Báo cáo Y học: Characterization of four substrates emphasizes kinetic similarity between insect and human C-domain angiotensin-converting enzyme pptx

Characterization of four substrates emphasizes kinetic similarity
between insect and human C-domain angiotensin-converting enzyme
Korneel Hens
1
, Anick Vandingenen
1
, Nathalie Macours
1
, Geert Baggerman
1
, Adriana Carmona
Karaoglanovic
2
, Liliane Schoofs
1
, Arnold De Loof
1
and Roger Huybrechts
1
1
Zoological Institute of the Catholic University of Leuven, Laboratory of Developmental Physiology and Molecular Biology, Leuven,
Belgium;
2
Universidade Federal de Sao Paulo, Escola Paulista de Medicina, Department of Biophysics, Sao Paulo, Brazil
Angiotensin converting enzyme (ACE) was already discov-
ered in insects in 1994, but its physiological role is still
enigmatic. We have addressed this problem by purifying
four new ACE substrates from the ovaries of the grey
fleshfly, Neobellieria bullata. Their primary structures were
identified as NKLKPSQWISLSD (Neb-ODAIF-1
1)13

),
NKLKPSQWI (Neb-ODAIF-1
1)9
), SLKPSNWLTPSE
(Neb-ODAIF-2) and LEQIYHL. Database analysis showed
significant homology with amino acid sequence stretches as
present in the N-terminal part of several fly yolk proteins. An
antiserum raised against Neb-ODAIF-1
1)9
immunostained
one out of three yolk protein bands of SDS/PAGE-separ-
ated fly haemolymph and egg homogenate, thus confirming
that these peptides originate from a yolk protein gene
product. Kinetic analysis of these peptides and of the
peptides Neb-ODAIF and Neb-ODAIF-1
1)7
with insect
ACE and human ACE show both similar and unique
properties for insect ACE as compared with human
C-domain ACE.
Keywords: ACE kinetics; domain specific substrates; insect
physiology; reproduction.
Insect ACE was first isolated from head membranes of the
housefly Musca domestica in 1994 [1], a long time after the
discovery of its mammalian counterpart in horse plasma in
1956. Since this discovery, and after cloning and purification
of several insect ACEs it has become clear that insect and
mammalian ACE, despite of their evolutionary distance, are
structurally remarkably similar. The molecular biological
analyses of insect ACEs revealed a high cDNA and amino

acid sequence conservation with mammalian ACE, especi-
ally around the active site [2]. The enzymatic activity is also
well conserved as insect ACE can hydrolyse mammalian
ACE substrates such as angiotensin I, substance P, lutein-
izing hormone releasing hormone, enkephalins and enkeph-
alinamides, hereby displaying the same exo- and
endopeptidase activities as mammalian ACE [3].
Mammalian ACE occurs in two isoforms. Somatic ACE
(sACE) has a wide tissue distribution and has two active
domains, probably generated by gene duplication of asmaller
ancestral gene. Testicular ACE is transcribed from the same
gene as sACE but from another, intragenic promotor [4]. It
has a single active domain. In Drosophila melanogaster two
isoforms of the enzyme have been found as well, namely
AnCE and ACER [5]. This suggests that gene duplication
has occurred in both Deuterostomia and Protostomia.
Another difference between the mammal and insect ACE
is the presence and absence, respectively, of a membrane
anchor at the C-terminal part of the enzyme. As a
consequence, mammalian ACE is mainly membrane bound
while insect ACE is soluble.
Mammalian sACE is involved in regulating blood pressure
and water and electrolyte homeostasis. Indications about the
role of insect ACE range from prohormone processing [6]
over immunity [7,8], to neurotransmitter inactivation [6].
Several reports indicate a role of ACE in insect reproduction
as well. In addition to impaired male fertility following ACE
gene knock-out in Drosophila [9], Schoofs et al. found ACE
immunoreactivity in the testis of Locusta migratoria, Neobel-
lieria bullata and Leptinotarsa decemlineata [6]. These find-

