Two protein bands 82 and 86 kDa were immunologically detected after PAGE and Western blot of cytosol or membrane prepared from transfected cells.. melanogaster DPP III DPP III activity w
Trang 1Characterization of a functionally expressed dipeptidyl
Claire Mazzocco1,*, Kayoko M Fukasawa2, Patrick Auguste3and Jacques Puiroux1,†
1
Laboratoire des Re´gulations Neuroendocriniennes, Universite´ Bordeaux I, Talence, France;2Department of Oral Biochemistry, Matsumoto Dental College, Shioriji, Nagano, Japan;3Laboratoire 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 S2cells
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 (Km 4 lM) In addition, low con-centration of the specific DPP III inhibitor tynorphin pre-vented proctolin degradation (IC50¼ 0.62 ± 0.15 lM) 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 (Km¼ 3.8 ± 1.1 lM) and enkephalins (Km¼ 4.2 ± 0.8 lM) 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-philaGenome 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: j.puiroux@lnr.u-bordeaux.fr
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)
Trang 2S2cells 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
S2cells was from Invitrogen
Stable transfection of S2cells with aD melanogaster
cDNA encoding a putative DPP III
A 2.484 k b HpaI fragment of the D 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
control of the metallothionein promoter Schneider S2cells
were cultured at 24C in Schneider S2 cell medium
containing L-glutamine (1 mM), Penicillin-Streptomycin
(25 mgÆmL)1) and 10% (v/v) heat-inactivated fetal bovine
serum Cells (106 cellsÆmL)1) were placed in Petri dishes
(35 mm) and incubated overnight at 24C before
transfec-tion The mixture for cell transfection was prepared by
mixing 36 lL of 2MCaCl2with 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 mM Hepes,
1.5 mMNaH2PO4, 280 mM NaCl, pH 7.1) and incubated
for 30 min at room temperature prior to transfection of S2
cells A control transfection was carried out using the
pMTA expression vector without the HpaI fragment of the
D melanogaster cDNA (mock transfection) After
over-night incubation at 24C, the transfected cells were rinsed
twice with 2 mL of complete S2 medium to remove the
precipitates and finally incubated at 24C with 2 mL of
complete S2medium 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
CuSO4(final concentration 0.5 mM) to hygromycin-selected
recombinant cells 48 h before measurements
Analysis of transfected S2cells The transfection of S2cells 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 HpaI fragment (5¢-AGCGGAAGT GTGATGCCG-3¢) were used with DNA from transfected,
mock transfected or untransfected S2 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, mock transfected or untransfected
S2 cells were used after DNAse I treatment to prepare cDNA templates
Immunocytochemistry of stable transfected S2cells Immunocytochemical studies were performed on stable recombinant S2cells 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/Pi(137 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, 1.8 mM KH2PO4,
pH 7.4) Then, the unfixed cells were incubated with anti-(rat liver DPP III) Ig (1/2000) in NaCl/Piwith 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/Pifor 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 mM Tris/HCl, pH 8) containing DAB (1.39 mM) for 5 min and finally revealed
by addition of 0.01% (v/v) H2O2 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 putativeD melanogaster DPP III expressed in S2cells
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/Pi (pH 7) The pellet was suspended in 0.5 mL ice-cold Hepes buffer (10 mMHepes, 5% (w/v) glycerol, 5 mM MgCl2, pH 7.2) and homogen-ized with a motor driven Teflon-glass homogenizer The homogenate was centrifuged (3000 g for 10 min at 4C in
a Beckman J2-MC centrifuge) The pellet was discarded and the supernatant was centrifuged (40 000 g for 20 min
at 4C) 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 4C) The final membrane pellet was suspended in 200 lL Hepes buffer and stored at )20 C A total of 8 L of stable trans-fected S2 cells induced for 48 h were thus prepared for chromatography and the total cytosolic sample contained
Trang 3about 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 4C under
constant agitation and the sample was then
ultracentri-fuged (100 000 g for 1 h at 4C, 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 mM in 25 min) generated with buffer B (Hepes
buffer containing 1MNaCl) 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
putativeD 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 lM, 6 nmol) as substrate The incubation was carried
out in presence of bestatin (100 lM) in a final volume of
150 lL for 15 min at 25C under constant stirring The
reaction was stopped by the addition of 5 lL of 2MHCl
and centrifugation (Hettich EBA 12R, 16 500 g at 4C for
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 CH3CN in MilliQ water)
delivering 1 mLÆmin)1under the following gradient
condi-tions: from 1 to 15% CH3CN for the first 5 min, then from
15 to 30% CH3CN 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 lM) and the effect of tynorphin concentrations (from
100 lMto 0.1 nM) on the degradation of proctolin (40 lM)
were examined with the putative DPP III partially purified
from cytosol or solubilized membrane sample The effect of
the divalent metal ions Zn2+ and Co2+was checked on DPP III activity contained in whole cell homogenates A curve-fitting computer program (CRICKET GRAPH)
to determine the Kmvalue for proctolin and the IC50for tynorphin
Cell surface degradation and internalization
of proctolin by stable transfected S2cells Transfected S2cells were previously induced for 48 h with CuSO4 (final concentration in culture medium 0.5 mM) Then, transfected cells were rinsed twice in NaCl/Pi 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)
5 lL HCl (2M) 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 lL HCl (2M)
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 S2cells not previously induced SDS/PAGE and Western blot analysis
The samples consisting of transfected, mock 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 mMTris/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-thickmini 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 mM Tris/HCl, 137 mM 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 37C under constant stirring Then, the membrane was incubated overnight at 4C with 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
