Upload
claire-mazzocco
View
215
Download
0
Embed Size (px)
Citation preview
Identification and characterization of two dipeptidyl-peptidase III isoforms in Drosophila melanogasterClaire Mazzocco1, Jennifer Gillibert-Duplantier2, Veronique Neaud2, Kayoko M. Fukasawa3,Stephane Claverol4, Marc Bonneu4 and Jacques Puiroux1
1 Laboratoire de Neurobiologie des Reseaux, CNRS-UMR 5816, Universite Bordeaux I, Talence, France
2 Groupe de Recherche pour l’Etude du Foie, INSERM E9917, Universite Victor Segalen Bordeaux II, Bordeaux, France
3 Department of Oral Biochemistry, Matsumoto Dental College, Nagano, Japan
4 Plateforme Genomique Fonctionnelle, Universite Victor Segalen Bordeaux II, Bordeaux, France
Dipeptidyl-peptidase (DPP) III (EC 3.4.14.4) has been
characterized in rat [1,2] and human [3] as a soluble
enzyme (molecular mass 82 kDa, SwissProt accession
numbers O55096 and Q9NY33-1), confirming the
results of their cloning and sequencing. This zinc met-
allopeptidase has also been reported to contain a speci-
fic HELLGH domain [4] which cleaves the second
bound peptide of enkephalins.
Current functional analyses of genomes have allowed
the identification of putative DPP IIIs in about 20
species. In most cases, they are deduced as � 700-amino
acid proteins containing the specific catalytic motif
HELLGH-52X-E. However, in a few cases, DPP IIIs
have been predicted despite the shorter presumed
DPP III and ⁄or the lack of the specific HELLGH
domain. In particular, the HELLGH domain is missing
from the hypothetical Caenorhabditis elegans DPP III
(NP492288, 682 residues). In humans, a truncated 317-
residue DPP III isoform (SwissProt accession number
Q9NY33-2), lacking � 420 amino acids including the
Keywords
dipeptidyl-peptidase III; enkephalinase;
neuropeptide; proctolin; proteomic
Correspondence
J. Puiroux, Laboratoire de Neurobiologie des
Reseaux, CNRS-UMR 5816, Universite
Bordeaux I, Avenue des Facultes,
33405 Talence Cedex, France
Fax: +33 540 002561
Tel: +33 540 002569
E-mail: [email protected]
(Received 2 November 2005, revised
4 January 2006, accepted 9 January 2006)
doi:10.1111/j.1742-4658.2006.05132.x
Dipeptidyl-peptidase III (DPP III) hydrolyses small peptides with a broad
substrate specificity. It is thought to be involved in a major degradation
pathway of the insect neuropeptide proctolin. We report the purification
and characterization of a soluble DPP III from 40 g Drosophila melanogas-
ter. Western blot analysis with anti-(DPP III) serum revealed the purifica-
tion of two proteins of molecular mass 89 and 82 kDa. MS ⁄MS analysis of
these proteins resulted in the sequencing of 45 and 41 peptide fragments,
respectively, confirming � 60% of both annotated D. melanogaster DPP III
isoforms (CG7415-PC and CG7415-PB) predicted at 89 and 82 kDa.
Sequencing also revealed the specific catalytic domain HELLGH in both
isoforms, indicating that they are both effective in degrading small pep-
tides. In addition, with a probe specific for D. melanogaster DPP III, nor-
thern blot analysis of fruit fly total RNA showed two transcripts at � 2.6
and 2.3 kb, consistent with the translation of 89-kDa and 82-kDa DPP III
proteins. Moreover, the purified enzyme hydrolyzed the insect neuropeptide
proctolin (Km � 4 lm) at the second N-terminal peptide bound, and
was inhibited by the specific DPP III inhibitor tynorphin. Finally, anti-
(DPP III) immunoreactivity was observed in the central nervous system of
D. melanogaster larva, supporting a functional role for DPP III in procto-
lin degradation. This study shows that DPP III is in actuality synthesized
in D. melanogaster as 89-kDa and 82-kDa isoforms, representing two
native proteins translated from two alternative mRNA transcripts.
Abbreviations
DPP III, dipeptidyl-peptidase III.
1056 FEBS Journal 273 (2006) 1056–1064 ª 2006 The Authors Journal compilation ª 2006 FEBS
catalytic motif HELLGH-52X-E, has also been deduced
in addition to the long isoform. The DPP activity of
such proteins is always questionable until they have
been expressed and purified and their activity tested.
