B, reflecting the site of expression and its localization at t he cytological locus 99B. Correspondingly the peptide is named DUP99B according to the standard Drosophila nomencla- ture. Based on the homologies o f DUP99B and SP in the Correspondence to E. Kubli, Zo o logisches Institut, U niversita ¨ tZu ¨ rich- Irchel, Winterthurerstrasse 190, CH-8 057 Zu ¨ rich, Switzerland. Fax: + 411 635 5909, Tel .: + 411 635 4892, E-mail: ekubli@zool.unizh.ch Abbreviations: SP, sex-peptide; DIG, digoxygenin; ED-OSS, ejaculatory duct ovulation stimulating substance. Note: The SWISS-PROT accession numbers for the sequences dis- cussed in t his paper are: DUP99B, P 81160 DUP99B DEJP-DROME; Sp, p 05623; sp swall, a70a_drome. Note: P. Saudan and K. Hauck contributed equally to this work. *Present address: Biology Department and Center for Complex Systems, Brandeis University, 415 South St, Waltham 02454, USA. Present address: F riedrisch Miescher Institut, PO Box 2543, CH-4002 Basel, Sw itz er la nd . (Received 10 September 2001, revised 6 December 2001, accepted 11 December 2001) Eur. J. Biochem. 269, 989–997 (2002) Ó FEBS 2002 signal sequences of their precursors and in the C-terminal parts of t he mature peptides, we consider the two peptides to be members of a new sex-peptide pheromone gene family. MATERIALS AND METHODS Fly stocks and bioassays for ovulation and receptivity Wild-type Oregon R flie s were bred in large quantities in plastic boxes on standard food at 25 °C [10]. Injection assays were performed on sexually mature 5-day-old virgin females as d escribed by Schmidt et al.[8]. Peptide isolation Flies of 13 to 14-days-old were collected, frozen in liquid nitrogen, vigorously shaken, and fractionated into heads, appendages, and abdomen + thorax, respectively, by siev- ing through nylon nets of different mesh sizes ( 800 and 400 lm, respectively). Separated heads (100 g per isolation) were homogenized and extracted with 80% m ethanol. DUP99B was isolated by sequential passage of the boiled extract over an anionic exchange column (50/20 Pharmacia; Acell Plus QMA) eluted with a gradient from 0 to 1molÆL )1 NaCl in 25 mmolÆL )1 Tris/HCl pH 8.45, and four distinct RP-HPLC columns: a Brownlee Aquapore C-8 column, 10 (i.d.) · 220 mm, eluted with a 0–95% acetonitrile (MeCN, Biosolve) gradient in 0 .05% trifluoro- acetic acid (Pierce); a Brownlee C-18 column, 10 (i.d.) · 220 mm, eluted with 0–80% MeCN in 0.1% heptafluorobutyric acid (Pierce); a Vydac C-8 pH-stable column, 4.6 (i.d.) · 220 mm, eluted with 0–80% MeCN in 0.1% ammonium acetate; and a Vydac C-18 column, 2.1 (i.d.) · 220 mm, eluted with 0– 95% MeCN in 0.05% trifluoroacetic acid. Active fractions were identified after each step by injection of aliquots into sexually mature, virgin females subsequently bio-assayed. Purity was checked by an API III + electrospray ionization triple- quadrupole mass spectrometer (Sciex). Enzymatic digestions Endo-Lys-C digest: 400 pmol DUP99B was denatured in 50 mmol ÆL )1 Tris/HCl pH 8.5, 3 molÆL )1 guanidine-HCl, 5mmolÆL )1 dithiothreitol and digested with 0.1 lg Endo-Lys-C (Boehringer Mannheim) in Tris/HCl (pH 8.5, 25 mmol ÆL )1 )/1 mmolÆL )1 EDTA for 12 h at 37 °C. The reaction was s topped by adding trifluoroacetic acid. Chymotrypsin digest: 2 lg DUP99B was digested with 20 ng chymotrypsin (Boehringer Mannheim) in 100 mmolÆL )1 Tris/HCl (pH 7.8), 10 mmolÆL )1 CaCl 2 for 6hat25°C. Asp-N digest: 1.1 lg DUP99B w as digested with 4% (w/w) Asp-N (Boehringer M annheim) a t 3 7 °Cfor 6h in 60lLNH 4 HCO 3 buffer (10 mmolÆL )1 ,pH7.8) under Argon. Reduction and S-carboxamidomethylation. Deglycosylation with N-glycosidase A The C-terminal peptide fragment resulting from the diges- tion of DUP99B with A sp-N was dissolved in 70 lL Tris/ HCl buffer (Sigma; 20 mmolÆL )1 , pH 8.4), reduced and S-carboxamidomethylated by a 200-fold excess of Tris 2-carboxyethylphosphine hydrochloride (Pierce) and a 500-fold excess of iodoacetamide (Fluka). The mixture was incubated under Argon in the dark for 2 h at room temperature. DUP99B (1.2 lg) was digested with 0.5 mU N-glyco- sidase A (Boehringer Mannheim) in sodium acetate buffer 10 mmol ÆL )1 pH 5.1, for 24 h at 37 °C. Mass spectrometry Peptides obtained b y e nzymatic digestion o r c hemical modification were separated and analysed by LC-MS. For reversed-phase chromatography, a Vydac C8 column, 1 (i.d.) · 250 mm, was u sed at a flow rate of 50 lLÆmin )1 and the effluent was monitored at 215 nm. Solvent A was 0.1% trifluoroacetic acid (v/v); solvent B c ontained 0.09% trifluoroacetic acid (v/v) in 80% MeCN. After elution with 5% solvent B for 5 min, a gradient