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 similaritybetween insect and human C-domain angiotensin-converting enzyme Korneel Hens1, Anick Vandingenen1, Nathalie Macours1, Gee

Characterization of four substrates emphasizes kinetic similarity

between insect and human C-domain angiotensin-converting enzyme

Korneel Hens1, Anick Vandingenen1, Nathalie Macours1, Geert Baggerman1, Adriana Carmona

Karaoglanovic2, Liliane Schoofs1, Arnold De Loof1and Roger Huybrechts1

1

Zoological Institute of the Catholic University of Leuven, Laboratory of Developmental Physiology and Molecular Biology, Leuven, Belgium;2Universidade 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-11)13),

NKLKPSQWI (Neb-ODAIF-11)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-11)9immunostained

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-11)7 withinsect ACE and human ACE show both similar and unique properties for insect ACE as compared withhuman 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 withmammalian ACE,

especi-ally around the active site [2] The enzymatic activity is also

well conserved as insect ACE can hydrolyse mammalian

ACE substrates suchas 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 a smaller

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 bullataand 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

M A T E R I A L S A N D M E T H O D S

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: korneel.hens@bio.kuleuven.ac.be

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)

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 mMBorax,

0.3MNaCl, 0.2M(NH4)2SO4, 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-11)13), NKLKPSQ

WISL (Neb-ODAIF-11)11), NKLKPSQWI

(Neb-ODAIF-11)9), NKLKPSQ (Neb-ODAIF-11)7), SLKPSNWLTPSE

(Neb-ODAIF-2) and LEQIYHL were from

ResearchGen-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 witha linear gradient of 0% to 50% ACN in

0.1% trifluoroacetic acid in 120 min and a flow rate of

2 mLÆmin)1 Two-ml fractions were automatically collected;

(b) C8 column (Supelco, LC-8DB, 4.6· 250 mm, 5 lm),

elution witha linear gradient of 0% to 50% ACN in 0.1%

trifluoroacetic acid in 120 min and a flow rate of

1 mLÆmin)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 witha 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 witha Waters

486 tunable absorbance detector

ACE inhibition screening

The ACE inihibition assay is based on the ACE activity

assay by Ryan et al [17], modified by Vandingenen et al

[11] Briefly, ACE-activity in diluted fly haemolymph is

measured witha synthetic, tritiated ACE substrate

p-[3H]benzoylglycylglycylglycine (Sigma) (¼ standard

con-dition) Adding 10 lM 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 withtheMAX-ENT3 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 (Km) 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 (0.1MTris/HCl, 0.05MNaCl, 10 lMZnCl2, pH 7.0) 1 mL final volume with1 lMfinal concentration of Abz-FRK-(Dnp)P for sACE, nACE and LomACE or 2.5 lM 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 y0+ 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 v0 Th e same experiment was repeated in the presence of a nonfluorogenic (dark) purified substrate (5 lM to 50 lM 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 vi Th e initial rate of cleavage of the fluorescent substrate is

described by the Michaelis–Menten equation: v0¼ kcat·

E0/(1 + Km/S) with kcatthe turnover number of fluorescent

substrate cleavage, E0 the concentration of ACE enzyme,

Km 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: vi¼ kcat· E0/

[1 + Km/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 viand v0can thus be described by: vi/v0¼ (1 + Km/

S)/[1 + Km/S· (1 + S¢/K¢m)] Since the parameters viand

v0 are measured and Km, S and S¢ are known, we can

calculate K¢mfrom this equation

Peptide interaction with ACE

To determine whether the purified peptide is a competitive

inhibitor or a substrate, 10 lMfinal concentration of peptide

Neb-ODAIF-11)13or LEQIYHL in Tris/HCl buffer (0.1M

Tris/HCl, 0.05MNaCl, 10 lMZnCl2, pH 7.0), 100 lL final

volume, were incubated with sACE After 5 h , th e reaction

was stopped by adding 10 lL captopril 10 lM

As a negative control, this experiment was repeated in the

presence of 1 lM 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 withan isocratic flow of MilliQ water with0.1%

formic acid at a flow rate of 10 lLÆmin)1After 2 min the

column switching valve was switched, placing the

precol-umn online withthe analytical capillary colprecol-umn, a Pepmap

C18, 3 lm 75 lm· 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-11)9was raised in New

Zealand white rabbits To improve the immunogenicity,

2 mg synth etic Neb-ODAIF 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 withthis 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 lL Neb-ODAIF-11)9in different concentrations (1 lg to 10 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 withthe 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 witha 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 withCoomassie blue staining or with the PAP-immunohistological technique as described [21] Bands corresponding to the Neobellieria yolk polypeptides were identified as described [23]

R E S U L T S

Purification of ACE-interactive peptides After prepurification of the crude ovary homogenate on a Megabond Elute C18cartridge, the activity was restricted to the 60% ACN fraction This fraction was further separated

on a Deltapak C18 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 C18 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-11)13and LEQIYHL) or five (Neb-ODAIF-11)9 and Neb-ODAIF-2) successive HPLC columns The final chromatograms are shown in Fig 1 withthe 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

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 withMS/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-11)13 Th e

second peptide, NKLKPSQWI is completely comprised in

Neb-ODAIF, but lacks the C-terminal dipeptide SL, so it

was called Neb-ODAIF-11)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-11)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-11)13 and Neb-ODAIF-11)9displayed 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

60% ACN fraction 25 · 100 mm, 15 lm in 150 min

3 Waters Xterra Supelco Supelcosil LC-8DB 0–50% ACN 20% ACN

4 Supelcosil LC-8DB Supelco Suplex PKB 100 0–50% ACN 20% ACN

3 Waters Xterra Supelco Supelcosil LC-8DB 0–50% ACN 19% ACN

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 withabsorbance at 214 nm

and elution gradient (% ACN) indicated.

