Báo cáo khoa học: Characterization of a functionally expressed dipeptidyl aminopeptidase III from Drosophila melanogaster doc

Characterization of a functionally expressed dipeptidyl
aminopeptidase III from
Drosophila melanogaster
Claire Mazzocco
1,
*, Kayoko M. Fukasawa
2
, Patrick Auguste
3
and Jacques Puiroux
1,
†
1
Laboratoire des Re
´
gulations Neuroendocriniennes, Universite
´
Bordeaux I, Talence, France;
2
Department of Oral Biochemistry,
Matsumoto Dental College, Shioriji, Nagano, Japan;
3
Laboratoire des Facteurs de Croissance et de la Diffe
´
renciation Cellulaire,
INSERM EPI-0113, Universite
´
Bordeaux I, Talence, France
A Drosophila melanogaster cDNA clone (GH01916)
encoding a putative 723-residue long (82 kDa) protein
(CG 7415) and displaying 50% identity with mammalian

cytosolic dipeptidyl aminopeptidase (DPP) III was func-
tionally expressed in Schneider S
2
cells. Immunocytochemi-
cal studies using anti-(rat liver DPP III) Ig indicated the
expression of this putative DPP III at the outer cell mem-
brane and into the cytosol of transfected cells. Two protein
bands (82 and 86 kDa) were immunologically detected after
PAGE and Western blot of cytosol or membrane prepared
from transfected cells. Western blot analysis of partially
purified D. melanogaster DPP III confirmed the over-
expression of these two protein bands into the cytosol and on
the membranes of transfected cells. Despite the identification
of six potential glycosylation sites, PAGE showed that these
protein bands were not shifted after deglycosylation experi-
ments. The partially purified enzyme hydrolysed the insect
myotropic neuropeptide proctolin (Arg-Tyr-Leu-Pro-Thr)
at the Tyr-Leu bond (K
m
 4 l
M
). In addition, low con-
centration of the specific DPP III inhibitor tynorphin pre-
vented proctolin degradation (IC
50
¼ 0.62 ± 0.15 l
M
).
These results constitute the first characterization of an evo-
lutionarily conserved insect DPP III that is expressed as a

cytosolic and a membrane peptidase involved in proctolin
degradation.
Keywords: enkephalinase; genome sequencing; insects;
neuropeptides; proctolin.
Mammalian DPP III was first discovered in the bovine
anterior pituitary gland [1] and it has been recently cloned
from rat liver as a 738-residue (82 kDa) cytosolic protein
[2,3]. This enzyme (EC 3.4.14.4) is a zinc metallopeptidase
containing a specific domain HELLGH-18X-E where a zinc
molecule is bound to both histidines [4]. DPP III is mainly
identified as a cytosolic peptidase, but DPP III was also
detected on membranes prepared from the brain of guinea-
pig [5] and rat [6]. Angiotensins and enkephalins constitute
the preferred substrates of the rat brain cytosolic DPP III
[7]. The routes of degradation of the insect myotropic
neuropeptide proctolin (Arg-Tyr-Leu-Pro-Thr) have been
compared to those of enkephalins. Indeed, a dipeptidyl
aminopeptidase activity appears as one major proctolin-
degrading peptidase, liberating the N-terminal Arg-Tyr
dipeptide [8–11]. This dipeptidyl aminopeptidase activity
was compared to the vertebrate DPP III [11] and is mainly
recovered as a cytosolic enzyme [8]. Interestingly, a proct-
olin-degrading DPP activity is also measured on membranes
[8,9] especially those obtained from insect proctolin-rich
tissues such as hindgut [10]. None of the presumed
proctolinases has been fully characterized yet.
We recently purified [12] from hindgut membranes of the
cockroach, Blaberus craniifer, a proctolin-degrading protein
(76 and 80 kDa) that removes the N-terminal dipeptide
from proctolin (K

m
¼ 3.8 ± 1.1 l
M
) and enkephalins
(K
m
¼ 4.2 ± 0.8 l
M
). The partial sequencing of this puri-
fied protein revealed a significant homology with the rat
liver cytosolic DPP III that was confirmed by the specific
detection of this purified insect protein with anti-(rat liver
DPP III) Ig in Western blot analysis. In addition, this
sequencing allowed the identification in Drosophila melano-
gaster of a set of homologous cDNA sequences and a
related genomic sequence (available at the Berkeley Droso-
phila Genome Project) that encode a deduced 723-residue
long protein (82 kDa) sharing 50% identity with mamma-
lian DPP III. This D. melanogaster DPP III is annotated
CG7415 [13]. Western blot analysis of crude membrane and
soluble material prepared from D. melanogaster showed the
presence of two protein bands (82 and 86 kDa) immuno-
logically related to vertebrate DPP III [12]. From these
results, it could be speculated that an evolutionarily
conserved DPP III is present in insects.
In the present study, the functional expression of the put-
ative D. melanogaster DPP III was attempted in Schneider
Correspondence to J. Puiroux, Laboratoire de Neurobiologie des
Re
´

seaux, CNRS-UMR 5816, Universite
´
Bordeaux 1, Avenue des
Faculte
´
s, 33405 Talence Cedex, France.
Fax: + 33 557 962561, Tel.: + 33 557 962569,
E-mail:
Abbreviations: BDGP, Berkeley Drosophila genome project;
DAB, diaminobenzidine; DPP, dipeptidyl aminopeptidase.
Enzyme: rat liver DPP III (EC 3.4.14.4).
*Present address: Groupe de Recherche pour l’Etude du Foie,
INSERM E 9917, Universite
´
Victor Segalen Bordeaux II,
146 rue Le
´
o Saignat, 33076 Bordeaux, France.
Present address: J. Puiroux, Laboratoire de Neurobiologie des
Re
´
seaux, CNRS-UMR 5816, Universite
´
Bordeaux 1, Avenue
des Faculte
´
s, 33405 Talence Cedex, France.
(Received 20 March 2003, revised 19 May 2003,
accepted 27 May 2003)
Eur. J. Biochem. 270, 3074–3082 (2003) Ó FEBS 2003 doi:10.1046/j.1432-1033.2003.03689.x