Regardless of the authenticity of the short human
DPP III, it is worth noting the presence of two DPP III
isoforms in this species. In a slightly different fashion,
two DPP III isoforms of 80 and 76 kDa have been iden-
tified in the cockroach Blaberus craniifer [5]. Thereafter,
an ORF corresponding to a 723-residue DPP III
(82 kDa) was deduced in Drosophila melanogaster [5],
and a single gene encoding DPP III was annotated in
the D. melanogaster genome as CG7415 [6]. This con-
trasted with the detection of 89-kDa and 82-kDa protein
bands in this insect [5] and in S2 cells stably transfected
with a CG7415-related cDNA clone [7]. The synthesis of
a longer putative DPP III of 786 amino acids (89 kDa)
in D. melanogaster was finally speculated to result from
alternative splicing of mRNA. In these conditions, the
expression of two putative DPP III isoforms at 89 and
82 kDa is consistent with the prediction of the two
DPP III proteins in D. melanogaster (786 and 723
amino acids) deduced from cDNA clones.
Gathering information from proteomic analysis is
at present necessary to validate gene annotation [8].
Unusually, the analysis of the D. melanogaster genome
suggested that DPP III is probably expressed as two
long isoforms, both including the catalytic domain
HELLGH required for enzyme activity, but this nee-
ded to be validated. Therefore, we here describe the
characterization of two DPP III proteins actually syn-
thesized in D. melanogaster as native 89-kDa and
82-kDa isoforms. Moreover, northern blot analysis
revealed two DPP III mRNA transcripts with the
expected sizes for the distinct translations of the two
isoforms. Substrate specificity and inhibition studies
confirmed that the purified enzyme displayed the cru-
cial features of DPP III. In addition, we report the
localization of DPP III immunoreactivity in the central
nervous system of D. melanogaster.
Results
Purification of soluble fruit fly DPP III
D. melanogaster soluble DPP III was purified by chro-
matography by the method used to purify putative
D. melanogaster DPP III functionally expressed in S2
cells [7] and described in Experimental procedures. The
overall recovery of DPP III activity, as verified by
reversed-phase chromatography of met-enkephalin
degradation products produced during incubation with
a fraction aliquot, was 7% and corresponded to a
46-fold purification of about 400 lg protein. Analysis
of pooled active fractions by SDS ⁄PAGE and western
blotting with an antibody to rat liver DPP III revealed
only two protein bands, at � 89 and 82 kDa (Fig. 1A,
lanes 1 and 2).
MS/MS analysis of purified enzyme
Purified DPP of fruit fly (6 lg) was electrophoresed on
a 10% polyacrylamide slab gel. After Coomassie Bril-
lant Blue staining, the protein bands at 89 and 82 kDa
were excised and processed for sequence analysis. After
two separate trypsin digestions and reversed-phase sep-
arations, 41 peptide fragments (437 amino acids) were
sequenced from the 82-kDa protein band and 45 pep-
tide fragments (483 amino acids) were sequenced from
the 89-kDa protein band, including the 41 sequences
previously mentioned. The four fragments (46 amino
acids) specifically sequenced from the 89-kDa protein
band were found along the N-terminal region of the
786-residue D. melanogaster DPP III (SwissProt No
Q9VHR8-1), and matched 46 out of the first 63 amino
acids with 100% identity. The remaining 41 sequenced
A
B
Fig. 1. SDS ⁄ PAGE and western blot of purified D. melanogaster
DPP III and northern blot analysis of D. melanogaster DPP III tran-
scripts. (A) 10-lL aliquots of pooled active fractions obtained from
Superdex chromatography were separated on SDS ⁄ PAGE and sil-
ver-stained (lane1) or analysed by western blotting with DPP III
antibody (lane 2). Two major bands at 89 and 82 kDa (left arrows)
were identified with both methods. Molecular mass markers are
indicated in kDa on the right. (B) Total RNA was extracted from
fruit flies and fractionated on a 1.5% agarose gel, blotted to a
Hybond N+ membrane and hybridized using Ultrahyb solution with
the a32P-labeled D. melanogaster DPP III PCR probe (783 bp). Two
transcripts were visualized at 2.3 and 2.6 kb (right arrows). RNA
molecular mass markers are indicated in kilobase pairs on the left.
C. Mazzocco et al. Two DPP III isoforms in the fruit fly
FEBS Journal 273 (2006) 1056–1064 ª 2006 The Authors Journal compilation ª 2006 FEBS 1057
peptides were distributed in the protein region between
amino acids 64 and 786, representing the 723-residue
long DPP III isoform (SwissProt No Q9VHR8-2),
starting at the methionine at position 64 of the longest
D. melanogaster DPP III. Of the 45 sequences avail-
able, almost 62% of the long D. melanogaster DPP III
isoform were identified and almost 61% of the shorter
conceptual DPP III protein were thus verified.
Furthermore, MS ⁄MS suggested that none of the
sequenced peptide fragments displayed either glycosy-
lation or phosphorylation. For instance, four N-X-T
or N-X-S potential glycosylation sites out of six were
recovered as nonglycosylated among the sequenced
fragments of D. melanogaster DPP III. Finally, the
catalytic site HELLGH-52X-E was found among the
sequenced fragments of both D. melanogaster DPP III
isoforms, corroborating the DPP III activity measured
in the purified enzyme. In addition, after northern blot
analysis of fruit fly total RNA using a specific D. mel-
anogaster DPP III probe (783 bp), two bands at 2.6
and 2.3 kb were revealed (Fig. 1B).