ings were complemented by measurements of enzyme activity
in Locusta migratoria [10] and Neobellieria bullata [11].
A major problem in resolving a physiological role for
insect ACE is the lack of known in vivo substrates. In this
respect we observed that fly ovaries are a rich source of
ACE-competitive substances [11]. Hence, we describe the
purification and characterization of four novel ACE
substrates from fleshfly ovaries and discuss their possible
physiological functions.
MATERIALS AND METHODS
Animals, haemolymph and tissue collection
The grey fleshfly Neobellieria bullata was reared as described
[12]. Staging of ovarian development was done according to
Correspondence to K. Hens, K. U. Leuven, Laboratory of Develop-
mental Physiology and Molecular Biology, Naamsestraat 59, 3000
Leuven, Belgium. Fax: +32 16 32 39 02, Tel.: +32 16 32 42 60,
E-mail:
Abbreviations: Abz, aminobenzoic acid; ACE, angiotensin converting
enzyme; ACN, acetonitrile; cACE, C-domain of human angiotensin
converting enzyme; Dnp, dinitrophenyl; ESI Q TOF MS, electrospray
ionization quadrupole time of flight mass spectrometry; nACE,
N-domain of human angiotensin converting enzyme; Neb, Neobellieria
bullata; Lom, Locusta migratoria; ODAIF, ovary derived
angiotensin converting enzyme interactive factor; PAP, peroxidase
antiperoxidase; PVDF, polyvinylidene difluoride; sACE, somatic
angiotensin converting enzyme; tACE, testicular angiotensin
converting enzyme; TMOF, trypsin modulating oostatic factor.
(Received 21 February 2002, revised 5 June 2002,
accepted 8 June 2002)
Eur. J. Biochem. 269, 3522–3530 (2002) Ó FEBS 2002 doi:10.1046/j.1432-1033.2002.03043.x

Pappas and Fraenkel [13]. For collection of haemolymph, a
leg was amputated of anaesthetized flies, the haemolymph
was drawn from the resulting wound with a capillary and
diluted immediately in ice-cold borate buffer [50 m
M
Borax,
0.3
M
NaCl, 0.2
M
(NH
4
)
2
SO
4
, pH 7.5].
The desert locust Locusta migratoria was raised as
described [14]. Only yellow coloured, sexually mature males
were used for collection of testes.
Synthetic peptides
NKLKPSQWISLSD (Neb-ODAIF-1
1)13
), NKLKPSQ
WISL (Neb-ODAIF-1
1)11
), NKLKPSQWI (Neb-ODAIF-
1
1)9
), NKLKPSQ (Neb-ODAIF-1

1)7
), SLKPSNWLTPSE
(Neb-ODAIF-2) and LEQIYHL were from Research Gen-
etics Inc. (Huntsville, AL, USA). Synthesis and character-
istics of the internally quenched fluorescent ACE substrate
O-aminobenzoic acid-Phe-Arg-Lys-2,4-dinitrophenyl-
Pro[Abz-FRK-(Dnp)P] was as described previously [15].
Purification of mammalian and insect ACE enzymes
Chinese hamster ovary cells expressing recombinant human
sACE, cACE and nACE were a kind gift of P. Corvol and
A. Michaud (Institut National de la Sante
´
at de la
Recherche Medicale, Paris). Cell culture and ACE purifi-
cation were as described [16].
Purification of Locusta migratoria testicular ACE was as
described [10].
Tissue extraction and HPLC purification
Eight thousand mid-vitellogenic (stage 4B) or late-vitello-
genic (stage 4C) fly ovaries were dissected, extracted and
prepurified as described [11]. Columns and operating
conditions of subsequent HPLC (Gilson) fractionations
were: (a) Xterra C18 (Waters Xterra RP18, 7.5 · 300 mm,
7 lm), elution with a linear gradient of 0% to 50% ACN in
0.1% trifluoroacetic acid in 120 min and a flow rate of
2mLÆmin
)1
. Two-ml fractions were automatically collected;
(b) C8 column (Supelco, LC-8DB, 4.6 · 250 mm, 5 lm),
elution with a linear gradient of 0% to 50% ACN in 0.1%

trifluoroacetic acid in 120 min and a flow rate of
1mLÆmin
)1
. Fractions according to absorbance peaks were
manually collected; (c) PKB 100 column (Supelco, PKB
100, 4.6 · 250 mm, 5 lm), elution and collection as (b);
(d) Hypercarb column (Thermoquest, Hypercarb,
3 · 250 mm, 5 lm), elution with a linear gradient of 0%
to 81% ACN in 0.01% trifluoroacetic acid in 90 min and a
flow rate of 0.3 mLÆmin
)1
. Fractions were manually collec-
ted. Absorbance was measured at 214 nm with a Waters
486 tunable absorbance detector.
ACE inhibition screening
The ACE inihibition assay is based on the ACE activity
assaybyRyanet al. [17], modified by Vandingenen et al.
[11]. Briefly, ACE-activity in diluted fly haemolymph is
measured with a synthetic, tritiated ACE substrate
p-[
3
H]benzoylglycylglycylglycine (Sigma) (¼ standard con-
dition). Adding 10 l
M
final concentration of captopril
(Sigma) served as a negative control. Captopril is a strong
and specific ACE inhibitor. Only the activity that could be
inhibited by captopril was regarded as ACE activity. To find
out if the HPLC-fractions contain an inhibitor for ACE,
appropriate amounts of lyophilized fraction material were