S
2
cells to investigate its apparent dual cellular location and
to examine the characteristics of this enzyme. Immunocyto-
chemical studies, Western blot analysis using anti-(rat liver
DPP III), partial purification and proctolin degradation
studies demonstrate the overexpression of the D. melano-
gaster DPP III on membranes and into the cytosol of
transfected cells. These results confirm that DPP III can be
attached to cell membranes, and they represent the first
identification and characterization of an insect DPP III that
plays a major role in proctolin degradation.
Experimental procedures
Materials
Diaminobenzidine (DAB), Hepes, hydrogen peroxide,
enkephalins, the enkephalin N-terminal dipeptide Tyr-Gly
and proctolin were purchased from Sigma (France). All
chromatographic materials were from Pharmacia (Uppsala,
Sweden). The specific DPP III inhibitor tynorphin (Val-
Val-Tyr-Pro-Trp) was a gift from K. Fukasawa (Matsu-
moto Dental University, Nagano, Japan). The anti-(rat liver
DPP III) was prepared as described by Fukasawa et al.[2].
Goat anti-rabbit Ig with peroxidase labelling was from
Boehringer-Mannheim. The proctolin fragments Arg-Tyr
and Leu-Pro-Thr were a gift from B. G. Loughton (York
University, Ontario, Canada). The expression system in
S
2
cells was from Invitrogen.
Stable transfection of S

2
cells with a
D. melanogaster
cDNA encoding a putative DPP III
A2.484kbHpaIfragmentoftheD. melanogaster cDNA
clone GH01916 (in pOT2 vector) coding a putative DPP III
was subcloned into the EcoRV site of pMTA/V5-His B
expression vector previously dephosphorylated with calf
intestinal alkaline phosphatase
1
. This vector is under the
control of the metallothionein promoter. Schneider S
2
cells
were cultured at 24 °C in Schneider S
2
cell medium
containing
L
-glutamine (1 m
M
), Penicillin-Streptomycin
(25 mgÆmL
)1
) and 10% (v/v) heat-inactivated fetal bovine
serum. Cells (10
6
cellsÆmL
)1
) were placed in Petri dishes

(35 mm) and incubated overnight at 24 °C before transfec-
tion. The mixture for cell transfection was prepared by
mixing 36 lLof2
M
CaCl
2
with 19 lg of expression vector
pMTA/DPP III and 1 lg of selection vector pCoHYGRO
coding for hygromycin B and adjusted to a final volume of
300 lL with sterilized MilliQ water. The mixture was added
dropwise to 300 lL Hepes sterile buffer (50 m
M
Hepes,
1.5 m
M
NaH
2
PO
4
,280m
M
NaCl, pH 7.1) and incubated
for 30 min at room temperature prior to transfection of S
2
cells. A control transfection was carried out using the
pMTA expression vector without the HpaIfragmentofthe
D. melanogaster cDNA (ÔmockÕ transfection). After over-
night incubation at 24 °C, the transfected cells were rinsed
twice with 2 mL of complete S
2

medium to remove the
precipitates and finally incubated at 24 °Cwith2mLof
complete S
2
medium. Stable recombinant cells were selected
by adding hygromycin B (300 lgÆmL
)1
) 2 days post-trans-
fection. The protein expression was induced by addition of
CuSO
4
(final concentration 0.5 m
M
) to hygromycin-selected
recombinant cells 48 h before measurements.
Analysis of transfected S
2
cells
The transfection of S
2
cells by the expression vector pMTA/
DPP III was verified by PCR using a Perkin Elmer
GeneAmp PCR System 2400 thermocycler. A sense primer
designed against the sequence of the pMTA vector
(5¢-GGGGATCTAGATCGGGGT-3¢) and an antisense
primer specific for the HpaIfragment(5¢-AGCGGAAGT
GTGATGCCG-3¢) were used with DNA from transfected,
ÔmockÕ transfected or untransfected S
2
cells as template.

These primers and a second set of primers (sense primer
5¢-GAATTCGAGGGCTTCGTGGCC-3¢ and antisense
primer 5¢-AACGAGTCCTTCGCCTGCCT-3¢) specific
for the HpaI fragment were used for RT-PCR. Total RNAs
from transfected, ÔmockÕ transfected or untransfected
S
2
cells were used after DNAse I treatment to prepare
cDNA templates.
Immunocytochemistry of stable transfected S
2
cells
Immunocytochemical studies were performed on stable
recombinant S
2
cells 48 h after induction. Twenty milliliters
of cell culture were centrifuged (1000 g, 5 min) and the
pelleted cells were rinsed twice with 1 mL NaCl/P
i
(137 m
M
NaCl, 2.7 m
M
KCl, 10 m
M
Na
2
HPO
4
,1.8m