of 5–60% solvent B was applied for 60 min. The HPLC effluent was split, and  90% was collected for further analyses. The remaining 10% was directed on-line into the API III + mass spec- trometer for molecular mass determinations. A mass range from 300 Da to 2000 Da was scanned with a step size of 0.25–0.5 Da and a scan duration of 4–5 s. The t andem m ass spectrum of the glycopeptide resulting from endoprotease Lys-C digestion was obtained by mass-selection of t he triply charged precursor ion and collision-induced dissociation with Argon. Amino-acid analysis and Edman sequencing The amino-acid composition of the entire DUP99B and of selected fragments was determined u sing two different amino-acid analysers ( Amino Quant, Hewlett Packard; 420 A D/H Applied Biosystems). Sequence determinations by automated Edmann degra- dation were carried out on a model 477A sequencer (Applied Biosystems) equipped with an online phenyl- thiohydantoin amino-acid analyser (Model 120A , Applied Biosystems). PCR and cloning of the Dup99B genomic region PCR of genomic DNA (50 ng in 25 lL) was performed with AmpliTaq (Perkin E lmer) according to the manufac- turer’s instructions. Degenerate primers (25 pmol) were: SP(I-D), 5¢-CGGAATTCATHCARAGYCARAARGA-3¢; SP(R-C), 5¢-CGAATTCGNGARAARTGGTG-3¢ and AS (G-G), 5¢-GGAATTCCCCICCIARRTAIGGICC-3¢). Amplifications were carried out for 36 cycles (93 °C 60 s, 54 °C60s,72°C 60 s). Verification of genomic Dup99B sequences was carried out with primers GD6 (5¢-ATT CCAGTACAATTAGCTAGTTG-3¢)andGD7 (5¢-AG GAGTGTGCAATTTCTAAGG-3¢) for 30 cycles (94 °C40s,58°C60s,72°C 60 s). A mplification from a k-cDNA library (a gift of R. Graf [17]) was performed with primers AS(Y-R) (5¢-CGAATTCTAGGGGCCTAAGTT TAGCCG-3¢), AS(L-E) (5¢-CGAATTCAAGTTTAGCC GGCA CCACTTC-3¢), k-1 ( 5¢-ATTAACCCTCACTAAA GGGA AC-3¢)andk-2 (5¢-CCGCTCTAGAACTAGTGG ACT-3¢)on1.5lL of library (1.1 · 10 9 pfuÆmL )1 )anda subsequent nested PCR on 1/10 thereof for 3 7 cycles (93 °C 40 S , 58 °C60s,72°C 60 s ) w ith an initial 5 min denatur- 990 P. Saudan et al. (Eur. J. Biochem. 269) Ó FEBS 2002 ationstepat74°C. Products were cloned and sequenced according to standard methods [18]. Genomic Dup99B sequences were identified from P1 clones obtained from the European Drosophila Genome Project, subcloned, and sequenced according to standard methods [18]. P1 clones positive for Dup99B were DS07294, DS00322 and DS02922. In situ hybridization to polytene chromosomes. Northern-blot analysis and tissue in situ hybridization In situ hybridization to polytene chromosomes using digoxygenin (DIG)-labelled probes of genomic clones containing  600 bp of the promoter re gion were performed as describ ed by Langer-Safer et al. [19] with the following modifications: DNA was D IG-labelled using the D IG High Prime labeling Kit (Boehringer Mannheim), hybrids were detected by using antidigoxigenin-pod fab-fragments (Boehringer Mannheim) with diaminobenzidine as substrate. RNA was prepared according to the method of Chom- czynski and Sacchi [20], separated on 1% formaldehyde agarose gels [18] and blotted onto Geenscreen Plus (NEN) by vacuum blotting (Vacugene XL, Pharmacia). Filters were hybridized (50% formamide, 6 · SSPE, 5 · Den- hardt’s solution, 0.5% SDS, 0.1 mgÆmL )1 salmon sperm DNA, 10% dextransulfate) either with a 140 nucleotide Dup99B cDNA probe, a 200 nucleotide sex-peptide cDNA probe or with a fragment from the Drosophila ribosomal protein 49 (rp49) gene [21], which was random prime labelled according to the instructions of the manufacturer (Pharmacia). Exposed filters were analysed by a phospho- image system (Molecular Dynamics). Whole mount in situ hybridizations to male abdomens and brains were performed according to the protocol of Tautz and Pfeifle [22] by using antisense DIG labelled in vitro transcripts performed according to the instructions of the manufacturer (Boehringer Mannheim). As control for hybridization, DIG-labelled sense in vitro transcripts were used. RESULTS DUP99B elicits the two postmating responses when injected into virgin females The peptide was initially isolated from D. melanogaster heads during a search for oviposition-stimulating substances possibly localized in the pars intercerebralis of adult flies, a brain region known to house a variety of neurosecretory cells containing numerous neuropeptides [23–25]. Substan- ces eliciting oviposition were found in extracts from both sexes. However, the active component of th e female extract elutes in a different fraction and is unstable. It has not been characterized at a molecular level. Hence, we decided to isolate the active principle from male