Identification and characterization of two dipeptidyl-peptidase III isoforms in Drosophila melanogaster

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.


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.



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-



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).



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



(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



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).

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Identification and characterization of two dipeptidyl-peptidase III isoforms in Drosophila melanogaster