37C) Staining was obtained by incubation with a fresh solution of 1.39 mMDAB (in 50 mMTris/HCl, pH 7.3 for
5 min at room temperature) added with H2O2(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
Trang 4Sequence 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
usingSIGNALPfrom the Expasy proteomic tools andTMat
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 S2cells expressing a putative
D melanogaster DPP III
S2 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 S2
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 S2cells 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 mock transuntrans-fected S2cells, and also in transfected
S2cells 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 S2cells
after 48 h induction By contrast, soluble DPP III related
protein could not be detected by Western blot analysis (data
not shown) in transfected S2cell 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 S2 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 S2
cells were stained than in the absence of detergent (data not
shown) By comparison, no labeling could be observed after
immunocytochemical treatment of untransfected S2cells or
transfected S cells not previously induced (data not shown)
After a 5-min incubation of proctolin (25 nmol) with stably transfected and induced S2cells in NaCl/Picontaining the aminopeptidase inhibitor bestatine (100 lM), 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 HpaI fragment of D 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
mock 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 mock 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
mock 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 mock 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).
Trang 5S2cells contained only traces of proctolin and Arg-Tyr after
5 min incubation A significant peakof 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
S2cells not previously induced, homogenates of uninduced
S2 cells contained proctolin-degrading activities including
a weakdipeptidyl aminopeptidase activity of 0.07 ± 0.45
nmol proctolinÆmg protein)1Æmin)1 By contrast, a
signifi-cant DPP activity was measured in homogenates of
trans-fected and Cu2+-induced S2 cells using met-enkephalin
(data not shown) or the insect neuropeptide proctolin
as substrate (1.66 ± 0.35 nmol proctolinÆmg protein)1Æ
min)1) When divalent metal ions (Zn2+) 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)1Æmin)1) By contrast, the
expressed dipeptidyl aminopeptidase activity contained in
S2cell homogenates was strongly increased by the presence
of Co2+(13.6 ± 1.4 nmol proctolinÆmg protein)1Æmin)1)
Given these results, cytosol and membranes were prepared
separately from stably transfected and induced S2 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 S2
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 S2cells
Cytosolic and membrane DPP activities expressed after
48 h induction in stable transfected S 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 lM)
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 Kmvalues of 4.1 ± 0.7 lMfor the cytosolic enzyme and 3.0 ± 0.6 lM for the membrane enzyme They both hydrolysed met-enkephalin at the Gly-Gly bond (data not shown) An IC50of 0.62 ± 0.15 lM was obtained with the DPP III inhibitor tynorphin (40 lM)
of proctolin degradation induced with D melanogaster DPP III partially purified from the cytosol of transfected cells (data not shown)
The contamination of transfected S2 cell membranes was investigated by the addition of partially purified cytosolic DPP III to membranes prepared from trans-fected S2 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 contaminated 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 S2 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).
Trang 6metazoan 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 S2 cells After 48 h induction, transfected S2 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 melanogasterDPP 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 Kmvalues of the recombinant D melanogaster DPP III for proctolin (4 lMand 3 lM) are very similar to the Kmvalue (also 4 lM)
of the presumed DPP III purified in cockroach [12] The potency of tynorphin against recombinant DPP III (IC50¼ 0.62 lM) and that purified in cockroach (0.68 lM) 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-gasterDPP III cDNA in S2cells 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 cells and recovered as
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 lg protein 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.
Trang 7However, 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 S2 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 S2 cells Both bands are also identified after partial purification from transfected S2cells 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 S2 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 S2cells with or without detergent indicated that D melanogaster DPP III is possibly expressed at the cell surface Even though the contamination of transfected S2cell membrane preparations from transfected S2 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 S2cell 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 S2 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 Km 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)1min)1) Purification factor
Trang 8The 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 S2 cell
culture media as verified by Western blot analysis
Furthermore, no trace of DPP activity could be measured
in ultrafiltered culture media of transfected S2 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 melanogasterDPP 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 S2 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 S2 cells (Fig 3C) These results provide evidence that D melanogaster DPP III is expressed in S2 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 S2 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 melanogasterDPP 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 Blackframes 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.
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