Báo cáo Y học: Ductus ejaculatorius peptide 99B (DUP99B), a novel Drosophila melanogaster sex-peptide pheromone docx

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.. It follows from comparison of the mole

Ductus ejaculatorius peptide 99B (DUP99B), a novel

Philippe Saudan1, Klaus Hauck1, Matthias Soller1,*, Yves Choffat1, Michael Ottiger1, Michael Spo¨rri1, Zhaobing Ding1, Daniel Hess2,†, Peter M Gehrig2, Stefan Klauser2, Peter Hunziker2and Eric Kubli1 1

Zoologisches Institut Universita¨t Zu¨rich-Irchel, Zu¨rich, Switzerland;2Biochemisches Institut der Universita¨t Zu¨rich-Irchel, Zu¨rich, Switzerland

We have characterized a glycosylated, 31 amino-acid

pep-tide of 4932 Da isolated from Drosophila melanogaster

males The mature peptide contains a sugar moiety of

1184 Da at a NDT consensus glycosylation site and a

disulfide bond It is synthesized in the male ejaculatory duct

via a 54 amino-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 melanogastergenome 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-glycosylated 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 melanogastersex-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 synthesized in the male 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 egg production comparable to SP [10,11]

In accord with this finding, SP stimulates juvenile hormone synthesis in corpora allata/corpora cardiaca 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 nervous system and also in the 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 other peptides in inducing oviposition [15,16] Although SP is sufficient to elicit the two 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 The expression of the gene was studied in both sexes by Northern blot analysis and whole mount

in situ hybridization The gene was named Dup99B, reflecting the site of expression and its localization at the cytological locus 99B Correspondingly the peptide is named DUP99B according to the standard Drosophila nomencla-ture Based on the homologies of DUP99B and SP in the

Correspondence toE Kubli, Zoologisches Institut, Universita¨t

Zu¨rich-Irchel, Winterthurerstrasse 190, CH-8057 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 this 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: Friedrisch Miescher Institut, PO Box 2543, CH-4002

Basel, Switzerland.

(Received 10 September 2001, revised 6 December 2001, accepted 11

December 2001)

signal sequences of their precursors and in the C-terminal

parts of the mature peptides, we consider the two peptides to

be members of a new sex-peptide pheromone gene family

M A T E R I A L S A N D M E T H O D S

Fly stocks and bioassays for ovulation and receptivity

Wild-type Oregon R flies 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 described 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% methanol

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

1 molÆL)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 mmolÆL)1Tris/HCl pH 8.5, 3 molÆL)1 guanidine-HCl,

5 mmolÆL)1 dithiothreitol and digested with 0.1 lg

Endo-Lys-C (Boehringer Mannheim) in Tris/HCl (pH 8.5,

25 mmolÆL)1)/1 mmolÆL)1EDTA for 12 h at 37°C The

reaction was stopped by adding trifluoroacetic acid

Chymotrypsin digest: 2 lg DUP99B was digested with

20 ng chymotrypsin (Boehringer Mannheim) in

100 mmolÆL)1Tris/HCl (pH 7.8), 10 mmolÆL)1CaCl2for

6 h at 25°C Asp-N digest: 1.1 lg DUP99B was digested

with 4% (w/w) Asp-N (Boehringer Mannheim) at 37°C for

6 h in 60 lL NH4HCO3 buffer (10 mmolÆL)1, pH 7.8)

under Argon

Reduction and S-carboxamidomethylation

Deglycosylation with N-glycosidase A

The C-terminal peptide fragment resulting from the

diges-tion of DUP99B with Asp-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 mmolÆL)1pH 5.1, for 24 h at 37°C

Mass spectrometry Peptides obtained by enzymatic digestion or chemical modification were separated and analysed by LC-MS For reversed-phase chromatography, a Vydac C8 column,

1 (i.d.)· 250 mm, was used 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 contained 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 tandem mass spectrum of the glycopeptide resulting from endoprotease Lys-C digestion was obtained by mass-selection of the 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 using 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 theDup99B genomic region PCR of genomic DNA (50 ng in 25 lL) was performed with AmpliTaq (Perkin Elmer) 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°C 60 s, 72 °C 60 s) Verification of genomic Dup99B sequences was carried out with primers GD6 (5¢-ATT CCAGTACAATTAGCTAGTTG-3¢) and GD7 (5¢-AG GAGTGTGCAATTTCTAAGG-3¢) for 30 cycles (94°C 40 s, 58 °C 60 s, 72 °C 60 s) Amplification 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¢) and k-2 (5¢-CCGCTCTAGAACTAGTGG ACT-3¢) on 1.5 lL of library (1.1 · 109pfuÆmL)1) and a subsequent nested PCR on 1/10 thereof for 37 cycles (93°C