Active fractions are indicated withan arrow.

several Drosophila species Entering Neb-ODAIF-2 in the

searchyielded yp3 of Musca domestica and yp1 and yp2 of

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 and with yp3 of Calliphora

is given in Fig 2 The peptides align N-terminally with the

yps Interestingly, Neb-ODAIF1–13and Neb-ODAIF-2 align

at the same position within the yps suggesting that they are

peptides derived from two different yps in Neobellieria Th e

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-11)11, Neb-ODAIF-11)9 and Neb-ODAIF-11)7were 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-11)13 and LEQIYHL are inhibitors or true substrates, these peptides were incubated for 5 hwithsACE 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 )

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).

captopril For both Neb-ODAIF-11)13and 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-11)13and LEQIYHL are either inhibitors or

substrates witha very low turnover number

Kmdetermination of the purified peptides

The Kmvalues of ACE for the purified peptides and for

Neb-ODAIF-11)11and Neb-ODAIF-11)7were determined

withrecombinant 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 lMfinal concentration of fluorogenic substrate was used

For cACE, 2.5 lM 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 lM final concentration being the highest concentration

used For Neb-ODAIF-11)7, 25 lMwas 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

withthe different types of ACE Using SPSS SIGMAPLOT,

we performed nonlinear regression to the function f(x)¼

y0+ a[1) exp(–bx)] describing an exponential increase to

a maximum The parameters a and b were used to calculate

the initial cleavage rate v0of 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, eachACE type was inhibited differently by different peptides, Neb-ODAIF-11)11being the best overall substrate All peptides tested were significantly better recognized by the C domain then by the N domain Neb-ODAIF-11)7and Neb-ODAIF-2 are almost not recognized

by nACE and are not included in Fig 3B For Neb-ODAIF-11)7this might be explained by the fact that only

25 lMwas tested The Kmof sACE for Neb-ODAIF-11)11is half the value of Kmfor LEQIYHL The Kmof nACE is nearly the same, indicating that Neb-ODAIF-11)11is more C-domain specific than LEQIYHL Neb-ODAIF-11)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-11)9were 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-11)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-riafemales, 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) 50 l M

Neb-ODAIF 1–13 , (e) 50 l M Neb-ODAIF 1–9 ,

(f) 50 l M LEQIYHL and (g) 50 l M

Neb-ODAIF

PVDF membrane replica of the separated proteins was

immunostained in a PAP-experiment using the

anti-Neb-ODAIF-11)9antiserum in a dilution series [1 : 2500 (A),

1 : 5000 (B)] (Fig 4) A second PVDF replica was stained

withCoomassie blue (C) in order to visualize all protein

bands Yp bands are marked on the Coomassie blue-stained

membrane (C) witharrows 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-11)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

D I S C U S S I O N

Anti-Neb-ODAIF-11)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-11)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 migh t be th e cause of anti-Neb-ODAIF-11)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 suchas 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 withthe 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-11)13and 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 withan enzyme withsuchbroad 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 suggested to be released by th e ovaries and to be transported through the haemolymph to the midgut Here, Neb-TMOF terminates the protein meal-induced trypsin biosynthesis

Th is results in an impaired blood digestion and a lack of 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-11)13, Neb-ODAIF-11)11, Neb-ODAIF-11)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-11)11 is very well recognized by insect testis ACE (Lom testis ACE: Km¼ 2 lM) and may be a 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 withmammalian 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 h aemolymph(3) fly egg h omogenate 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.

between purified peptides and recombinant human sACE,

nACE and cACE will provide key information about the

enzymatic similarity of insect ACE withthe 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-11)11and 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-11)11is an excellent substrate for Lom testis ACE

(Km¼ 2 lM), the peptide LEQIYHL, also very well

recognized by cACE, is almost not recognized by Lom

testis ACE This may indicate that Neobellieria ACE and

LocustaACE have different enzymatic properties However,

it is more plausible that testes of locusts contain a different

ACE isoform th an th e ACE in circulation Th e same migh t

be true for the fly as in Drosophila, two isoforms of ACE

withdifferent 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

A C K N O W L E D G E M E N T S

We thank L Vanden Bosch for technical assistance with HPLC and

sequencing, I Bongaers for assistance withscreening 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 Instituut voor de

Aanmoed-iging van Innovatie door Wetenschap en Technologie in Vlaanderen

Vlaanderen, 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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