heads. Peptide extracts prepared from D. melanogaster male heads were fractionated by FPLC and HPLC, and subse- quently injected into sexually mature, virgin females for an oviposition assay [8]. One fraction induced oviposition reproducibly to the same degree as injected synthetic SP used as a control (Fig. 1). The same fraction was a lso able t o reduce the receptivity of virgin females [26]. The peptide purified from the active fraction has a molecular mass of 4932 Da (SP: 4428 Da [5]); and an amino-acid composition different from that of SP (data not shown). About 1500 pmol DUP99B were isolated from 100 g male heads. In later s tages of the project, after showing that the DUP99B gene was t ranscribed in the ductus ejaculatorius, the peptide was isolated from abdomen of mass-reared flies of both sexes. Calculations reveal that  25 nmol DUP99B can be isolated from 100 g of abdomen. The molecular properties of DUP99B are independent of the source of the peptide. DUP99B is a glycosylated peptide of 31 amino acids The sequence of the peptide was determined in three steps. First, we sequenced several C-terminal p eptide fragments. This information was used to design appropriate primers to isolate and sequence a part of the genomic DNA. Primers derived from the genomic sequence were then used to i solate cDNAs which served to derive the N-terminus of the peptide and thus to complete the sequence. The final results are presented in Figs 2 a nd 3. To purify native DUP99B (nDUP99B) for sequencing purposes, peptide extracts were prepared from heads of mass-reared adult flies o f both s exes. Native DUP99B was isolated by subsequent ru ns of peptide extracts on FPLC- and HPLC-column. As the intact peptide was resistant t o Edman degradation, it was digested with various proteases. The resulting fragments were analysed by LC-MS and selected peptide fragments were subjected to Edman degra- dation (Table 1). The longest continuous amino-acid sequences were obtained from two chymotryptic fragments of 1317 Da and 1419 Da and from a 2250-Da fragment of the Asp-N digest. The cysteines in the Asp-N fragment had been reduced and carboxamidomethylated, allowing detec- tion of the two cysteine residues by Edman sequencing. In addition, reduction and S-carboxamidomethylation of this fragment resulted in a mass increase of exactly 116.2 Da , which implies that the cysteines of the un modified peptide form a disulfide bridge. Confirmatory evidence for the presence of a disulfide bond was obtained from a 1437-Da fragment of the chymotryptic digest (Table 1). Edman sequencing clearly indicated that this fragment consisted of two peptide chains linked by a disulfide bond. Several other relatively abundant peptides of various digests Fig. 1. Purification of the peptide DUP99B. HPLC chromatogram of a crude male head extract after FPLC-fractionation a nd results of injections into females ( shaded columns). O viposition is strongly stimulated by a f raction elu ting at 54 min. The other fraction s d o not stimulate oviposition above the background egg-laying rate. Ó FEBS 2002 DUP99B, a novel Drosophila sex-peptide (Eur. J. Biochem. 269) 991 were found to b e inaccessible to E dman degradation, apparently due to a modified N-terminus. Taken together the re sults of all digests y ield a sequence of 23 amino acids representing the C-terminal end of the mature peptide (Figs 2A and 3 ). Based on this peptide sequence, degenerated oligonucleotides were designed to PCR-amplify a sequence from genomic DNA. The PCR products were cloned and sequenced. This latter sequence was used to derive a nondegenerated, unambiguous primer for t he isolation of cDNAs coding for the N-terminus of the peptide. Together with a k-primer this oligonucleotide was used to PCR-amplify a partial cDNA sequence of Dup99B from a k-ZAP-cDNA library prepared from fractionated heads of both sexes as starting materi al. The sequence of the isolated Dup99B cDNA revealed an open reading frame encoding a signal peptide and the N-terminus of the mature DUP99B peptide. Mature DUP99B contains the sequence NDT, a consensus site for N-glycosylation. The existence of the modification was also suggested by t he fact that the measured molecular mass o f isolated DUP99B (4932 Da) did not fit any calculated mass of the peptide fragments deduced from the DNA sequence. The nature of the modification and the ambiguity o f the signal peptide c leavage site was resolved by d e-glycosylation of mature DUP99B. As we suspected a1–3 fucosylation of the asparagine-linked G lcNAc, which had been shown to inhibit de-glycosylation of g lycopeptides or glycoproteins by