M
KH
2
PO
4
,
pH 7.4). Then, the unfixed cells were incubated with anti-
(rat liver DPP III) Ig (1/2000) in NaCl/P
i
with or without
0.1% (w/v) Tween 20 for 1 h at room temperature under
slow constant agitation. The cells were centrifuged (1000 g,
5 min) and rinsed with 1 mL NaCl/P
i
for 15 min under slow
agitation. Cells were then incubated for 1 h at room
temperature with horseradish peroxidase labeled secondary
antibody (1/1000). Cells were rinsed as described above,
then incubated in Tris buffer (50 m
M
Tris/HCl, pH 8)
containing DAB (1.39 m
M
) for 5 min and finally revealed
by addition of 0.01% (v/v) H
2
O
2
. Cells were rinsed twice
and mounted on glass slide in 80% (w/v) glycerol and

observed with a microscope (Reichert–Jung model Polyvar)
and image analysis software (Spot RT, Diagnostic Instru-
ments, USA).
Partial purification of a putative
D. melanogaster
DPP III expressed in S
2
cells
Typically, 50 mL of cell culture were centrifuged (1000 g,
5 min, room temperature) and the pelleted cells were
rinsed twice with 1 mL NaCl/P
i
(pH 7). The pellet was
suspended in 0.5 mL ice-cold Hepes buffer (10 m
M
Hepes,
5% (w/v) glycerol, 5 m
M
MgCl
2
, pH 7.2) and homogen-
ized with a motor driven Teflon-glass homogenizer. The
homogenate was centrifuged (3000 g for 10 min at 4 °Cin
a Beckman J2-MC centrifuge). The pellet was discarded
and the supernatant was centrifuged (40 000 g for 20 min
at 4 °C). The final supernatant (cytosolic sample) was
separated from the membrane pellet, filtered (0.45 lm
pore size syringe filter) and stored at )80 °C. The
membrane pellet was suspended in 1 mL Hepes buffer
and recentrifuged (40 000 g for 20 min at 4 °C). The final

membrane pellet was suspended in 200 lL Hepes buffer
andstoredat)20 °C. A total of 8 L of stable trans-
fected S
2
cells induced for 48 h were thus prepared for
chromatography and the total cytosolic sample contained
Ó FEBS 2003 Characterization of D. melanogaster DPP III (Eur. J. Biochem. 270) 3075
about 50 mg protein. The pooled membrane preparations
contained approximately 45 mg proteins and this mem-
brane homogenate (1 mg proteinÆmL
)1
) was solubilized by
adding a concentrated Chaps solution (10% w/v in cold
Hepes buffer) dropwise to a final concentration of 1%.
Solubilization was carried out for 1 h at 4 °C under
constant agitation and the sample was then ultracentri-
fuged (100 000 g for 1 h at 4 °C, Beckman L8-55
centrifuge). The supernatant (solubilized sample) was
filtered, added with 90 mL Hepes buffer (to reduce the
detergent concentration) and processed for purification.
The partial purification of expressed D. melanogaster
putative DPP III was performed according to the method
already described in [14]. Briefly, cytosolic or solubilized
sample was first loaded on a 5-mL HiTrap Q Sepharose
cartridge connected to a Pharmacia AKTA FPLC system
(Pharmacia, Uppsala, Sweden) delivering 5 mLÆmin
)1
.
The cartridge was then rinsed with Hepes buffer (con-
taining 0.1% w/v Chaps for the solubilized sample) and

proteins were eluted by a regular NaCl gradient (from 10
to 500 m
M
in 25 min) generated with buffer B (Hepes
buffer containing 1
M
NaCl). Fractions were collected every
minute and the presence of DPP III was identified by
degradation studies using proctolin as substrate and by
Western blot analysis with anti-(rat liver DPP III) Ig. The
enzyme fractions were pooled and concentrated by ultrafil-
tration (Macrosep Pall Filtron cartridge, molecular mass cut
off
2
¼ 10 000 spun at 4000 g for 90 min at 4 °C). The protein
content and the DPP III activity were measured and the
concentrated sample was applied to two Superdex HR 200
10/30 columns (connected in line). The isocratic separation
of proteins was obtained with Hepes buffer delivered at
0.25 mLÆmin
)1
. The enzyme fractions were identified as
above. Both separations were monitored at 280 nm.
Degrading activity of the functionally expressed
putative
D. melanogaster
DPP III
DPP III activity was measured in homogenates of whole
transfected cells, in cytosolic or solubilized membrane
samples and in partially purified fractions with proctolin

(40 l
M
, 6 nmol) as substrate. The incubation was carried
out in presence of bestatin (100 l
M
) in a final volume of
150 lLfor15minat25°C under constant stirring. The
reaction was stopped by the addition of 5 lLof2
M
HCl
and centrifugation (Hettich EBA 12R, 16 500 g at 4 °Cfor
8 min). The degradation products were separated on a
Pharmacia PepRPC HR 5/5 reversed-phase column con-
nected to an FPLC system (pump A: 0.1% v/v trifluoro-
acetic acid in MilliQ water; pump B: 0.1% v/v
trifluoroacetic acid, 60% v/v CH
3
CN in MilliQ water)
delivering 1 mLÆmin
)1
under the following gradient condi-
tions: from 1 to 15% CH
3
CN for the first 5 min, then from
15 to 30% CH
3
CN for the next 15 min. The detection of
neuropeptide fragments was monitored at 206 and 280 nm.
The proctolin fragments Arg-Tyr and Leu-Pro-Thr were
identified by coelution with standard solutions of di- and

tripeptide. The effect of proctolin concentrations (from 1 to
250 l
M
) and the effect of tynorphin concentrations (from
100 l
M
to 0.1 n
M
) on the degradation of proctolin (40 l
M
)
were examined with the putative DPP III partially purified
from cytosol or solubilized membrane sample. The effect of
the divalent metal ions Zn
2+
and Co
2+
was checked on
DPP III activity contained in whole cell homogenates. A
curve-fitting computer program (
CRICKET GRAPH
)
3
was used
to determine the K
m
value for proctolin and the IC
50
for
tynorphin.