added to the standard condition setup. Addition of an ACE
inhibitor or an ACE substrate results in competition with
the tritium-labelled substrate for ACE and appears as a
reduction in ACE activity.
Identification of the isolated peptides
Nanoflow electrospray ionization (ESI) quadrupole (Q)
orthogonal acceleration TOF-MS was performed on a
Q-TOF system (Micromass, UK). An appropriate volume
of the pure active fraction was dried and redissolved in
10 lL of ACN/water/acetic acid (30 : 69.9 : 0.1, v/v/v).
One microliter of this sample was loaded in a gold-coated
capillary (Protana L/Q nanoflow needle). This sample was
sprayed at a typical flow rate of 30 nLÆmin
)1
giving
extended analysis time in which an MS spectrum as well
as several tandem MS (MS/MS) spectra was acquired.
During MS/MS fragment ions are generated from a selected
precursor ion by collision induced dissociation [18]. Because
not all peptide ions fragment with the same efficiency, the
collision energy is typically varied between 20 and 35 eV so
that the parent ion is fragmented in a satisfying number of
different daughter ions. Needle voltage was set at 850 V,
cone voltage was 35 V. The fragmentation spectra obtained
were combined and transformed into their single charged
state by treatment with the
MAX
-
ENT
3 software (Masslynx

3.5 software; Micromass, UK).
N-Terminal amino acid sequencing was performed on an
Applied Biosystems Procise protein sequencing system
(Edman degradation) running in the pulsed liquid mode.
Kinetic studies of the purified peptides
Michaelis–Menten (K
m
) constants of ACE for the purified
peptides were determined using a competition-based assay.
ACE activity was measured using the internally quenched
fluorogenic ACE-substrate Abz-FRK-(Dnp)P as described
[15]. Briefly, ACE activity was monitored in Tris/HCl buffer
(0.1
M
Tris/HCl, 0.05
M
NaCl, 10 l
M
ZnCl
2
,pH7.0)1mL
final volume with 1 l
M
final concentration of Abz-FRK-
(Dnp)P for sACE, nACE and LomACE or 2.5 l
M
final
concentration for cACE. Cleavage of the fluorogenic
substrate at the R–K bond removes the quencher Dnp
from the fluorogenic group Abz resulting in the appearance

of fluorescence at 420 nm after excitation at 320 nm which
was followed continuously for 10 min in a PerkinElmer
LS-50B fluorimeter. The initial cleavage rate was deter-
mined by nonlinear regression of the data to the function
f(x) ¼ y
0
+a[1) exp(–bx)] describing an exponential
increase to a maximum using the software package
SPSS
SIGMAPLOT
. The parameters a and b were used to calculate
the slope of the tangent of this function through the origin,
corresponding to the initial cleavage rate v
0
.Thesame
experiment was repeated in the presence of a nonfluorogenic
(dark) purified substrate (5 l
M
to 50 l
M
final concentra-
tion) in a competition experiment. As we use only the initial
cleavage rate, the dark substrate acts as a competitive
inhibitor as described by Xie et al. [19]. Hence, the initial
cleavage rate in presence of a dark substrate is called v
i
.The
initial rate of cleavage of the fluorescent substrate is
Ó FEBS 2002 Ovary-derived ACE substrates and insect physiology (Eur. J. Biochem. 269) 3523
described by the Michaelis–Menten equation: v

0
¼ k
cat
·
E
0
/(1 + K
m
/S)withk
cat
the turnover number of fluorescent
substrate cleavage, E
0
the concentration of ACE enzyme,
K
m
the Michaelis–Menten constant for cleavage of the
fluorescent substrate and S the concentration of fluorescent
substrate. As the nonfluorescent substrate is a competitive
inhibitor in these experimental conditions, we can describe
the fluorescent substrate cleavage as: v
i
¼ k
cat
· E
0
/
[1 + K
m
/S · (1 + S¢/K¢

m
)] with S¢ the concentration of
nonfluorescent substrate and K¢
m
the Michaelis–Menten
constant for cleavage of the nonfluorescent substrate. The
ratio of v
i
and v
0
can thus be described by: v
i
/v
0
¼ (1 + K
m
/
S)/[1 + K
m
/S · (1 + S¢/K¢
m
)]. Since the parameters v
i
and
v
0
are measured and K
m
, S and S¢ are known, we can
calculate K¢

m
from this equation.
Peptide interaction with ACE
To determine whether the purified peptide is a competitive
inhibitor or a substrate, 10 l
M
final concentration of peptide
Neb-ODAIF-1
1)13
or LEQIYHL in Tris/HCl buffer (0.1
M
Tris/HCl, 0.05
M
NaCl, 10 l
M
ZnCl
2
, pH 7.0), 100 lL final
volume,wereincubatedwithsACE.After5h,thereaction
was stopped by adding 10 lL captopril 10 l
M
.
As a negative control, this experiment was repeated in the
presence of 1 l
M
captopril. Ten microliters of the hydro-
lysation products were separated and analysed using
capillary liquid chromatography/tandem MS. These experi-
ments were conducted using an Ultimate HPLC pump, a
column switching device (Switchos) and a Famos autosam-