N-glycosidase F [27], N-glycosidase A was used for de-glycosylation. The molecular mass of de-glycosylated DUP99B was determined as 3748 Da by LC-MS, leaving 1184 Da for the sugar moiety of the molecule. The structure of the N-glycan is described below. It follows from comparison of the molecular mass of de-glycosylated DUP99B with masses p redicted from the cDNA and gene sequences that the s ignal peptide contains 21 amino acids and that the mature DUP99B peptide starts with a pyroglutamic acid at its N-terminal end. The conversion of the N-terminal glutamine to pyroglutamic acid explains why intact DUP99B peptide as well a s several N-terminal proteolytic fragments thereof were r esistant to Edman degradation. Although the cDNA is not complete at its 3¢ end, in combination with the results from the peptide sequence analysis mentioned above, and sequencing of genomic DNA (see below), we conclude that the mature DUP99B peptide contains 31 amino acids (Figs 2 A and 3). Structure of the glycosyl group The mass of the N-linked oligosaccharide moiety ( 1184 Da) indicates the presence of two N-acetylhexoses, three hexoses and two fucoses. All N-linked glycans share the common core structure Mana1–3(Mana1–6)Manb(1–4)GlcNAcb1– 4GlcNAc-Asn. On e fucose residue in a1–6 linkage to the innermost N-acetylglucosamine residues is a commonly found substituent, while the presence of a second fucose residue is rather unusual. In order to establish the linkage positions of the fucoses, the 2815 Da fragment from the endoprotease Lys-C digest containing the glycan was subjected to MS /MS a nalysis. The fragment ions produced by the glycopeptide were derived predominantly from cleavage of the glycosidic l inkages with charge retention on the peptide. Fragmentation of the p eptide moiety was minimal. The MS/MS spectrum exh ibited various fragment ions consisting of the peptide and p arts of the glycan which are consistent with the glycan structure shown in Fig. 3. A doubly charged fragment ion at m/z 1064 corresponds to the peptide containing one acetylglucosamine and two fucose residues, implying that both fucoses are attached to the asparagine-linked N-acetylglucosamine. The precursor peptide of DUP99B contains a signal peptide and two additional amino acids at its C-terminus. The gene contains an intron at the same site as the sex-peptide gene As the isolated Dup99B cDNA terminated prematurely, we decided to clone the genomic regions. First, Dup99B was cytologically localized on polytene salivary gland chromo- somes prepared from D. melanogaster larvae. Only the cytological region 99B was labelled (Fig. 4G), suggesting that the Dup99B sequence is localized at only one site in the D. melanogaster genome. Subsequently, P1 clones f rom this cytological r egion w ere screened f or Dup99B and the genomic region was cloned and sequenced. Fig. 2. Sequence of the DUP99B precursor peptide and the Dup99B gene. DUP99B i s synthesized via a precursor peptide with a 21-amino- acid signal peptide and tw o additional amino-acid r esidues at the C-terminus that are cleaved off during the pe ptide maturation process. (A) Seq uence of the DUP99B pre cursor peptide and c omparison w ith the sex-peptide (SP) precurso r. Identical amin o acids are indicated by vertical bars. Filled triangles, sites of c leavage of signal pe ptides; open triangles, sites of insertion of the introns in the genomic sequence; filled arrow, cleavage site of the two C-terminal amino-acid residues of the DUP99B precursor; open arrow, g lycosylation site in the mature DUP99B peptide; filled circle, pyroglutamine; overlined amino acids, glycosylation consensus sequence; s tars, hydroxyprolines. (B) Com- bined Dup99B cDNA and gene seq uence. Underlined, intron sequence; asterisk, stop codon. 992 P. Saudan et al. (Eur. J. Biochem. 269) Ó FEBS 2002 The sequence of the genomic DNA reveals an open reading frame encoding 54 amino acids (Fig. 2B). Hence, DUP99B is synthesized via a precursor peptide with a 21 amino-acid signal peptide and two additional amino-acid residues (RK) at the C-terminus. As we found two additional amino acids encoded at the C-terminus which were not present in the purified pep tide, we PCR-amplified genomic Dup99B DNA from t he stock that was originally used for purification of the peptide. This DNA does also encode the two add itional amino acids (RK), hence they must be cleaved off during maturation. These sequence d ata were later confirmed by the sequences published by the Drosophila Sequencing Project [28]. A comparison with the SP gene [29] shows that an intron is localized in the two genes at exactly the same