Cell surface degradation and internalization
of proctolin by stable transfected S
2
cells
Transfected S
2
cells were previously induced for 48 h with
CuSO
4
(final concentration in culture medium 0.5 m
M
).
Then, transfected cells were rinsed twice in NaCl/P
i
and
incubated (50 000 cells per tube) in Hepes degradation
buffer (100 lL) with proctolin (25 nmol) as substrate. At
the end of incubation, the supernatant (incubation
medium) was separated from the cells by centrifugation
(1000 g for 8 min at room temperature)
4
andaddedwith
5 lLHCl(2
M
). Then, the cell pellet was rinsed twice
before cell disruption and centrifugation (13 000 g for
15 min) to isolate the cytosolic sample also added with
5 lLHCl(2
M
)

5
. Proctolin degradation was analyzed by
reversed-phase separation of the incubation medium in
order to estimate the metabolism of proctolin at the cell
surface. The reversed-phase separation of cytosolic samples
indicated the rate of neuropeptide degradation after
internalization. These results were compared to those
obtained with transfected S
2
cells not previously induced.
SDS/PAGE and Western blot analysis
The samples consisting of transfected, ÔmockÕ transfected or
untransfected cells, cytosol or membrane preparations
(30 lg per lane) or partially purified expressed enzyme (1
or 2 lg) were prepared in sample buffer (62.5 m
M
Tris/HCl,
pH 6.8, 0.025% bromophenol blue, 10% w/v glycerol and
1% w/v SDS) and electrophoresed [15] on a 7.5% acryl-
amide 1 mm-thick mini gel at 20 mA for 150 min. Then the
gel was stained with Coomassie Brillant Blue R-250, silver
stained (Silver Stain Plus Kit, Bio-Rad), or transferred to a
nitrocellulose membrane (Hybond C extra, pore size
0.45 lm, Amersham) for 1 h using a mini-trans-blot
apparatus (Bio-Rad) for Western blot analysis. The mem-
brane was soaked in Tris buffered saline with Tween-20
(TBS Tween, 20 m
M
Tris/HCl, 137 m
M

NaCl, 0.1% v/v
Tween-20, pH 7.3) with 5% (w/v) dry low fat milk (used as
blocking agent) for 30 min at 37 °C under constant stirring.
Then, the membrane was incubated overnight at 4 °Cwith
rabbit polyclonal anti-(rat liver DPP III) Ig (1/2000 in TBS
Tween under constant slow agitation) and then with goat
anti-rabbit Ig (Boehringer-Mannheim) conjugated with
horseradish peroxidase (1/2000 in TBS Tween, 30 min at
37 °C). Staining was obtained by incubation with a fresh
solution of 1.39 m
M
DAB (in 50 m
M
Tris/HCl, pH 7.3 for
5 min at room temperature) added with H
2
O
2
(0.1 lLÆmL
)1
Tris buffer). Deglycosylation experiments were carried out
on partially purified D. melanogaster DPP III using an
enzymatic deglycosylation kit (Bio-Rad, including NANase
II, O-glycosidase DS and PNGase F) according to the
manufacturer’s instructions. Samples were checked before
and after deglycosylation by SDS/PAGE, followed by
staining with Coomassie Blue, using fetuin as a glycosylated
protein standard.
3076 C. Mazzocco et al.(Eur. J. Biochem. 270) Ó FEBS 2003
Sequence comparisons

The coding sequences of D. melanogaster and rat liver
DPP III were aligned with homologous coding sequences
from the worm Caenorhabditis elegans and the protozoan
Leishmania major using
CLUSTALW
1.8. In addition, the
corresponding protein sequences were analyzed for the
presence of a signal peptide and transmembrane regions
using
SIGNALP
from the Expasy proteomic tools and
TM
at
the EMBNET [16].
Measurement of protein content
Membrane, cytosolic and purified proteins were measured
using a commercial protein assay reagent kit (Bio-Rad) with
BSA as a standard [17]. Proteins in solubilized samples were
measured according to the instructions of a commercial
detergent-compatible reagent kit (Bio-Rad), again with
BSA as the standard.
Results
Stable transfection of S
2
cells expressing a putative
D. melanogaster
DPP III
S
2
cells were double transfected with the pMTA/DPP III

and pCoHygro vectors and then selected for their resistance
to hygromycin. The transfection of hygromycin-resistant S
2
cells with the pMTA/DPP III vector was first verified by
PCR, using a sense primer specific for pMTA and an
antisense primer designed against the putative D. melano-
gaster DPP III HpaI fragment (Fig. 1A) to amplify a
1017 bp fragment (Fig. 1B). The transcription of specific
mRNAs was deduced from the amplification of the 1017 bp
fragment after RT-PCR with cDNA templates prepared
from total RNA of transfected S
2
cells previously induced
for 48 h (Fig. 1C, lane 5). When RT-PCR was carried out
with these cDNA templates and a second set of primers
specific to the core D. melanogaster DPP III sequence
(Fig. 1A), a 783 bp fragment was also amplified (Fig. 1C,
lane 5). By contrast, the results of RT-PCR experiments
with the second set of primers clearly indicated no
significant transcription of endogenous DPP III in untrans-
fected and ÔmockÕ transfected S
2
cells, and also in transfected
S
2
cells that have not been previously induced (Fig. 1C).
These results were corroborated by SDS/PAGE
(Fig. 2A) and Western blot analysis (Fig. 2B) with anti-
(rat liver DPP III) Ig, revealing two protein bands in the
expected range of 82 and 86 kDa only in transfected S