Activity assay, kinetic studies and inhibition
stimulation of the purified enzyme
The purified D. melanogaster enzyme efficiently hydro-
lysed proctolin (40 lm) over 1 h incubation as indica-
ted by reversed-phase separation of the degradation
Fig. 2. Degrading activity and kinetic studies of purified D. melanogaster DPP III. (A,B) Elution profiles (280 nm) obtained after reversed-
phase separation of samples prepared from proctolin (20 lM) incubated with purified D. melanogaster DPP III for 15 min (A) and 60 min (B).
Proctolin (peak 2) was eluted at �8 min, and only traces are detected after 1 h incubation. The N-terminal dipeptide Arg-Tyr (peak 1) was
eluted at � 5 min and significantly increased according to the duration of incubation. (C,D) Increasing concentrations of proctolin (C; 2.5–
40 lM) and met-enkephalin (D; 5–300 lM) were incubated for 30 min with purified D. melanogaster DPP III. The samples were separated by
reversed-phase chromatography to identify and quantify the remaining proctolin or met-enkephalin and their metabolites produced during
incubation. The results of saturation are the mean of three independent measurements and are expressed as lmol neuropeptide degra-
dedÆmin)1Æ(mg purified enzyme))1 (corresponding Lineweaver–Burk plots are included).
Two DPP III isoforms in the fruit fly C. Mazzocco et al.
1058 FEBS Journal 273 (2006) 1056–1064 ª 2006 The Authors Journal compilation ª 2006 FEBS
products. Only the N-terminal dipeptide Arg-Tyr
(monitored at 280 nm, Fig. 2A,B, and 206 nm,
Fig. 3A,C) and the C-terminal tripeptide Leu-Pro-Thr
(detected at 206 nm, Fig. 3A,C) were liberated from
proctolin incubated with the purified enzyme, in the
presence of bestatin (100 lm) to inhibit aminopepti-
dase activity. After a 2 h incubation, no other degrada-
tion products could be detected other than the
dipeptide Arg-Tyr (Fig. 2B) and the tripeptide (not
shown). We further characterized the degrading activ-
ity of the purified D. melanogaster enzyme by incuba-
tion with increasing concentrations of the insect
neuropeptide proctolin (from 2.5 to 40 lm) and met-
enkephalin (from 5 to 300 lm). A Km of 4.5 lm was
calculated for proctolin (Fig. 2C) and 41.9 lm for
met-enkephalin (Fig. 2D). Vmax values were 0.14 lmol
proctolinÆmin)1Æ(mg protein))1 and 0.11 lmol met-
enkephalinÆmin)1Æ(mg protein))1.
Compared with the rate of proctolin degradation in
control conditions (Fig. 3A), the addition of the metal
chelator EGTA (1 mm) to the incubation medium pre-
vented 96% of proctolin degradation (Fig. 3B). In
these inhibiting conditions, the addition of 1 mm Zn2+
restored and slightly increased (110%) the proctolin-
degrading activity (Fig. 3C). When the bivalent ion
Zn2+ was tested at 0.1 mm on the purified enzyme, a
slight inhibition of proctolin-degrading activity (81%
of control) was measured (Fig. 3D). At higher concen-
trations (1 mm), Zn2+ almost completely prevented
(� 0.2%) the hydrolysis of proctolin (Fig. 3D). In
Fig. 3. Inhibition and stimulation of the puri-
fied D. melanogaster DPP III. (A–C) Elution
profiles (206 nm) obtained after reversed-
phase separation of samples prepared from
proctolin (250 lM) incubated with purified
D. melanogaster DPP III for 10 min in con-
trol conditions (A), in the presence of 1 mM
EGTA (B) or in the presence of 1 mM EGTA
and 1 mM ZnSO4 (C). The Arg-Tyr dipeptide
(peak 1) and the Leu-Pro-Thr tripeptide (peak
2) were detected after the control incuba-
tions. In contrast, the presence of the metal
chelator in the incubation medium preven-
ted the degradation of proctolin (peak 3),
and no dipeptide or tripeptide could be
detected. When proctolin was incubated
with EGTA and ZnSO4, the enzyme activity
was restored and slightly increased (110%
compared with control conditions) as indica-
ted by the detection of both Arg-Tyr (peak 1)
and Leu-Pro-Thr (peak 2). (D) Proctolin
(250 lM) was incubated with purified
D. melanogaster DPP III for 60 min in con-
trol conditions (C), or after preincubation
with 0.1 or 1 mM ZnSO4 (Zn2+) or with 0.1
or 1 mM CoCl2 (Co2+) or with the specific
DPP III inhibitor tynorphin (Tyn.) at 10 lM.
The results are mean ± SD from three
experiments.