40 S, 58°C 60 s, 72 °C 60 s) with an initial 5 min

denatur-ation step at 74°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 tissuein situ hybridization

In situ hybridization to polytene chromosomes using

digoxygenin (DIG)-labelled probes of genomic clones

containing 600 bp of the promoter region were performed

as described by Langer-Safer et al [19] with the following

modifications: DNA was DIG-labelled using the DIG 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

Dup99BcDNA 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

vitrotranscripts performed according to the instructions of

the manufacturer (Boehringer Mannheim) As control for

hybridization, DIG-labelled sense in vitro transcripts were

used

R E S U L T S

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 the 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 also able to

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 stages of the project, after showing that the DUP99B gene was transcribed 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 peptide 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 isolate 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 and 3

To purify native DUP99B (nDUP99B) for sequencing purposes, peptide extracts were prepared from heads of mass-reared adult flies of both sexes Native DUP99B was isolated by subsequent runs of peptide extracts on FPLC-and HPLC-column As the intact peptide was resistant to 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 unmodified 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 and results of injections into females (shaded columns) Oviposition is strongly stimulated by a fraction eluting at 54 min The other fractions do not stimulate oviposition above the background egg-laying rate.

were found to be inaccessible to Edman degradation,

apparently due to a modified N-terminus

Taken together the results of all digests yield 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 the 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

material

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 the fact that the measured molecular mass of

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 of the signal peptide cleavage site was resolved by de-glycosylation

of mature DUP99B As we suspected a1–3 fucosylation of the asparagine-linked GlcNAc, which had been shown to inhibit de-glycosylation of glycopeptides 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 predicted from the cDNA and gene sequences that the signal 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 as several N-terminal proteolytic fragments thereof were resistant 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 2A 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 One 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 analysis The fragment ions produced

by the glycopeptide were derived predominantly from cleavage of the glycosidic linkages with charge retention

on the peptide Fragmentation of the peptide moiety was minimal The MS/MS spectrum exhibited various fragment ions consisting of the peptide and parts 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 melanogastergenome Subsequently, P1 clones from this cytological region were screened for Dup99B and the genomic region was cloned and sequenced

Fig 2 Sequence of the DUP99B precursor peptide and the Dup99B

gene DUP99B is synthesized via a precursor peptide with a

21-amino-acid signal peptide and two additional amino-21-amino-acid residues at the

C-terminus that are cleaved off during the peptide maturation process.

(A) Sequence of the DUP99B precursor peptide and comparison with

the sex-peptide (SP) precursor Identical amino acids are indicated by

vertical bars Filled triangles, sites of cleavage of signal peptides; 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, glycosylation site in the mature

DUP99B peptide; filled circle, pyroglutamine; overlined amino acids,

glycosylation consensus sequence; stars, hydroxyprolines (B)

Com-bined Dup99B cDNA and gene sequence Underlined, intron sequence;

asterisk, stop codon.

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 peptide, we PCR-amplified

genomic Dup99B DNA from the stock that was originally

used for purification of the peptide This DNA does also

encode the two additional amino acids (RK), hence they

must be cleaved off during maturation These sequence data

were later confirmed by the sequences published by the

DrosophilaSequencing 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 control 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 signal is seen only in the lanes containing RNA isolated from male abdomen or total male RNA (Fig 4A–C) As expected the SP probe lights up the lanes containing RNA from wild-type males and the transgenic females (due to the presence of fat 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

The site of Dup99B 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 4D–F) The staining is cytoplasmic As the peptide was initially isolated from male heads, male brains were also investigated However, no signal was detected 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 biological activity of the peptide lacking the glycosyl group was demonstrated 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 the postmating responses does not depend on the 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 for 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 Some

functions may be shared by the two peptides but based on different

structures, some may be performed by both 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 small letters denote amino acids derived

from the DUP99B gene 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 mass 1437 Da

consists of two peptides linked by a disulfide 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

Fig 4 The Dup99B gene is expressed in the ductus ejaculatorius of the male genital tract, and the gene is localized at the cytological region 99B (A–C) Northern-blots with (A) Dup99B cDNA (B) Sex-peptide cDNA, and (C) ribosomal protein 49 (rp49) cDNA as probes H, RNA extract from heads; T, from thoraces; A, from abdomen; X, from whole body extracts #, males; $, mated females; , virgin females Mated female RNAs 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 situ hybridization of a DIG-labelled Dup99B probe to male 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 duct Same region as shown in (E) (G) In situ hybridiza-tion of a DIG-labelled genomic clone of Dup99B to polytene salivary gland chromo-somes The probe labels the cytological region 99B.