site. On the protein level a high homology with SP is found in the N-terminal parts of the signal sequences and the parts encoded by the second exon of each gene (Fig. 2A). The Dup99B open reading frame, however, codes for two additional amino acids at its 3¢ end which are not encoded in this part of the SP gene [29]. The C-terminal parts of the signal peptides and the N-terminal parts of the mature peptides differ in most amino acids. Dup99B is expressed in the ejaculatory duct of the male The site of expression of the Dup99B gene was determined by Northern-blot analysis with RNA isolated from male heads, thoraces, and abdomen. As a control we extracted RNA from heads and whole wild-type virgin females, and, furthermore, from virgin females of a transgenic line. The latter strain contains a transgenic SP gene expressed under the contr ol of a yolk protein 1 promoter [7], i.e. SP is constitutively expressed in the fat body of adult females. As probes we used random primed, radioactively labelled Dup99B cDNA. cDNAs coding for SP and D. melanogaster ribosomal protein 49 (rp 49) were used as loading controls. With the Dup99B probe the s ignal is seen only in the lanes containing RNA isolated f rom male abdomen or total male RNA (Fig. 4 A–C). As e xpected the SP probe lights up t he lanes containing RNA from wild-type m ales and the transgenic females (due to the p resence of f at body tissue in the head of adults, SP is also expressed in the head in this line), and the rp 49 probe all lanes. We conclude that the Dup99B gene is transcribed in the male abdomen. ThesiteofDup99B transcription in the male abdomen was determined by whole mount in situ hybridization of dissected male abdomens with a DIG-labelled Dup99B probe. Strong staining was found in the ejaculatory duct (Fig. 4 D–F). The staining is cytoplasmic. As the peptide was initially isolated from male heads, male brains were also investigated. However, no signal was detecte d with this method in whole mount incubations (results not shown). Synthetic, un-glycosylated DUP99B elicits the two postmating responses. The presence of the glycosyl group reduces the critical concentration in a bioassay The biolo gical activity of t he peptide lacking the glycosyl group was d emonstrated by injecting synthetic DUP99B (sDUP99B, s for synthetic; sDUP99B is not glycosylated) into the hemolymph of sexually mature, virgin females. Both postmating responses are elicited by this peptide as with the native DUP99B (nDUP99B) purified from adult flies (Fig. 5 [14,26]);. The same results were also obtained by injecting enzymatically de-glycosylated nDUP99B. Because only little material was obtained after de-glycosylation we tested only ovulation. Eighty per cent ovulation was observed 3.5 h after injection of 2 pmol de-glycosylated nDUP99B. Therefore, in the bioassay, stimulation of t he postmating responses does not depend on th e presence of the glycosyl group. The influence of the glycosyl group was further investi- gated by determining the critical concentration needed to induce the two postmating responses by nDUP99B and sDUP99B, respectively (Fig. 5). Significantly different criti- cal concentrations are needed to elicit 50% ovulation: 0.6 pmol f or sDUP99B/female (the corresponding value for Fig. 3. Compilation of known and assumed functions of DUP99B and sex-peptide. Results from in vitro and in vivo experiments. Som e functions may be shared by the two peptides but based on different structures, some may be p erformed by bo th peptides with almost identical structures, and some functions are unique to sex-peptide. Table 1. Molecular masses and amino-acid sequences of proteolytic fragments of DUP99B. Capital letters indicate amino acids identified by Edman sequencing, and s mall letters denote amino acids derived from th e DUP99B g ene sequence (Fig . 2). The cysteine residues in the Endo-Asp-N fragment are carboxamidomethylated; the cysteines in all other peptides form a disulfide bond. The fragment of m ass 1437 D a consists of two peptides linked by a d isulfide bond. <q ¼ pyroglutamine. Digest Measured mass [Da] Calculated mass [Da] Amino-acid sequence Chymotrypsin 1316.8 1316.7 IQSQKDREKW 1419.0 1418.7 cRLNLGPYLGGRc 1437.2 1436.7 cRLNLGpy LGGRc Endo-Asp-N 2250.3 2250.6 DREKWCRLNlGPYLGGRC Endo-Lys-C 2815.4 2815.8 <qdrndtewiqsqk 1604.8 1604.8 wcrLNLGPYLGGrc Ó FEBS 2002 DUP99B, a novel Drosophila sex-peptide (Eur. J. Biochem. 