pler (all LC Packings, the Netherlands) coupled to a Q-TOF
hybrid quadrupole/TOF mass spectrometer (Micromass,
UK). Chromatography was performed using a guard
column (l-guard column MGU-30 C18, LC-Packings, the
Netherlands) acting as a reverse phase support to trap the
peptides. Ten microliters of the sample was loaded on the
precolumn with an isocratic flow of MilliQ water with 0.1%
formic acid at a flow rate of 10 lLÆmin
)1
After 2 min the
column switching valve was switched, placing the precol-
umn online with the analytical capillary column, a Pepmap
C18, 3 lm75lm · 150 mm nano column (LC Packings).
Separation was conducted using a linear gradient from 95%
solvent A, 5% solvent B to 5% A, 95% B in 55 min (solvent
A: water/acetonitrile/formic acid, 94.9 : 5 : 0.1, v/v/v;
solvent B: water/acetonitrile/formic acid, 19.9/80/0.1,
v/v/v). The flow rate was set at 150 nLÆmin
)1
.
The Ultimate capillary liquid chromatography was
connected in series to the electrospray interface of the
Q-TOF mass spectrometer. The column eluent was directed
through a metal coated fused silica tip (Picotip type FS360-
75-10 D; New Objective, USA). Needle voltage was set at
1400 V, cone voltage at 30 V. Nitrogen was used as
nebulizing gas. Tandem MS was carried out in an automa-
ted fashion. Peptide masses of interest were automatically
selected for fragmentation during the nano-LC tandem MS
separation. Argon was used as a collision gas, collision

energy was set at 15–35 eV depending on the selected mass.
Preparation of polyclonal antibodies
An antiserum against Neb-ODAIF-1
1)9
was raised in New
Zealand white rabbits. To improve the immunogenicity,
2mgsyntheticNeb-ODAIF
1–9
was coupled to 25 mg bovine
thyroglobulin as a carrier using the carbodiimide method as
described [20]. The resulting conjugate was dissolved in
physiological saline and supplemented with an equal volume
of complete Freund’s adjuvant (first immunization) or
incomplete Freund’s adjuvant (subsequent immunizations).
Rabbits were injected subcutaneously with this conjugate at
2-weekly intervals for 20 weeks. Prior to the first immuniza-
tion, the rabbits were bled to obtain preimmune serum. The
antiserum was characterized in a dot-spot assay. One lLNeb-
ODAIF-1
1)9
in different concentrations (1 lgto10 pg)was
spotted on to a nitrocellulose membrane and immobilized by
baking. The spots were incubated with different dilutions of
antiserum (1 : 100, 1 : 500 and 1 : 1000). The spots are
visualized with the peroxidase antiperoxidase (PAP)-immu-
nohistological technique as described [21].
SDS/PAGE
SDS/PAGE using a polyacrylamide gradient (9–12%) in
vertical slab gels in combination with a discontinuous
buffering system was performed according to Laemmli [22].

The resulting separated proteins were transferred to a
polyvinylidene difluoride (PVDF) membrane by electro-
blotting. Protein bands were visualized with Coomassie blue
staining or with the PAP-immunohistological technique as
described [21]. Bands corresponding to the Neobellieria yolk
polypeptides were identified as described [23].
RESULTS
Purification of ACE-interactive peptides
After prepurification of the crude ovary homogenate on a
Megabond Elute C
18
cartridge, the activity was restricted to
the 60% ACN fraction. This fraction was further separated
on a Deltapak C
18
while absorbance was followed at
214 nm. Thirty ovary equivalents of each fraction were
tested for inhibition activity, revealing inhibition activity in
almost every fraction tested. One equivalent is the amount
of a sample that would contain the material present in one
ovary. The active fraction that eluted after 82 min, corres-
ponding to elution at 33% ACN, was selected for further
purification because of its high ACE inhibiting capacity
(30% inhibition) by applying it on an Xterra C
18
semi-
preparative column. This time 60 ovary equivalents were
tested in the inhibition assay. The activity divided into
several different fractions. The further purification scheme is
described in Table 1. Absorbance was always monitored at