2
cells
after 48 h induction. By contrast, soluble DPP III related
protein could not be detected by Western blot analysis (data
not shown) in transfected S
2
cell culture medium after 48 h
induction. When immunocytochemical studies were per-
formed with anti-(rat liver DPP III) Ig on unfixed trans-
fected and induced S
2
cells in the absence of detergent, a
significant and presumably surface labeling was observed
(Fig. 3A,B). When cells were treated with 0.1% Tween 20,
approximately twice as many transfected and induced S
2
cells were stained than in the absence of detergent (data not
shown). By comparison, no labeling could be observed after
immunocytochemical treatment of untransfected S
2
cells or
transfected S
2
cells not previously induced (data not shown).
After a 5-min incubation of proctolin (25 nmol) with
stably transfected and induced S
2
cells in NaCl/P
i
containing

the aminopeptidase inhibitor bestatine (100 l
M
), 6.25 nmol
of proctolin and 1.75 nmol Arg-Tyr (and Leu-Pro-Thr,
not shown) were recovered into the incubation medium as
determined after reversed-phase separation (Fig. 3C). When
incubation was carried out for 10 min, only 5 nmol
proctolin and 2.75 nmol Arg-Tyr (and Leu-Pro-Thr, not
shown) were measured. After 20 min incubation, 2.7 nmol
proctolin and 4.55 nmol Arg-Tyr (and Leu-Pro-Thr, not
shown) were quantified, indicating a correlated degradation
of extracellular proctolin via DPP activity over the 20 min
incubation. By comparison, cytosolic contents of induced
Fig. 1. Schematic model of the pMTA/DPP III construction vector and
verification of the transfection of S
2
cells with this vector. (A) Hatched
area indicates the 2.484 kb HpaIfragmentofD. melanogaster cDNA
clone GH 01916 coding for a putative DPP III. This fragment was
inserted in the pMTA/V5-His B expression vector. Arrows represent
the primer pairs (1 and 2; 3 and 4) used in the PCR and RT-PCR
experiments, and the approximative sequence area expected to be
amplified (1 kb and 0.8 kb, respectively). (B) Transfection of S
2
cells
with pMTA/DPP III vector was verified by PCR with DNA from
ÔmockÕ transfected and from stable transfected S
2
cells amplified using
a set of primers specific for the pMTA/DPP III construction vector

(primers 1 and 2). A 1.0 kb fragment (expected size 1.02 kb) was
observed after separation on a 0.8% (w/v) agarose gel of PCR prod-
ucts obtained with DNA of stable transfected and induced S
2
cells
(lane 2). No amplification product could be detected from control PCR
with DNA from ÔmockÕ transfected S
2
cells (lane 1). (C) Transcription
of putative D. melanogaster DPP III mRNA in transfected S
2
cells was
verified by RT-PCR experiments carried out on RNAs extracted from
ÔmockÕ transfected and stable transfected S
2
cells using a second set of
primers (3 and 4) specific for the HpaI fragment encoding a putative
DPP III. Control amplification of a 0.8 kb product (expected size
0.78 kb) was obtained with DNA of the D. melanogaster clone
GH 01916 as observed after separation on a 0.8% (w/v) agarose gel
(lane 1). No amplification product was observed after RT-PCR using
RNA from ÔmockÕ transfected S
2
cells (lane 2). A 0.8 kb fragment was
visualized after separation of RT-PCR products performed with
RNAs from stably transfected and induced S
2
cells (lane 3). When RT
was omitted (control for contaminant DNA extracted from stable
transfected S

2
cells) the 0.8 kb PCR product was not detected (lane 4).
When using the first set of primers (1 and 2), the 1.0 kb fragment was
amplified after RT-PCR using RNA from transfected S
2
cells after
induction (lane 5).
Ó FEBS 2003 Characterization of D. melanogaster DPP III (Eur. J. Biochem. 270) 3077
S
2
cells contained only traces of proctolin and Arg-Tyr after
5 min incubation. A significant peak of tyrosine (about
15 nmol) was detected and suggested a complete and rapid
degradation of the 65–70% internalized neuropeptide
despite the utilization of bestatine (data not shown). In
addition, no DPP activity could be measured into concen-
trated (ultrafiltration) culture medium (data not shown)
using proctolin as a substrate.
Although no trace of putative D. melanogaster DPP III
could be noticed after Western blot analysis of transfected
S
2
cells not previously induced, homogenates of uninduced
S
2
cells contained proctolin-degrading activities including
a weak dipeptidyl aminopeptidase activity of 0.07 ± 0.45
nmol proctolinÆmg protein
)1
Æmin