269) 993 Fig. 4. The Dup99B gene is expressed in the ductus ejaculatorius of the male genital tract, and the gene is localized at the c ytological region 99B. (A–C) Northern-blots with (A) Dup99B cDNA (B) Sex-peptide cDNA, and (C) ribosomal protein 49 (rp49)cDNAas probes.H,RNAextractfromheads;T,from thoraces;A,fromabdomen;X,fromwhole body extracts. #,males;$,matedfemales; , virgin females. Mated female RNA s were extracted from a transgenic stock expressing the sex-peptide gene in the fat body (see Materials and methods). Molecular mass markers are on the left side of the figure. (D) Whole mount in s it u hybridization of a DIG- labelled Dup99B probe to m ale genital tracts. ag, Accessory glands; be, bulbus ejaculatorius; de, ductus ejaculatorius; te, testes. Scale bar ¼ 0.2 mm (E) Same as (D) but region of ductus ejaculatorius enlarged. Scale bar ¼ 0.01. (F) DAPI staining of nuclei of the ductus ejaculatorius. Note the large cells in the upper part of the ejaculatory d uct. Same region as shown in (E). (G) In situ hybridiz a- tion of a DIG-labelled genomic clone of Dup99B to polytene salivary gland chromo- somes. Th e probe labels the c ytological region 99B. Fig. 5. Dose–response of native and synthetic DUP99B. The critical concentration need ed to elicit ovulation is lower fo r native D UP99B (n DUP) than synthetic DUP99B (sDU P; Probit analysis in SPSS [45]: Chi-squared parallelism test ¼ 1101.162; DF ¼ 1; P < 0.0001). The values for the receptivity response are not significantly different (Chi-squared parallelism test ¼ 0.00 0; DF ¼ 1; P ¼ 1.000). Each point represents the mean ± SD of thre e experiments with at least 2 0 virgin females in each experiment. (A) Ovulation response (% of females ovulating). (B) Receptivity response (% of the total females mated). Native DUP99B contains a pyroglutamic acid at its N-terminal end and is glycosylated. Synthetic DUP99B contains a pyroglutamic acid at its N-terminal end but is not glycosylated. 994 P. Saudan et al. (Eur. J. Biochem. 269) Ó FEBS 2002 SP is 0.6 pmol per female for all responses [8]), and 0.2 pmol nDUP99B per f emale. The c ritical c oncentrations needed to reduce the receptivity are not significantly different. This discrepancy c ould b e due to the fact that the ovulation bioassay is more ÔrobustÕ than the receptivity assay. Thus, nDUP99B, when injected into virgin females, does induce ovulation at lower concentrations than sDUP99B and SP. DISCUSSION Sex-peptides The DUP99B purification scheme was b ased on the SP bioassay as a functional test, hence, it is not surprising to find nearly identical sequences in the C-terminal parts of the two mature peptides (Fig. 2A). Indeed, this part of SP had been shown to be essential to e licit the two postmating responses [8,14]. It is also c onserved in S P sequences of other Drosophila species [30–32] (T. Schmidt & E. Kubli, unpub- lished data). However, the N-terminal parts of the mature peptides are different. Comparison of the amino-acid sequence and composition of the matu re DUP99B peptide with the gen omic sequence revealed that DUP99B is synthesized via a 54-amino-acid precursor with a signal peptide of 21 amino acids (Fig. 2A). Furthermore, an arginine and a lysine residue are cleaved off from the C-terminal end of the precursor, i.e. the mature peptide contains 31 amino acids. Sex-peptide is synthesized via a 55-amino-acid precursor containing a cleaved off signal peptide of 19 amino acids, and the C-terminal end is not processed. The secreted mature SP contains 36 amino acids. Both genes contain one intron inserted at the s ame s ite. The second exon encodes the conserved C-terminal regions. Evolutionarily, these findings could be interpreted as a sign of exon shuffling. H owever, sequence homologies are also found in the N-terminal parts of the precursors. In the signal peptide of DUP99B, 10 out of 12 amino acids are homologous to the corresponding SP signal sequence (Fig. 2A). I t is unlikely that this fact is due solely to the general hydrophobicity of the a mino acids characteristic f or signal s equences. Therefore, we suggest that Dup99B and SP evolved from a common a ncestor gene, and we consider the genes coding for DUP99B and SP a s members of a new SP gene family. Although the sequence of the Dup99B gene is included in the DNA sequence published b y the Drosophila Genome Project [28], it was not identified as a gene. This is probably due to the fact that the p rotein identification programs used have difficulty in finding genes encoding small peptides. Indeed,  8% of the peptides encoded by a male accessory gland cDNA library were mis sed as peptide coding genes by the Drosophila Genome Sequencing Project [33]. Thus, identification of peptide coding genes via cDNA libraries, or biochemical isolation and characterization of peptides, have not become