214 nm. Due to material loss during purification and
screening, an increasing number of equivalents of the
fractions had to be screened after each purification step.
After the first HPLC purification 30 equivalents were tested,
after the second 60 equivalents, then 120 equivalents, then
240 and finally 480 equivalents resulting in a pure active
fraction after four (Neb-ODAIF-1
1)13
and LEQIYHL) or
five (Neb-ODAIF-1
1)9
and Neb-ODAIF-2) successive
HPLC columns. The final chromatograms are shown in
Fig. 1 with the final active fractions indicated.
Identification of the purified peptides
ESI-TOF MS confirmed the purity of the fractions and
yielded the mass of the purified peptides summarized in
3524 K. Hens et al. (Eur. J. Biochem. 269) Ó FEBS 2002
Table 2. Fragmentation of the ion in a subsequent
collision induced dissociation experiment resulted in a
partial amino acid sequence by a clear series of b and y¢¢
type ions (data not shown). In addition, the amino acid
sequence was determined by automated N-terminal
sequencing, resolving leucine/isoleucine and lysine/gluta-
mine ambiguities with MS/MS sequencing. The first
sequence obtained was NKLKPSQWISLSD, a peptide
that completely comprises the previously purified
Neb-ODAIF (A. Vandingenen, personal communication)
but with the extension of the dipeptide SD at the
C-terminus. Hence it was called Neb-ODAIF-1

1)13
.The
second peptide, NKLKPSQWI is completely comprised in
Neb-ODAIF, but lacks the C-terminal dipeptide SL, so it
was called Neb-ODAIF-1
1)9
. SLKPSNWLTPSE is the
sequence of the third purified peptide. This peptide
resembles Neb-ODAIF but is not completely identical, so
it was called Neb-ODAIF-2. The last sequence obtained
was LEQIYHL, which shares no sequence similarity with
Neb-ODAIF and hence it will not be given an abbreviated
name. Neb-ODAIF will be called Neb-ODAIF-1
1)11
to
avoid confusion.
For sequence comparison, the sequences were submitted
to protein databases using
BLAST
at NCBI. All entries
yielded, among hits with other proteins, stretches of amino
acids as present in yolk proteins (yps) of different fly species
as the most abundant hits. Neb-ODAIF-1
1)13
and Neb-
ODAIF-1
1)9
displayed the highest sequence similarity with
a yolk protein (yp3) of the bluebottle fly Calliphora vicina,
yp3 and yp2 of the housefly Musca domestica and yp1 of

Table 1. Purification procedure of several ACE-competitive peptides. HPLC purification of several ACE-competitive peptides from an extract of
8000 ovary equivalents from the grey flesh fly, Neobellieria bullata. Elution conditions and active fractions are indicated and the sequence of the
purified peptide is given.
Step Active fraction Next purification step
Resulting
active fractions
Peptide
identified
1 Megabond Elute Waters Deltapak C18 0–60% ACN 33% ACN
60% ACN fraction 25 · 100 mm, 15 lm in 150 min
2 Deltapak C18 Waters Xterra RP18 0–50% ACN 26% ACN
33% ACN 7.8 · 300 mm, 7 lm in 120 min 27.5% ACN
3 Waters Xterra Supelco Supelcosil LC-8DB 0–50% ACN 20% ACN
26% ACN 4.6 · 250 mm, 5 lm in 120 min
4 Supelcosil LC-8DB Supelco Suplex PKB 100 0–50% ACN 20% ACN
20% ACN 4.6 · 250 mm, 5 lm in 120 min 21% ACN
5 Suplex PKB 100 Termoquest Hypercarb 0–80% ACN 51.5% ACN NKLKPSQWI
20% ACN 3 · 250 mm, 5 lm in 90 min
5 Suplex PKB 100 Termoquest Hypercarb 0–80% ACN 31.5% ACN SLKPSNWLTPSE
21% ACN 3 · 250 mm, 5 lm in 90 min
3 Waters Xterra Supelco Supelcosil LC-8DB 0–50% ACN 19% ACN
27.5% ACN 4.6 · 250 mm, 5 lm in 120 min 21% ACN
4 Supelcosil LC-8DB Supelco Suplex PKB 100 0–50% ACN 22% ACN LEQIYHL
19% ACN 4.6 · 250 mm, 5 lm in 120 min
4 Supelcosil LC-8DB Supelco Suplex PKB 100 0–50% ACN 19% ACN NKLKPSQWISLSD
21% ACN 4.6 · 250 mm, 5 lm in 120 min
Fig. 1. Final HPLC chromatograms of the
purified peptides. Chromatograms of the
HPLC runs that resulted in the final purifi-
cation of (A) Neb-ODAIF