Fig 5 Dose–response of native and synthetic DUP99B The critical concentration needed to elicit ovulation is lower for native DUP99B (nDUP) than synthetic DUP99B (sDUP; 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 0.000; DF ¼ 1; P ¼ 1.000) Each point represents the mean ± SD

of three experiments with at least 20 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.

SP is 0.6 pmol per female for all responses [8]), and 0.2 pmol

nDUP99B per female The critical concentrations needed to

reduce the receptivity are not significantly different This

discrepancy could be 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

D I S C U S S I O N

Sex-peptides

The DUP99B purification scheme was based 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 elicit the two postmating

responses [8,14] It is also conserved in SP sequences of other

Drosophilaspecies [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 mature DUP99B peptide with the genomic 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 same site

The second exon encodes the conserved C-terminal regions

Evolutionarily, these findings could be interpreted as a sign

of exon shuffling However, 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) It is unlikely that this fact is due solely to the

general hydrophobicity of the amino acids characteristic for

signal sequences Therefore, we suggest that Dup99B and SP

evolved from a common ancestor gene, and we consider the

genes coding for DUP99B and SP as members of a new SP

gene family

Although the sequence of the Dup99B gene is included in

the DNA sequence published by the Drosophila Genome

Project [28], it was not identified as a gene This is probably

due to the fact that the protein 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 missed 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 mature peptides differ in several respects The

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 Man3GlcNAc2 core [35,36] Native DUP99B induces ovulation at lower 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 and, probably, a hydroxylated leucine residue [5] However, biological functions have not been assigned for any of the SP modifications [5,8]

DUP99B was initially isolated from male head extracts (see Materials and methods) Northern-blots and whole mount in situ hybridization revealed expression of the Dup99Bgene in the ductus ejaculatorius (Fig 4) A recent promoter analysis [37], with lacZ as a reporter gene, also yielded a strong expression in the ejaculatory duct, confirming our results However, Rexhepaj [37] found that the reporter gene was also expressed in the 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 producing 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 occurrence of the 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 biarmipes (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 gene encoding ED-OSS has not been isolated) However, both contain a glycosylation signal in this part of the peptide, but for ED-OSS it is not known whether it is glycosylated in its native form Interestingly, when ED-OSS is injected into the hemolymph of virgin

D biarmipes females, 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 glycosylated nDUP99B, induces the two responses at a lower critical concentration than sDUP99B or SP (Fig 5) Incubation of125I-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 cervical connective, to discrete parts of the thoracic ganglion, and to the genital tract [14] These findings suggest 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

Because no null mutants are available for the two genes

so far, the question of redundancy can only be answered

indirectly One approach is to study in 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 kills

the main cells of the accessory glands, thus 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 synthesized in the ejaculatory duct of the

DTA-males are transferred into the reproductive tract of feDTA-males

[39] either DUP99B is not synthesized in the ejaculatory

duct, DUP99B is not transferred without sperm, or

DUP99B is transferred but has no effect The 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 proliferation 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 during 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 oviposition,

reduction of receptivity was not observed However, a

recent detailed behavioural analysis showed that after

copulation with prdRes males, females 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 partial increase

of egg laying Nevertheless, after 1 h of rejection, all females

have re-copulated, as 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 responses The interpretation of the experiments

of Kalb et al [39] and Xue & Noll [40] is complicated

further by the fact that no accessory gland fluid is

transferred by these transgenic males The function(s) of

the remaining seminal fluid 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) This region of SP is also

conserved in SPs 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 amino acids of SP are also essential 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 be the molecular basis of the sperm 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 persistence is lost and the two responses fade away after 2 days [44] Hence, the presence

of sperm provides the persistency, possibly via bound SP and DUP99B

A summary of known and 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 on different structures Finally, some functions may be performed by both peptides with almost identical structures

A C K N O W L E D G E M E N T S

We thank R Sack for amino-acid analysis, N Birchler for peptide sequence analysis, R Bruggmann 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 This research has been supported by the Kanton Zu¨rich, the Hescheler-Stiftung, the Julius Klaus-Stiftung, Pro Scientia, and the Swiss National Science Foundation (grants no 31-42 067.94 and 31 52440.97 to E K).

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