)1
. By contrast, a signifi-
cant DPP activity was measured in homogenates of trans-
fected and Cu
2+
-induced S
2
cells using met-enkephalin
(data not shown) or the insect neuropeptide proctolin
as substrate (1.66 ± 0.35 nmol proctolinÆmg protein
)1
Æ
min
)1
). When divalent metal ions (Zn
2+
) were added to
the incubation medium, the rate of met-enkephalin (data
not shown) or proctolin degradation was similar (1.59 ±
0.45 nmol proctolinÆmg protein
)1
Æmin
)1
). By contrast, the
expressed dipeptidyl aminopeptidase activity contained in
S
2
cell homogenates was strongly increased by the presence
of Co
2+

(13.6 ± 1.4 nmol proctolinÆmg protein
)1
Æmin
)1
).
Given these results, cytosol and membranes were prepared
separately from stably transfected and induced S
2
cells,
electrophoresed and analyzed with anti-(rat liver DPP III) Ig
after Western blotting. Two protein bands at 82 and 86 kDa,
already visualized from whole transfected and induced S
2
cells, were clearly detected in cytosolic samples (Fig. 4A,B).
Both protein bands were also observed in membrane samples
(data not shown). Although an equivalent amount of
cytosolic or membrane protein was initially loaded, the
cytosolic 82 and 86 kDa protein bands were approximately
twice as intense as those observed from membrane samples,
indicating that a proportionally larger amount of over-
expressed D. melanogaster DPP III is present into the
cytosol than on the membranes of transfected cells.
Partial purification and characterization of the putative
D. melanogaster
DPP III expressed in S
2
cells
Cytosolic and membrane DPP activities expressed after
48 h induction in stable transfected S
2

cells were partially
purified, first using a 5-mL Hitrap Q cartridge then two size
exclusion columns (Table 1). The DPP fractions were
identified by degradation studies using proctolin (40 l
M
)
as the substrate. SDS/PAGE analysis of fractions contain-
ing partially purified DPP activitiy revealed two major
bands in the range of 82 and 86 kDa in both cytosolic and
membrane samples (Fig. 5). Western blot analysis of both
partially purified samples confirmed the presence of two
protein bands with similar molecular masses (82 and
86 kDa) that were immunologically related to rat liver
DPP III (Fig. 5). Both partially purified D. melanogaster
DPP III removed Arg-Tyr from the N terminus of proctolin
(data not shown) with K
m
values of 4.1 ± 0.7 l
M
for the
cytosolic enzyme and 3.0 ± 0.6 l
M
for the membrane
enzyme. They both hydrolysed met-enkephalin at the Gly-
Gly bond (data not shown). An IC
50
of 0.62 ± 0.15 l
M
was obtained with the DPP III inhibitor tynorphin (40 l
M

)
of proctolin degradation induced with D. melanogaster
DPP III partially purified from the cytosol of transfected
cells (data not shown).
The contamination of transfected S
2
cell membranes
was investigated by the addition of partially purified
cytosolic DPP III to membranes prepared from trans-
fected S
2
cells not previously induced. After a routine
twice washing of the membrane pellet, Western blot
analysis of this membrane sample revealed only traces of
DPP III proteins that are not proportionally related to
the amount of partially purified DPP III added to this
sample. Furthermore, DPP III activity was measured on
these ÔcontaminatedÕ membranes when no DPP III acti-
vity could be detected on control membranes (transfected
cells not previously induced), but this contamination
accounted for only a third of the total DPP activity
recovered on membranes prepared from transfected S
2
cells after induction.
Sequence analysis
The comparison of protein sequences identified in animals
as putative or fully characterized DPP III showed variable
regions at both extremities and consensus regions in the core
sequence (Fig. 6). An ancestral molecule of 679 amino
acids, found in the protozoan Leishmania major, includes a

potential signal peptide with a possible cleavage site between
residues 23 and 24 that is not recovered in the studied
Fig. 2. Expression of a putative D. melanogaster DPP III protein in stably transfected S
2
cells. (A) Whole cell homogenates (30 lg per lane) prepared
from stably transfected cells (cell lines 1a and 1b) after 48 h induction (lanes 1 and 3) or not previously induced (lanes 2 and 4) for the synthesis of
D. melanogaster DPP III were separated by SDS/PAGE on a 7.5% acrylamide gel and stained with Coomassie Brillant Blue. The arrow indicates
the region where two protein bands at 82–86 kDa are specifically detected in induced cells. (B) Western blot analysis of cell line 1a was performed
with anti-(rat liver DPP III) Ig. Transfected S
2
cells were induced for 48 h (lane 2) or not previously induced (lane 1).
3078 C. Mazzocco et al.(Eur. J. Biochem. 270) Ó FEBS 2003
metazoan enzymes. In addition, a C-terminal extension of
about 20 amino acid residues is found in metazoan DPP III
but no specific function could be attributed to this region.
Among metazoa, the analysis of D. melanogaster DPP III
revealed the presence of six potential glycosylation sites
compared with two sites on rat DPP III. However, no
significant shift of the partially purified 86 kDa D. melano-
gaster DPP III could be observed on SDS/PAGE after
deglycosylation experiments (data not shown).
In addition, the analysis of D. melanogaster DPP III
indicated four strong putative transmembrane fragments,
including two adjacent hydrophobic regions, located near
the N terminus (Fig. 6), that could be involved in membrane
anchorage of D. melanogaster DPP III. By comparison, the
analysis of mammalian DPP III resulted in the identifica-
tion of two distant transmembrane regions with lower
significance (data not shown).
Discussion