C. Mazzocco et al. Two DPP III isoforms in the fruit fly
FEBS Journal 273 (2006) 1056–1064 ª 2006 The Authors Journal compilation ª 2006 FEBS 1059
contrast, the DPP III activity of the purified enzyme
was strongly increased (516% and 758%) by, respect-
ively, 0.1 mm and 1 mm Co2+ (Fig. 3D). As expected,
the specific DPP III inhibitor tynorphin (10 lm) pre-
vented 99% of proctolin degradation induced by the
purified D. melanogaster DPP III (Fig. 3D).
Central nervous system immunoreactivity
The central nervous system of third-instar larvae of
D. melanogaster was studied with antibody to DPP III.
Cell bodies of nerve cells were positively stained in the
compound ventral ganglion (Fig. 4). The most strongly
immunoreactive cells were visualized at the posterior
end of the ventral ganglion (Fig. 4A). At this location,
most of the cell bodies exhibited significant DPP III
immunoreactivity (Fig. 4B–E), the size of somata ran-
ging from 8 to 10 lm in diameter. Confocal analyses
at high magnification clearly showed that DPP III
immunoreactivity was restricted to the cytosol and
possibly the cell membranes (Fig. 4C). Although the
cell bodies in the ventral ganglion showed strong
DPP III immunoreactivity, stained neurites were scar-
cely observable. Analysis of a series of confocal micro-
scopy slices across the dorsoventral axis of the ventral
ganglion indicated that the somata of stained cells
were uniformly localized at the cortical region of the
ventral ganglion (Fig. 4C–E). When the confocal stud-
ies were focused at the median dorsoventral level, the
segmental neuropil regions were clearly visualized as
bilateral and symmetrical dark masses (Fig. 4C). In
addition, some cortical cell bodies of the posterior
end of the ventral ganglion were visualized by both
Nomarski optic analysis [9] and fluorescence analysis
(Fig. 4F).
Discussion
We here report the characterization of two soluble
DPP III isoforms purified from 40 g fruit flies by three
steps of chromatography. Our results demonstrate that
DPP III is actually expressed in D. melanogaster as
two potently active isoforms and validate both pre-
sumed DPP IIIs annotated from the D. melanogaster
genome [5,6].
In invertebrates, DPP III was first characterized in
Blaberus craniifer as two isoforms of 80 and 76 kDa
[5,10]. Two DPP III-related proteins were also detected
in D. melanogaster at 89 and 82 kDa [5]. Progress in
the sequencing of the D. melanogaster genome allowed
the prediction of first an 82-kDa DPP III [5,6] and
then an 89-kDa DPP III isoform [6]. Both presumed
D. melanogaster DPP III isoforms were functionally
expressed and, indeed, displayed genuine DPP III
activity [7]. We characterized the DPP IIIs actually
detected in D. melanogaster in order to elucidate the
relationship between the 89-kDa and 82-kDa isoforms.
The purified soluble DPP III from D. melanogaster
exhibited similar properties to those of purified B. cra-
niifer DPP III, particularly those of the presumed
D. melanogaster DPP III functionally expressed. When
analysed by electrophoresis and western blotting with
antibody to rat liver DPP III, two major bands at 89
and 82 kDa were detected (Fig. 1A) in the active frac-
tions from size exclusion chromatography. MS ⁄MS
analysis resulted in the sequencing of � 60% of both
isoforms, and 100% identity was observed with the
conceptual D. melanogaster DPP IIIs deduced from
the CG7415 gene [6], confirming the results obtained
from the functional expression of the GH01916 clone,
encoding one presumed D. melanogaster DPP III [7].
None of the 45 fragments of D. melanogaster
DPP III examined by MS ⁄MS displayed either phos-
phorylation or glycosylation. The molecular masses
deduced from the 786 and 723 residue theoretical
D. melanogaster DPP IIIs (89 195 and 81 937 Da,
respectively) are closely related to the apparent
molecular masses calculated from SDS ⁄PAGE of the
actual D. melanogaster DPP III proteins (89 and
82 kDa). So, the difference in molecular mass between
the two isoforms, formerly hypothesized to be the
result of post-translational processing of the predicted
82-kDa DPP III protein [5], may be explained by the
sole N-terminal extension of 63 residues retrieved on
the 89-kDa isoform and partly sequenced. In addition,
northern blot analysis of fruit fly total RNAs with a
D. melanogaster DPP III-specific probe revealed two
bands at 2.6 and 2.3 kb (Fig. 1B) that might corres-
pond to the two speculated alternative mRNA tran-
scripts presumably encoding the long or the short
DPP III in this species [6]. Finally, western blot analy-
sis with an anti-rat liver polyclonal antibody never
highlighted a 7-kDa peptide, precluding the 82-kDa
D. melanogaster DPP III resulting from hydrolysis of
the 89-kDa isoform. From these results, it can be con-
cluded that the two D. melanogaster DPP III isoforms
are probably synthesized as two independent native
proteins (89 and 82 kDa), translated from two distinct
alternative mRNAs (2.6 and 2.3 kb) transcribed from
a single gene (CG7415).