1-13
(B) Neb-
ODAIF
1–9
(C) Neb-ODAIF-2 and
(D) LEQIYHL with absorbance at 214 nm
and elution gradient (% ACN) indicated.
Active fractions are indicated with an arrow.
Ó FEBS 2002 Ovary-derived ACE substrates and insect physiology (Eur. J. Biochem. 269) 3525
several Drosophila species. Entering Neb-ODAIF-2 in the
search yielded yp3 of Musca domestica andyp1andyp2of
several Drosophila species. Finally, LEQIYHL was most
similar to Drosophila yp1.
A multiple alignment of the purified peptides with yp1, yp2
and yp3 of Musca and Drosophila andwithyp3ofCalliphora
is given in Fig. 2. The peptides align N-terminally with the
yps. Interestingly, Neb-ODAIF
1–13
and Neb-ODAIF-2 align
at the same position within the yps suggesting that they are
peptides derived from two different yps in Neobellieria.The
peptide LEQIYHL aligns a bit further in the yps but still
quite close to the N-terminus of the yps.
Peptide interaction with ACE
The peptides Neb-ODAIF-1
1)11
, Neb-ODAIF-1
1)9
and
Neb-ODAIF-1

1)7
were already shown to be true substrates
by Vandingenen (A. Vandingenen, Zoological Institute of
the Catholic University of Leuven, Laboratory of Develop-
mental Physiology and Molecular Biology, Belgium,
personal communication).
To determine whether the peptides Neb-ODAIF-1
1)13
and LEQIYHL are inhibitors or true substrates, these
peptides were incubated for 5 h with sACE. The reaction
was stopped by addition of the specific ACE-inhibitor
Table 2. Interaction of the purified peptides with different kinds of ACE. Protonated mass as determined by MS and K
m
values (l
M
) of the cleavage of
the purified peptides with sACE, nACE, cACE and locust testis ACE.
Peptide
Protonated
mass
K
m
(l
M
)
sACE nACE cACE Lom testes ACE
ODAIF
1–13
1515.73 136.7 673.8 231.4 459
ODAIF

1–11
1313.74 17.0 150.5 6.9 2.7
ODAIF
1–9
1114.71 81.5 523.7 62.2 147.2
ODAIF
1–7
813.9 90.5 No competition 75.3 112.6
ODAIF*
2–13
1359.12 311.7 No competition 108.0 233.8
LEQIYHL 915.64 34.7 159.2 15.7 412.5
Fig. 2. Multiple alignment of the purified pep-
tides with several fly yps. Multiple alignment of
the purified peptides with several fly yps,
namely Drosophila melanogaster (Drome),
Musca domestica (Musdo) and Calliphora
vicina (Calvi).
3526 K. Hens et al. (Eur. J. Biochem. 269) Ó FEBS 2002
captopril. For both Neb-ODAIF-1
1)13
and LEQIYHL, no
hydrolysation products could be detected using Q-TOF MS
after capillary liquid chromatography.
Furthermore, comparison of the absorbance peak cor-
responding to the intact peptide showed no significant
difference between the control condition and the experi-
mental condition (data not shown). These results show that
Neb-ODAIF-1
1)13

and LEQIYHL are either inhibitors or
substrates with a very low turnover number.
K
m
determination of the purified peptides
The K
m
values of ACE for the purified peptides and for
Neb-ODAIF-1
1)11
and Neb-ODAIF-1
1)7
were determined
with recombinant human sACE, cACE and nACE and with
the purified Locusta migratoria testis ACE using a compe-
tition based assay. The cleavage of a fluorogenic ACE-
substrate was followed in the absence and in the presence of
the test peptide. For sACE, nACE and locust testis ACE,
1 l
M
final concentration of fluorogenic substrate was used.
For cACE, 2.5 l
M
final concentration was used as this
cACE stock was less active. Different concentrations of
peptides were tested to obtain a clear inhibitory effect with
50 l
M
final concentration being the highest concentration
used. For Neb-ODAIF-1

1)7
,25l
M
was used as the highest
concentration because of the limited amounts of this
peptide available. In Fig. 3 the results of the assays are
shown for one representative concentration of peptide
with the different types of ACE. Using
SPSS SIGMAPLOT
,
we performed nonlinear regression to the function f(x) ¼
y
0
+a[1) exp(–bx)] describing an exponential increase to
a maximum. The parameters a and b were used to calculate
the initial cleavage rate v
0
of the fluorogenic peptide. The
initial cleavage rates were used to calculate the Michaelis–
Menten constants of ACE for the tested peptides. As shown
in Table 2, each ACE type was inhibited differently by
different peptides, Neb-ODAIF-1
1)11
being the best overall
substrate. All peptides tested were significantly better
recognized by the C domain then by the N domain. Neb-
ODAIF-1
1)7
and Neb-ODAIF-2 are almost not recognized
by nACE and are not included in Fig. 3B. For Neb-