A D. melanogaster cDNA clone coding for a putative
DPP III of 82 kDa (CG 7415) was stably transfected in S
2
cells. After 48 h induction, transfected S
2
cells specifically
synthesized two protein bands detected at 82 and 86 kDa
after SDS/PAGE. Western blot analysis using anti-(rat liver
DPP III) Ig confirmed that these protein bands were
immunologically related to rat liver DPP III. This putative
D. melanogaster DPP III expressed in transfected cells after
induction was partially purified by chromatography accord-
ing to the measurement of dipeptidyl aminopeptidase
activity and the detection of immunologically related
DPP III material in the enzyme fractions. The partially
purified enzyme is similar to mammalian DPP III in that it
hydrolyses small neuropeptides such as met-enkephalin and
the insect myotropin proctolin, from which it removes the
N-terminal dipeptide, and because it is inhibited by the
specific DPP III inhibitor tynorphin [18]. The K
m
values of
the recombinant D. melanogaster DPP III for proctolin
(4 l
M
and 3 l
M
) are very similar to the K
m
value (also 4 l

M
)
of the presumed DPP III purified in cockroach [12]. The
potency of tynorphin against recombinant DPP III
(IC
50
¼ 0.62 l
M
) and that purified in cockroach (0.68 l
M
)
are also very close. In addition, D. melanogaster DPP III is
sensitive to divalent metal ions in a similar manner to
vertebrate DPP III [19–21]. The deduced amino acid
sequence of the putative D. melanogaster DPP III predicted
a strong sequence identity ( 50%) with mammalian
DPP III [12]. The functional expression of the D. melano-
gaster DPP III cDNA in S
2
cells does, indeed, confirm that
the translated protein product is a genuine DPP III.
Fig. 3. Immunocytochemistry of transfected S
2
cells and proctolin-
degrading activity of transfected S
2
cells. (A,B) Anti-(rat liver DPP III)
Ig was used to probe transfected S
2
cells (see Experimental proce-

dures). After 48 h induction, transfected S
2
cells were stained without a
treatment with detergent. Arrows indicate the positively stained cells.
(C) Proctolin (25 nmol) was incubated in NaCl/P
i
with stably trans-
fected S
2
cells. After different time intervals, proctolin and its N-ter-
minal dipeptide Arg-Tyr were measured in the incubation medium by
reversed-phase chromatography. The results are the mean of three
experiments ± SEM. Two thirds of this proctolin (approximately
16–17 nmol) were rapidly internalized into S
2
cellsandrecoveredas
tyrosine (not shown).
Fig. 4. SDS/PAGE and Western blot analysis of transfected S
2
cells.
Cytosolic samples were prepared from transfected S
2
cells after
induction and electrophoresed. (A) Cytosolic sample (30 lgprotein
per lane) was separated on a 7.5% (w/v) acrylamide gel and stained
with Coomassie Brillant Blue. Arrows indicate both bands corres-
ponding to overexpressed D. melanogaster DPP III proteins visualized
at 82 and 86 kDa. (B) Western blot analysis of cytosolic sample probed
with anti-(rat liver DPP III) that allowed the detection of both bands
at 82 and 86 kDa.

Ó FEBS 2003 Characterization of D. melanogaster DPP III (Eur. J. Biochem. 270) 3079
However, a number of features of the D. melanogaster
DPP III are different from the mammalian DPP III. For
instance, cytosolic rat liver DPP III is expressed in E. coli as
a single 82 kDa protein [2,22] that is in agreement with the
expected size and coincides with the molecular mass of the
DPP III protein deduced after purification from rat [3].
Although the transfection of S
2
cells was also carried out
with a single D. melanogaster DPP III cDNA sequence, two
major protein bands at 82 and 86 kDa are expressed and
detected in transfected S
2
cells. Both bands are also
identified after partial purification from transfected S
2
cells.
The expected molecular mass of the D. melanogaster
DPP III is 81 937, which coincides with the lower protein
band revealed at about 82 kDa after Western blot analysis
using anti-(rat liver DPP III). The presence of six predicted
putative glycosylation sites on the insect DPP III compared
with only two sites on rat liver DPP III suggested that the
heavier D. melanogaster DPP III protein may result from
post-transductional processing of the 82 kDa DPP III to
raise the molecular mass of the D. melanogaster DPP III up
to 86 kDa. Deglycosylation experiments performed on
partially purified D. melanogaster DPP III and verified by
SDS/PAGE were inconclusive in demonstrating conven-

tional glycosylation of the 86 kDa expressed protein. The
structure of the 86 kDa expressed DPP III is not yet
elucidated but it cannot be inferred from the expression in S
2
cells because similar bands at 82 and 86 kDa have been
already detected in soluble samples and membranes pre-
pared from fruit flies [12]. In addition, control SDS/PAGE
and Western blot experiments were performed with rat liver
samples that resulted in the detection of a single protein
band at 82 kDa as previously reported [2,3,23].
The expression of the insect DPP III as a membrane
protein represents the second major difference with mam-
malian DPP III, typically recovered as a cytosolic peptidase.
Immunocytochemical studies of transfected S
2
cells with or
without detergent indicated that D. melanogaster DPP III is
possibly expressed at the cell surface. Even though the
contamination of transfected S
2
cell membrane preparations
from transfected S
2
cell cytosol containing overexpressed
D. melanogaster DPP III was established, this accounted
for only 30% of the total membrane DPP III activity
recovered from transfected S
2
cell membranes. These results
are in line with the detection of native DPP III after