The catalytic motif HELLGH-52X-E sequence (all
but three amino acids) was sequenced for both purified
D. melanogaster DPP III isoforms, suggesting that
both are effective in degrading small neuropeptides
such as met-enkephalin and proctolin, although the
89-kDa and 82-kDa isoforms were not assayed sepa-
Two DPP III isoforms in the fruit fly C. Mazzocco et al.
1060 FEBS Journal 273 (2006) 1056–1064 ª 2006 The Authors Journal compilation ª 2006 FEBS
rately in degradation studies. This is the first example
of two presumably active DPP III isoforms in a spe-
cies. The purified D. melanogaster DPP III was incuba-
ted with increasing concentrations of proctolin or
met-enkephalin as substrates (Fig. 2C,D). It is clear
that proctolin is more efficiently metabolized (Km ¼4.5 lm) than met-enkephalin (Km ¼ 41.9 lm). These
Km values are closely related to those of B. craniifer
Fig. 4. Detection of D. melanogaster DPP III
by immunohistochemistry in the central ner-
vous system of the third instar larva of fruit
fly. The expression of protein immunologi-
cally related to DPP III is shown in whole-
mounted ventral nerve cord (viewed from
the dorsal side) of D. melanogaster after
confocal observations. (A) Anti-(DPP III)
histochemistry displayed in a horizontal
30-lm-thick medioventral slice of the com-
pound ventral ganglion. The arrows point to
cell bodies of cortical neurons. Dark central
areas correspond to neuropil regions of the
compound ganglion. (B) Details of anti-
(DPP III) immunohistochemistry observed at
the posterior extremity of the ventral gan-
glion. The confocal acquisition represents a
50-lm-thick medioventral slice of the ventral
ganglion and shows several cell bodies of
positively stained neurons (arrows). (C–E)
Serial 10-lm-thick horizontal acquisitions
beginning in the medioventral region (C) and
almost ending at the ventral side of the
compound ganglion (E). Arrows indicate
positively stained neurons with fluorescence
localized in the cytosol. (F) A single 0.5-lm-
thick medioventral slice (compare with C)
was acquired for fluorescence and by phase
interference (Nomarsky) to delimit cell body.
The two analyses are superimposed and
clearly demonstrate the presence of the
fluorescent signal in the cytosol of two dis-
tinct cell bodies (arrows).
C. Mazzocco et al. Two DPP III isoforms in the fruit fly
FEBS Journal 273 (2006) 1056–1064 ª 2006 The Authors Journal compilation ª 2006 FEBS 1061
DPP III [5] and those of the functionally expressed
D. melanogaster DPP III [7]. The inhibiting effect of
EGTA, the restoring effect of Zn2+ and the strong
increasing effect of Co2+ on the activity of purified
D. melanogaster DPP III (Fig. 3) are in line with the
properties of human placental DPP III [11] and are
consistent with the expected properties of the M49
family of metallopeptidases to which DPP III belongs.
Finally, the specific DPP III inhibitor tynorphin [12]
almost completely abolished the activity of the purified
D. melanogaster DPP III (Fig. 3D), supporting the
identification of the purified proteins as a DPP III.
Proctolin-degrading DPP III activity has previously
been reported in several tissues of locust [13] and fur-
ther detailed in the central nervous system of Schistoc-
erca gregaria [14], but little is known about the
immunohistochemical localization of DPP III in inver-
tebrates. The immunohistochemical studies reported
here showed significant staining of DPP III in the cen-
tral nervous system of D. melanogaster larva, widely
spread in the cortex. The cytosol and possibly the
outer cell membrane of nerve cells were strongly
stained. Given these immunohistochemical results and
taking into account the fact that the DPP III purifica-
tion reported here was from a soluble source, DPP III
is undoubtedly expressed in D. melanogaster as a cyto-
solic enzyme functioning in peptide degradation, cata-
bolism and ⁄or processing.From this characterization of the purified D. melano-
gaster DPP III, it can be concluded that this peptidase is
actually synthesized in the insect as two native isoforms
of 89 and 82 kDa, translated from two distinct mRNA
transcripts, confirming the annotation of the D. melano-
gaster gene CG7415. In contrast with other models,
both D. melanogaster DPP III isoforms include the
catalytic motif HELLGH and are presumably active.
Experimental procedures
Materials
Bestatin, diaminobenzidine, Hepes, met-enkephalin and
proctolin were purchased from Sigma (St Louis, MO,
USA). All chromatographic materials were from Amersham
Pharmacia Biotech (Orsay, France). Rat liver DPP III anti-
bodies and the specific DPP III inhibitor tynorphin were
provided by K. M. Fukasawa (Matsumoto Dental College,
Nagano, Japan).