ODAIF-1
1)7
this might be explained by the fact that only
25 l
M
was tested. The K
m
of sACE for Neb-ODAIF-1
1)11
is
half the value of K
m
for LEQIYHL. The K
m
of nACE is
nearly the same, indicating that Neb-ODAIF-1
1)11
is more
C-domain specific than LEQIYHL. Neb-ODAIF-1
1)11
proved to be an excellent substrate for locust testis ACE,
confirming that this ACE is kinetically more related to the
human C-terminal ACE. The peptide LEQIYHL, however,
is a good inhibitor for the C domain of human ACE, but
not for locust testes ACE, indicating that locust testis ACE
shares some but not all of the kinetic properties with C-
domain ACE.
Western blotting
Polyclonal antibodies against Neb-ODAIF-1
1)9

were raised
in New Zealand white rabbits as described in Materials and
methods. The resulting antibodies were tested using dot spot
methods. A 1 : 100 dilution of the antiserum was able to
recognize 0.05 pmol of Neb-ODAIF-1
1)9
(data not shown).
As the bulk of the yps is synthesized in the female fat body
and transported by the haemolymph to be taken up by the
developing oocytes, haemolymph of vitellogenic Neobellie-
ria females, egg homogenate and haemolymph of male flies
as a negative control were subjected to SDS/PAGE. A
Fig. 3. Interaction of the purified peptides with
different kinds of ACE. Competition assay
for (A) sACE (B) nACE (C and D) cACE and
(E) locust testis ACE of the fluorogenic
substrate Abz-FRK-(Dnp)P and (a) 0 l
M
-test
peptide, (b) 50 l
M
Neb-ODAIF*
2)13
,
(c) 25 l
M
Neb-ODAIF
1–7
,(d)50l
M

Neb-ODAIF
1–13
,(e)50l
M
Neb-ODAIF
1–9
,
(f) 50 l
M
LEQIYHL and (g) 50 l
M
Neb-ODAIF
1)11
.
Ó FEBS 2002 Ovary-derived ACE substrates and insect physiology (Eur. J. Biochem. 269) 3527
PVDF membrane replica of the separated proteins was
immunostained in a PAP-experiment using the anti-Neb-
ODAIF-1
1)9
antiserum in a dilution series [1 : 2500 (A),
1 : 5000 (B)] (Fig. 4). A second PVDF replica was stained
with Coomassie blue (C) in order to visualize all protein
bands. Yp bands are marked on the Coomassie blue-stained
membrane (C) with arrows. The antibody-stained mem-
branes showed a single band corresponding to a yp band,
and in the lower molecular weight range additional bands
were stained. These lower molecular weight bands corres-
pond to degradation products of the yp, that are likely to
contain the Neb-ODAIF-1
1)9

sequence. The membranes
were photographed, digitalized and enlarged so that the
distance between the top of the membrane and the yp bands
could be measured accurately. The antibody-stained band
corresponds to the lowest yp band (yp3) on the Coomassie
blue-stained membrane. Hence it can be concluded that we
have developed an antiserum that is specific for Neobellieria
yp3. This is a strong indication that the purified Neb-
ODAIF-1 sequences are derived from yp3. Neb-ODAIF-2
resembles Neb-ODAIF-1, but is not completely identical.
This peptide is probably derived from the same position in
one of the two other yps present in Neobellieria bullata.
DISCUSSION
Anti-Neb-ODAIF-1
1)9
antibodies, apart from some yp
degradation products, specifically immunostain yp3 and did
not recognize yp1 or yp2. As the same antibodies did not
reveal any positive protein bands in male haemolymph, it
can be concluded that the Neb-ODAIF-1
1)9
peptide is
derived from a yp3 gene product. No data are available at
this moment on the mechanisms by which the peptides are
liberated from yp3. One possibility explaining the yp3 origin
of the purified peptides, is that these are the products when
the pinocytosed vitellogenins are transformed into vitellins.
Perhaps, the N-terminally cleaving off of a small part of yp3
promotes the nearly crystalline packing of vitellins in the
yolk platelets. Logically, when this hypothesis is correct, one

should not expect immunoreactivity in mature ovarian
extracts as vitellins would lack the Neb-ODAIF sequence.
However, as follicle cells also synthesize endogenous
vitellogenins [24], these follicle cell-derived vitellogenins
mightbethecauseofanti-Neb-ODAIF-1
1)9
immunoreac-
tivity observed in our ovarian extract. Alternatively, these
peptides might also be produced by yolk degradation in the
prospect of complete hydrolysis during subsequent embry-
onic development. Several proteases such as cathepsin and
acid phosphatase [25], capable of generating peptide frag-
ments, have been identified in insect yolk granules and the
proteasome complex that is identified in Drosophila
embryos [26] is also thought to break down yps to peptide
fragments [27]. Identification of the proteases present in the
vitellogenic follicles in combination with the elucidation of
the full sequence of the Neobellieria yps will allow unrave-
ling the exact digestive pathways of yps. The fact that we
purified two substrates from the same location in two
different yps (Neb-ODAIF-1
1)13
and Neb-ODAIF-2), sug-
gests that the yps are processed in a controlled manner.
Since no peptide has been proven to be an in vivo substrate
for insect ACE to date, assumptions about ACE physiology
in insects have to be made very carefully, especially when
dealing with an enzyme with such broad substrate specificity
as ACE. One potentially endogenous ACE substrate is
already known, namely the trypsin modulating oostatic

