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

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:
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. This research has been supported by the
Kanton Zu
¨
rich, the Hescheler-Stiftung, the Julius Klaus-Stiftung, Pro
Scientia, and the Swiss National S cience Fo undation (grants no. 31-42

067.94 and 31 52440.97 to E. K).
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