Western blot analysis of soluble and membrane samples
prepared from D. melanogaster [12] and with the purifica-
tion of a presumed DPP III from gut membranes in
cockroach [12,14]. Our results also agree with the dual
localization of DPP III previously reported in rat [6] and
guinea-pig [5] brain cytosol and membranes. The partial
purification of D. melanogaster DPP III expressed in
transfected S
2
cells was achieved from both cytosol and
solubilized membranes after thorough washing, and the
characterization of both D. melanogaster DPP III partially
purified from cytosol and membrane of transfected
cells showed similar K
m
values to met-enkephalin and
proctolin.
Fig. 5. SDS/PAGE and Western blot analysis of overexpressed
D. melanogaster DPP III. (A) D. melanogaster DPP III partially
purified from cytosol of transfected S
2
cells (1 lg) was electrophoresed
on a 7.5% (w/v) acrylamide gel and silver stained (lane 1) revealing the
presence of two protein bands at 82 and 86 kDa. After Western blot
analysis using anti-(rat liver DPP III) Ig, both bands were specifically
detected (lane 2). (B) An aliquot from the different steps of separation
of solubilized membranes from transfected S
2
cells was electrophored
on a 7.5% (w/v) acrylamide gel and silver stained. Solubilized mem-

branes of transfected S
2
cells (18 lg, lane 1), positive fractions resulting
from anion exchange separation (5 lg, lane 2) and from size exclusion
separation (2 lg, lane 3) were electrophoresed to confirm the isolation
of two protein bands at 82 and 86 kDa during the process of separ-
ation. (C) Western blot analysis of overexpressed D. melanogaster
DPP III partially purified from membranes (2 lg, lane 1) probed with
anti-(rat liver DPP III) Ig. Both bands at 82 and 86 kDa were thus
detected.
Table 1.
6
Purification of D. melanogaster cytosolic DPP III overexpressed in S
2
cells.
DPP III loaction Purification steps Proteins (mg)
Total activity
(nmolÆmin
)1
)
Specific activity
(nmolÆmg
)1
min
)1
) Purification factor
Cytosol Filtrate 49.32 39.45 0.799 1
Hi-Trap Q 17 207 12.2 15
Superdex 200 2 122.98 61.49 75
Membrane Filtrate 76 380 4.98 1

Hi-Trap Q 7 150 21.44 4.3
Superdex 200 0.25 81.9 312.7 62.79
3080 C. Mazzocco et al.(Eur. J. Biochem. 270) Ó FEBS 2003
The examination of several DPP III sequences indicated
that this enzyme was highly conserved during the
evolution process from protozoa to mammals and that
the extremities of the sequences are mostly variable. No
signal peptide could be identified in the D. melanogaster
DPP III and this was supported by the absence of
detection of recombinant DPP III in transfected S
2
cell
culture media as verified by Western blot analysis.
Furthermore, no trace of DPP activity could be measured
in ultrafiltered culture media of transfected S
2
cells. The
D. melanogaster DPP III is not secreted and is not a
circulating DPP. The fact that D. melanogaster DPP III
contains a putative N-terminal membrane anchor seq-
uence with a significantly higher probability than that
calculated for mammalian DPP III corroborates our
results that argue for a membrane DPP III in insects
when it is mainly identified as a cytosolic peptidase in
mammals. Furthermore, the analysis of the orientation of
the two putative transmembrane helices identified in
D. melanogaster DPP III (amino acid residues 44–62 and
81–106) indicates that the resulting loop between 63 and
80 is presumably on the inner side of the membrane,
whereas the other protein regions of D. melanogaster

DPP III are on the outer side. In these conditions and
considering the anchorage of the D. melanogaster DPP III
on the plasma membrane, the six potential glycosylation
sites and the HELLGH active site of the protein would be
exposed on the outer surface of the cell. Thus, one can
expect a significant cell surface-located DPP III activity in
transfected S
2
cells. This was confirmed by the detection
of a substantial amount of the proctolin N-terminal
dipeptide Arg-Tyr in the incubation medium (degradation
buffer) following incubation of the neuropeptide proctolin
with whole transfected and induced S
2
cells (Fig. 3C).
These results provide evidence that D. melanogaster
DPP III is expressed in S
2
cells as a cytosolic enzyme
and also as a membrane peptidase.
In terms of proctolin degrading activity, a dipeptidyl
aminopeptidase activity was shown to hydrolyse proctolin
in insects and was referred as to mammalian DPP III
[11] but this was not further investigated. In another
study, a dipeptidyl aminopeptidase activity was identified
in locust synaptosomes as a major proctolin-degrading
enzyme [8]. In this example, two thirds of DPP activity
was measured in the mitochondrial (cytosolic) fraction
and one third was recovered in the synaptosome
(membrane) fraction. In transfected S

2
cells, D. melano-
gaster DPP III is roughly expressed in these proportions
in the cytosol and on membranes as an efficient
proctolin-degrading enzyme.
In conclusion, our results confirm that the putative
D. melanogaster DPP III is a genuine DPP III, expressed in
the cytosol and on the membranes of transfected S2 cells.
The D. melanogaster DPP III herein identified represents
the first fully characterized peptidase involved in proctolin
degradation.
Fig. 6. Alignment of D. melanogaster and rat DPP III with putative homologous proteins identified in the worm C. elegans and the protozoan
L. major. Black frames correspond to the identity of residues in at least three sequences, grey frames indicate similarity between residues and dots
represent gaps inserted for optimal alignment with
CLUSTAL W
. Both squares correspond to potential transmembrane segments in D. melanogaster
DPP III as determined with
TM
software at the EMBNET.
Ó FEBS 2003 Characterization of D. melanogaster DPP III (Eur. J. Biochem. 270) 3081
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