Dissections and immunocytochemistry
Larvae of D. melanogaster (Canton strain) were dissected in
insect saline (140 mm NaCl, 5 mm KCl, 5 mm CaCl2, 4 mm
NaHCO3, 1 mm MgCl2, 5 mm trehalose, 100 mm sucrose,
adjusted to pH 7.2 with 5 mm Tris). Brain hemispheres and
the ventral ganglion were dissected with the peripheral nerves
cut short after removal of imaginal discs and the gut. Ner-
vous tissues were fixed in cold paraformaldehyde (4%) for
� 12 h. The rat liver DPP III-specific antiserum [1] was used
for immunocytochemistry. Labeling was demonstrated using
fluorescein-labeled goat anti-rabbit IgG (Vector Laboratories
Inc., Burlingame, CA, USA). The immunocytochemical
results were based on confocal analyses using a Bx51 Fluo-
view 500 confocal microscope (Olympus France, Rungis,
France) equipped with an argon laser light source.
Purification of enzyme
Fruit flies (40 g) were homogenized in 50 mL cold Hepes
buffer [buffer A: 10 mm Hepes, pH 7.2, 5% (w ⁄ v) glycerol,5 mm MgCl2 and 1 mm phenylmethanesulfonyl fluoride]
using a motor driven Potter-Elvehjem homogenizer with a
Teflon pestle. The sample was prepared from soluble pro-
teins obtained from the homogenate after centrifugation at
30 000 g for 20 min at 4 �C. The supernatant was vaccum
filtered (Millex-GV Millipore 0.22 lm) and the protein con-
centration was adjusted to 1 mgÆmL)1 in buffer A (total
volume of 800 mL). This large volume was processed in
four independent separations (200 mL each) loaded on to a
5-mL prepacked Hi-Trap Q Sepharose cartridge previously
equilibrated with buffer A delivered at 5 mLÆmin)1 with an
AKTA FPLC system. Elution was obtained with a gradient
of buffer B (buffer A containing 1 m NaCl, 2% NaClÆmin)1) and was monitored at 280 nm. Aliquots (100 lL) offractions (5 mL) were analyzed for DPP activity (see under
Activity assay, kinetic studies and inhibition-stimulation of
purified enzyme). Then, the fractions containing DPP activ-
ity were pooled (60 mL), and aliquots were tested for DPP
activity and total protein content. The pooled sample was
fourfold diluted with buffer A to reduce the concentration
of NaCl and loaded on to a second 5-mL prepacked Hi-
Trap Q Sepharose cartridge used as above but with a shal-
lower gradient (0.5% NaClÆmin)1). Aliquots (100 lL) of
fractions (2.5 mL) were tested for DPP activity. Fractions
containing DPP activity were pooled (20 mL) and concen-
trated (� 750 lL) by ultrafiltration (Macrosep; molecular
mass cut-off point 10 kDa; Pall Filtron, Northborough,
MA, USA) at 4000 g (J2-MC, rotor JA 20; Beckman Coul-
ter, Roissy, France) at 4 �C for 90 min, and aliquots
(2.5 lL) were analyzed for DPP activity and protein con-
tent. The remaining pooled fractions were loaded on to a
Superdex 200 HR 10 ⁄ 30 column equilibrated with buffer A
at a constant flow rate of 0.25 mLÆmin)1. Fractions
(250 lL) were collected and analyzed for DPP activity in
5-lL aliquots. Fractions containing DPP activity were
pooled and stored at )20 �C. Proteins were measured at
the different steps of purification with a commercial reagent
Two DPP III isoforms in the fruit fly C. Mazzocco et al.
1062 FEBS Journal 273 (2006) 1056–1064 ª 2006 The Authors Journal compilation ª 2006 FEBS
(Bio-Rad, Marne la Coquette, France) based on the Brad-
ford method [15].
Activity assay, kinetic studies and inhibition
stimulation of purified enzyme
During the purification process, the presence of DPP activ-
ity was verified by incubating aliquots of fractions or aliqu-
ots of pooled and concentrated fractions in a final volume
of 150 lL buffer A containing met-enkephalin (Tyr-Gly-
Gly-Phe-Met, 40 lm) as substrate with constant stirring at
20 �C. In addition, degradation studies were performed by
incubating 1 lL purified enzyme in a final volume of 75 lLwith the insect neuropeptide proctolin (Arg-Tyr-Leu-Pro-
Thr) as substrate, tested at concentrations of 2.5–40 lm, orwith met-enkephalin, tested at 5–300 lm. Samples were pre-
incubated for 15 min in the presence of the aminopeptidase
inhibitor bestatin (100 lm), then the neuropeptide was
added for an additional 30-min incubation. Alternatively,
the specific DPP III inhibitor tynorphin (Val-Val-Tyr-Pro-
Trp, 10 lm) or the metal chelator EGTA (1 mm) or the
bivalent metal ions Zn2+ (0.1 or 1 mm ZnSO4) or Co2+
(0.1 or 1 mm CoCl2) was preincubated for 15 min (with
bestatin, 100 lm) before the addition of proctolin (250 lm).The incubations were stopped by the addition of 30 or
15 lL HCl (1 m) and centrifugation at 16 500 g at 4 �C for
5 min (Hettich EBA 12R, Saint-Herblain, France). The
determination of enzyme activity was based on the separ-
ation of the products of neuropeptide hydrolysis by
reversed-phase chromatography. Supernatants were loaded
on a Pharmacia PepRPC HR 5 ⁄ 5 reversed-phase column
connected to an AKTA FPLC system delivering 0.1% tri-
fluoroacetic acid in MilliQ water with pump A and 0.1%
trifluoroacetic acid ⁄ 60% acetonitrile in MilliQ water with
pump B at 1 mLÆmin)1. Degradation products were separ-
ated using the following gradient conditions: 3% aceto-
nitrileÆmin)1 for the first 5 min, then 0.6% acetonitrileÆmin)1
for the next 15 min. This was monitored at 206 and 280 nm.