factor Neb-TMOF [28]. This peptide is capable of regulating
vitellogenesis and is present in the ovaries. Neb-TMOF is
suggestedtobereleasedbytheovariesandtobetransported
through the haemolymph to the midgut. Here, Neb-TMOF
terminates the protein meal-induced trypsin biosynthesis.
Thisresultsinanimpairedblooddigestionandalackof
amino acids that are needed for yolk synthesis, thus
regulating ovarian development. TMOF has been shown
to be an in vitro substrate for ACE present in the fly
haemolymph [29] and captopril-feeding experiments indi-
cate that TMOF is a true endogenous substrate [30]. The
purification of several ACE interactive peptides (Neb-
ODAIF-1
1)13
, Neb-ODAIF-1
1)11
, Neb-ODAIF-1
1)9
, Neb-
ODAIF-2, LEQIYHL) from the fly ovary stresses a putative
regulatory role of ACE in vitellogenic or embryogenic
events even more. The purified peptides might serve to stop
yolk synthesis as the first batch of eggs reach maturity as
does Neb-TMOF. If these peptides indeed regulate vitello-
genesis, this would represent an autoregulation mechanism
based upon the generation of peptides during yp degrada-
tion. Neb-ODAIF-1
1)11
is very well recognized by insect
testis ACE (Lom testis ACE: K

m
¼ 2 l
M
)andmaybea
physiological substrate for insect ACE. If physiological
experiments substantiate this hypothesis, these peptides
might be used to interfere with insect reproduction and
could thus be used in insect pest management.
In contrast with mammalian ACE, only single-domain
insect ACE has been identified. Because insect ACE was
used in the inhibition assay, peptides that are best recog-
nized by insect ACE will be preferentially purified. There-
fore, comparison of the kinetic parameters of the interaction
Fig. 4. Western analysis of fly haemolymph and egg homogenate with
anti-Neb-ODAIF
1–9
antibodies. Western blot of (1) male fly haemo-
lymph (2) female fly haemolymph (3) fly egg homogenate and
(4) molecular weight marker stained with (A) 1 : 2500 dilution and
(B) 1 : 5000 dilution of anti-Neb-ODAIF
1–9
antiserum or (C) Coo-
massie brilliant blue. Yp1, yp2 and yp3 as indicated by arrows refer to
the three yolk polypeptide bands. The vertical bars refer to the location
of yp degradation products.
3528 K. Hens et al. (Eur. J. Biochem. 269) Ó FEBS 2002
between purified peptides and recombinant human sACE,
nACE and cACE will provide key information about the
enzymatic similarity of insect ACE with the recombinant
human ACEs. From the presented data, it is obvious that

the purified peptides are all more or less C-domain specific.
Indeed, Neb-ODAIF-1
1)11
and the peptide LEQIYHL are
recognized very well by cACE. This may indicate that the
circulating form of ACE of Neobellieria that we used to
screen the HPLC-fractions for competitive peptides is more
kinetically related to cACE. However, although Neb-
ODAIF-1
1)11
is an excellent substrate for Lom testis ACE
(K
m
¼ 2 l
M
), the peptide LEQIYHL, also very well
recognized by cACE, is almost not recognized by Lom
testis ACE. This may indicate that Neobellieria ACE and
Locusta ACE have different enzymatic properties. However,
it is more plausible that testes of locusts contain a different
ACEisoformthantheACEincirculation.Thesamemight
be true for the fly as in Drosophila, two isoforms of ACE
with different kinetic properties are already known [31]. The
fly ovaries are thus a rich source of domain-specific
substrates and inhibitors. These might be used as a model
to develop domain-specific inhibitors of ACE, which in turn
may contribute to better insights into the domain-specific
functions of human ACE. We have also purified a peptide
that might allow us to distinguish between different
isoforms of insect ACE, which again may be useful in the

investigation of ACE functionality in insects.
ACKNOWLEDGEMENTS
We thank L. Vanden Bosch for technical assistance with HPLC and
sequencing, I. Bongaers for assistance with screening and antibody
preparation, J. Puttemans and M. Christiaens for figure layout,
P. Corvol and A. Michaud (Institut National de la Sante
´
et de la
Recherche Me
´
dicale, Unite
´
36, College de France, Paris, France) for the
kind gift of Chinese hamster ovary cells expressing sACE, cACE and
nACE. This work was supported by the ÔInstituut voor de Aanmoed-
iging van Innovatie door Wetenschap en Technologie in Vlaanderen
VlaanderenÕ, by the Research Foundation of the K. U. Leuven (GOA/
2000/04) and by the FWO (G0356.98 and G0187.00).
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