redundant in the age of genomics [34]. The m ature p eptides differ in s everal respec ts. T he N-terminus of DUP99B is blocked by pyroglutamic acid, and a glycosyl group is located in the N-terminal region of the peptide. The structure of the latter (Fig. 3) corresponds to a particular difucosylated oligosaccharide structure described for honeybee venom phospholipase A [35] and for membrane glycoproteins from three lepidopteran cell lines [36]. Fucose residues were found in a1–3 and in a1–6 linkages to the innermost N-acetylglucosamine of the Man 3 GlcNAc 2 core [35,36]. Native DUP9 9B induces ovulation a t l ower concentrations than sDUP99B and SP. Thus, the glycosyl modification could increase the stability of the peptide and/or increase the affinity of the native peptide for the putative receptor(s). Sex-peptide contains five hydroxyprolines an d, probab ly, a hydroxylated leucine residue [5]. However, biological functions have not been assigned for any of the SP modifications [5,8]. DUP99B was i nitially isolated from male head extracts (see Materials and methods). Northern-blots and whole mount in situ hybridization revealed expression of the Dup99B gene in the ductus ejaculatorius (Fig. 4). A recent promoter analysis [37], with lacZ as a reporter g ene, also yielded a strong expr ession in the ejaculatory duct, confirming our results. However, Rexhepaj [37] found that the reporter gene was also expressed in t he cardia of both sexes. The cardia are regarded as having a dual function, being a sphincter to prevent regurgitation of the ventricular contents, and an organ producin g and moulding the peritrophic membrane. They are localized in the distal part of the oesophagus, but a thin sheet of cells extends into the proboscis [38]. Consistent with this localization, DUP99B synthesis in the head/thorax complex of males and females was demonstrated by the sensitive method of RT/PCR and with Western blots ([37]; J. Peng and E. Kubli, unpublished data). These findings explain the o ccurrence of t he peptide in male head extracts, the original source of the peptide. Thus, in contrast with the SP gene, Dup99B is expressed in both sexes [5,29,37] (A. Rexhepaj & E. Kubli, unpublished data). A peptide with strong homology to the C-terminal parts of DUP99B and SP has been isolated from the ejaculatory duct of D. biar mipes (ED-OSS, for ejaculatory duct ovula- tion stimulating substance [32]). As the N-terminal part of the mature ED-OSS does not show any homology to DUP99B or SP, it is not clear whether the encoding genes are homologous (the g ene encoding ED-OSS has not been isolated). However, both contain a glycosylation s ignal in this part of the peptide, but for ED-OSS it is not known whether it i s glycosylated in its native form. Interestingly, when ED-OSS is injected into the hemolymph o f virgin D. biarmipes female s, it stimulates oviposition only in certain strains. Imamura et al. [ 32] interpret this finding as a result of an ongoing conflict in reproductive interests between males and females. Are DUP99B and SP functionally redundant? Injection of DUP99B or SP into virgin females elicits the two postmating responses. Native, i.e. g lycosylated nDUP99B, induces the two responses at a lower critical concentration than sDUP99B or SP ( Fig. 5). Incubation of 125 I-iodinated peptides to cryostat sections of females revealed the same binding patterns. In adult females both, radiolabelled SP and sDUP99B, bind to peripheral nerves, the suboesoph- ageal ganglion, the c ervical connective, to discrete parts of the thoracic ganglion, and to t he genital tract [14]. These findings s uggest at least a partial redundancy concerning the functions of SP and DUP99B. However, both approaches may not reflect the in vivo situation, as it is not known whether the two peptides reach the same targets in vivo. Ó FEBS 2002 DUP99B, a novel Drosophila sex-peptide (Eur. J. Biochem. 269) 995 Because no null mutants are available for the two genes so far, the question of redundancy c an only be answered indirectly. One approach is to study i n females the effects of a copulation with males lacking accessory gland products. Two groups have constructed trangenic strains with males showing these properties [39,40]. Kalb et al. [39] produced transgenic males (DTA-males) which express the gene encoding the diphtheria toxin protein A under the control of a promoter active in male accessory glands. In these males the diphtheria toxin k ills the m ain cells of the accessory g lands, t hus none of their products are made. In some of the transgenic lines neither main cell products nor sperm are