The N-terminal Arg-Tyr and Tyr-Gly fragments generated
during the incubation of, respectively, proctolin and met-
enkephalin, and the C-terminal tripeptide Leu-Pro-Thr
fragmented from proctolin were identified by coelution with
standard solutions of these peptides. A curve-fitting compu-
ter program was used to determine the Km values for the
two neuropeptides, after computation of the saturations
curves.
SDS/PAGE and western blot analysis
of purified enzyme
Electrophoresis was carried out by the method of Lae-
mmli [16] on a 1 mm-thick separating mini-gel with 0.1%
SDS run at a constant 30 mA current in Tris ⁄ glycinebuffer (pH 8.3). Purified enzyme was prepared in SDS
sample buffer [62 mm Tris ⁄HCl, pH 6.8, containing 10%
(w ⁄ v) glycerol and 0.025% (w ⁄ v) bromophenol blue
added with 1% (w ⁄ v) SDS], heated for 5 min at 90 �C.Gels were silver-stained (Silver Staining Plus kit; Bio-
Rad) or processed for western blotting on nitrocellulose
membrane (Hybond C; Pharmacia) for 1 h using a semi-
dry transfer apparatus (Bio-Rad). The membrane was
soaked in Tris buffer ⁄ saline with Tween-20 [20 mm
Tris ⁄HCl, 137 mm NaCl, 0.1% (w ⁄ v) Tween-20, pH 7.3]
added with 5% (w ⁄ v) dry low fat milk for 1 h at room
temperature with constant stirring. The membrane was
then rinsed twice for 10 min at room temperature in Tris
buffer ⁄ saline ⁄Tween and incubated with rabbit rat liver
DPP III polyclonal antibody (1 : 2000) in Tris buffer ⁄saline ⁄Tween overnight at 4 �C with constant slow agita-
tion. The membrane was rinsed twice in Tris buf-
fer ⁄ saline ⁄Tween for 15 min at room temperature and
incubated with goat anti-rabbit IgG (1 : 1000) conjugated
with horseradish peroxidase (Roche Applied Science,
Meylan, France) in Tris buffer ⁄ saline ⁄Tween for 1 h at
room temperature with slow agitation. The membrane
was rinsed twice for 10 min at room temperature in Tris
buffer ⁄ saline ⁄Tween and incubated with 1.39 mm diamino-
benzidine in 50 mm Tris ⁄HCl, pH 7.3, for 5 min at room
temperature with constant agitation. Staining was per-
formed by incubating the membrane with a fresh solution
of 1.39 mm diaminobenzidine in Tris ⁄HCl added with
H2O2 (1 lL in 10 mL buffer).
MS/MS analysis of the purified enzyme
Purified enzyme (20 lL) was separated by SDS ⁄PAGE on a
10% polyacrylamide slab gel (1 mm thick). The gel was
stained with Coomassie Brillant Blue R-250. Two major
bands (89 and 82 kDa) were excised, extracted from the
gel, and trypsin digested. Protein fragments were analysed
by MS ⁄MS (LCQ DecaXPlus Thermofinnigan; San Juan,
Puerto Rico) at the Plateforme Genomique Fonctionnelle
(Universite Bordeaux II, France).