transferred. However, the ejaculatory duct of the DTA-males is intact and, thus, DUP99B should be synthesized, secreted, and transferred. Nevertheless, mating with DTA-males does not elicit any of the postmating responses in their mating partners. As esterases s ynthesized in the ejaculato ry duct of t he DTA- males are transferred into the reproductive tract of females [39] either DUP99B is not synthesized in the ejaculatory duct, DUP99B is not transferred without sperm, or DUP99B is transferred but has n o effect. T he results of Xue & Noll [40] described below support one of the first two interpretations. The pair-rule gene paired (prd) is necessary for the development of male accessory glands [40,41]. An early function of promoting cell p roliferation is necessary for accessory gland formation, and a late function of promoting cell differentiation is essential for accessory gland matura- tion [41]. paired mutants rescued to adulthood by a specific prd-rescue construct lack accessory glands completely. Hence, neither SP nor any other accessory gland product is transferred d uring mating. prdRes males have an intact ejaculatory duct [40], and, as Western blots have shown, they synthesize DUP99B [37]. Xue and Noll [40] reported that these males induce only an increase of o viposition, reduction of receptivity was not observed. However, a recent detailed behavioural analysis showed that after copulation with prdRes males, female s reject courting males vigorously [37]. Thus, the male genital tract content, and probably DUP99B, is transferred and able to initiate a reduction of female receptivity, along with a p artial increase of egg laying. Nevertheless, after 1 h of r ejection, all females have re-copulated, a s previously observed by Xue & Noll [40]. We conclude that only little DUP99B is transferred in this situation, and that in vivo DUP99B elicits both postmating r esponses. The interpretation of the experiments of Kalb et al. [39] a nd Xue & Noll [40] is complicated further by the fact that no accessory gland fluid is transferred by these transgenic males. The f unction(s) of the remaining seminal flu id might be affected by the lack of these components, some of them associated with sperm [42]. Support for a unique function of SP is provided by the experiments of Fan et al. [13]. Whereas SP can induce elevated juvenile hormone synthesis in isolated corpora allata/corpora cardiaca complexes, DUP99B cannot [13]. The responsible, active region of SP is the N-terminus of the mature peptide. It does not share any homology with DUP99B (Figs 2A and 3). T his region of SP is also conserved in S Ps of other Drosophila species [30–32] (T. Schmidt & E. Kubli, unpublished data). Thus, according to these in vitro experiments, SP has at least one function that cannot be performed by DUP99B. The first five a mino acids o f SP are also es sential for the binding of this peptide to sperm (S. Bu ¨ sser & E. Kubli, unpublished data; Fig. 3), neither the C-terminal part of SP nor full size DUP99B can compete with SP binding. DUP99B also binds to sperm (J. Peng & E. Kubli, unpublished data), possibly also with amino acids of the N-terminal region and, possibly, the glycosyl group. Thus, although both peptides bind to sperm, they do not use the same structures. Sex-peptide and DUP99B bound to sperm may b e the molecular basis of the s perm effect described by Manning [43,44]. After a mating with wild- type males the two postmating responses last for about 1 week. When sperm is not transferred in the seminal fluid, however, the p ersistence i s lost a nd the two responses fade away after 2 days [44]. H ence, the presence of sperm provides the persistency, possibly via bound SP and DUP 99B. A summary of known a nd putative functions for DUP99B and SP is presented in Fig. 3. Taken together the experimental evidence shows that some functions are unique to one peptide, and some functions may be shared by the two peptides but are based o n different structures. Finally, some functions may b e performed by both peptides with almost identical structures. ACKNOWLEDGEMENTS We thank R. Sack for amino-acid analysis, N. Birchler f or peptide sequence analysis, R. B ruggmann for help with cloning, W . Blancken- horn for statistical advice and help, and D. Hosken for comments on the manuscript. The European Drosophila Research project provided the P1 clones. 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Amplifications were carried out for 36 cycles. Ductus ejaculatorius peptide 99B (DUP99B), a novel Drosophila melanogaster sex -peptide pheromone Philippe Saudan 1 , Klaus Hauck 1 , Matthias Soller 1, *,