Northern blot analysis
Total RNAs were isolated from 200 mg D. melanogaster
homogenized in lysis buffer [4 m guanidine thiocyanate,
25 mm sodium citrate, 0.5% (w ⁄ v) sarkosyl and 0.1 m
2-mercaptoethanol, adjusted to pH 7) with a Mixer Mill
MM300 (Qiagen, Courtabœuf, France) using the method of
Chomczynski & Sacchi [17]. Total RNAs (0.5–2.5 lg) wereseparated on 1.5% agarose gel prepared in Mops buffer
(20 mm Mops, pH 7.0, 8.5 mm sodium acetate, 1 mm
EDTA and 0.2 m formaldehyde) containing ethidium bro-
mide (0.1 lgÆmL)1) and then transferred to a Hybond N+
membrane (Amersham Pharmacia Biotech) by downward
capillary transfer in Mops buffer. Blots were probed with
C. Mazzocco et al. Two DPP III isoforms in the fruit fly
FEBS Journal 273 (2006) 1056–1064 ª 2006 The Authors Journal compilation ª 2006 FEBS 1063
a 783-bp PCR fragment of D. melanogaster DPP III
(sense, 5¢-GAATTCGAGGGCTTCGTGGCC-3¢; antisense,5¢-AACGAGTCCTTCGCCTGCCTC-3¢), labeled with
[32P]dCTP[aP] by random priming using Ready-to-go DNA
labeling beads (Amersham Biosciences). Hybridizations
were performed using Ultrahyb solution (Ambion, Hun-
tingdon, Cambs., UK). The blots were washed in stringent
conditions (0.1 · NaCl ⁄Cit, 0.1% SDS at 42 �C and 65 �C)and analyzed (Instant Imager; Packard Instruments, Meri-
dien, CT, USA).
References
1 Fukasawa K, Fukasawa KM, Kanai M, Fujii S, Hirose
J & Harada M (1998) Dipeptidyl peptidase III is a zinc
metallo-exopeptidase: molecular cloning and expression.
Biochem J 329, 275–282.
2 Ohkubo I, Li YH, Maeda T, Yamamoto Y, Yamane T,
Du PG & Nishi K (1999) Dipeptidyl peptidase III from
rat liver cytosol: purification, molecular cloning and
immunohistochemical localization. Biol Chem 380,
1421–1430.
3 Hashimoto J, Yamamoto Y, Kurosawa H, Nishimura
K & Hazato T (2000) Identification of dipeptidyl pepti-
dase III in human neutrophils. Biochem Biophys Res
Commun 273, 393–397.
4 Fukasawa K, Fukasawa KM, Iwamoto H, Hirose J &
Harada M (1999) The HELLGH motif of rat liver
dipeptidyl peptidase III is involved in zinc coordination
and the catalytic activity of the enzyme. Biochemistry
38, 8299–8303.
5 Mazzocco C, Fukasawa KM, Raymond AA & Puiroux
J (2001) Purification, partial sequencing and characteri-
zation of an insect membrane dipeptidyl aminopeptidase
that degrades the insect neuropeptide proctolin. Eur J
Biochem 268, 4940–4949.
6 Adams MD (2000) The genome sequence of Drosophila
melanogaster. Science 287, 2185–2195.
7 Mazzocco C, Fukasawa KM, Auguste P & Puiroux J
(2003) Characterization of a functionally expressed
dipeptidyl aminopeptidase III from Drosophila melano-
gaster. Eur J Biochem 270, 3074–3082.
8 Hewes RS & Taghert PH (2001) Neuropeptides and
neuropeptide receptors in the Drosophila melanogaster
genome. Genome Res 11, 1126–1142.
9 Allen RD, David GB & Nomarski G (1969) The
Zeiss-Nomarski differential interference equipment for
transmitted-light microscopy. Z Wiss Mikrosk. 69,
193–221.
10 Mazzocco C & Puiroux J (2000) Purification of procto-
lin-binding protein from the foregut of the insect Bla-
berus craniifer. Eur J Biochem 267, 2252–2259.
11 Shimanori Y, Watanabe Y & Fujimoto Y (1988)
Human placental dipeptidyl aminopeptidase III: hydro-
lysis of enkephalins and its stimulation by cobaltous
ion. Biochem Med Metab Biol 40, 305–310.
12 Yamamoto Y, Hashimoto J-I, Shimamura M, Yamagu-
chi T & Hazato T (2000) Characterization of tynorphin,
a potent endogenous inhibitor of dipeptidyl peptidase
III. Peptides 21, 503–508.
13 Quistad GB, Adams ME, Scarborough RM, Carney RL
& Schooley DA (1984) Metabolism of proctolin, a
pentapeptide neurotransmitter in insects. Life Sci 34,
569–576.
14 Isaac RE (1987) Proctolin degradation by membrane
peptidases from nervous tissues of the desert locust
Schistocerca gregaria. Biochem J 245, 365–370.
15 Bradford MM (1976) A rapid and sensitive method for
the quantitation of microgram quantities of proteins
utilizing the principle of dye binding. Anal Biochem 72,
248–254.
16 Laemmli UK (1970) Cleavage of structural proteins
during the assembly of the head of bacteriophage T4.
Nature 227, 680–685.
17 Chomczynski P & Sacchi N (1987) Single-step method
of RNA isolation by acid guanidinium thiocyanate-
phenol-chloroform extraction. Anal Biochem 162,
156–159.
Two DPP III isoforms in the fruit fly C. Mazzocco et al.
1064 FEBS Journal 273 (2006) 1056–1064 ª 2006 The Authors Journal compilation ª 2006 FEBS