Angiotensin I-converting enzyme (ACE) activity of the tomato moth, Lacanobia oleracea: changes in levels of activity during development and after copulation suggest roles during metamorphosis and reproduction

Insect Biochemistry and Molecular Biology 33 (2003) 989–998 www.elsevier.com/locate/ibmb

Angiotensin I-converting enzyme (ACE) activity of the tomato moth, Lacanobia oleracea: changes in levels of activity during development and after copulation suggest roles during metamorphosis and reproduction U.V. Ekbote a, R.J. Weaver b, R.E. Isaac a,∗ a

Molecular and Cellular Biosciences, Faculty of Biological Sciences, University of Leeds, L.C. Miall Building, Clarendon Way, Leeds LS2 9JT, UK b CSL, Sand Hutton, York YO41 1LZ, UK Received 16 May 2003; received in revised form 17 June 2003; accepted 20 June 2003

Abstract Angiotensin I-converting enzyme (ACE) is a dipeptidyl carboxypeptidase that removes C-terminal dipeptides from relatively short oligopeptides, usually smaller than 15 amino acids. In mammals, the enzyme has several important roles in the metabolism of vasoactive peptides, but its physiological role in insects is not fully understood. We now report the properties of an ACE in a lepidopteran species (the tomato moth, Lacanobia oleracea) and suggest new physiological roles for the enzyme in this insect. ACE activity increases four-fold during the last stadium and in early pupae, a rise which, in its timing, is similar to what has been observed previously in the transition of larva to pupa in Drosophila melanogaster. This suggests that the increase in ACE activity might be of general importance for peptide metabolism during metamorphosis in holometabolous insects. High levels of ACE activity were found in the haemolymph of sixth stadium larvae and adult insects, and in the reproductive tissues of both male and female adults. Almost all of the ACE activity in the reproductive tissues was found in the accessory glands of the male and the spermatheca and bursa copulatrix of the female. The decline in accessory gland ACE in mated males and the concomitant rise in ACE activity in the spermatheca and bursa copulatrix of the female suggested the transfer of ACE from the male to the female during copulation. Using several convenient peptides as substrates, we have shown that the spermatophore/bursa copulatrix taken from mated female insects possess an aminopeptidase, a carboxypeptidase and a dipeptidase, in addition to high levels of ACE. These peptidases might be involved in the breakdown of proteins to peptides and eventually to amino acids in the spermatophore. Evidence for such a proteolytic pathway and its role in providing substrates for the TCA cycle has been obtained previously in a study of reproduction in Bombyx mori.  2003 Elsevier Ltd. All rights reserved. Keywords: Angiotensin-I converting enzyme; Lacanobia oleracea; Metamorphosis; Male accessory gland; Peptide metabolism; Peptidase; Reproduction

1. Introduction Extracellular peptidases have an important role in determining the half-life of regulatory peptides after their release from neurons, neurosecretory and endocrine cells (Kenny and Hooper, 1991). Several peptidase

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0965-1748/$ - see front matter  2003 Elsevier Ltd. All rights reserved. doi:10.1016/S0965-1748(03)00105-X

activities have been implicated in the inactivation of insect peptides, one of which is the dipeptidyl carboxypeptidase activity of angiotensin I-converting enzymes (ACE, peptidyl dipeptidase A, EC 3.4.15.1) (Isaac and Nassel, 2003; Isaac et al., 2002; Wilson et al., 2002). ACE activity, which cleaves the penultimate peptide bond at the C-terminus of peptides, has been found in the haemolymph and tissues of a range of species from several insect orders (Cornell et al., 1995; Isaac and Lamango, 1994; Wijffels et al., 1996; Zhu et al., 2001). Some of the best-studied insect ACEs are Drosophila

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melanogaster ANCE and ACER (Siviter et al., 2002a, b). ANCE has many structural and enzymatic properties in common with human ACE, a key enzyme involved in the regulation of blood pressure and ion balance (Corvol et al., 1995; Williams et al., 1996). Peptide substrates for human ACE include angiotensin I, which is converted to angiotensin II, and bradykinin, which is hydrolysed to inactive peptide fragments. Both ANCE and human ACE have broad substrate specificity and can even function as endopeptidases, cleaving dipeptideamides and tripeptideamides from the C-terminus of some peptides, e.g. mammalian substance P and the locust and cockroach tachykinins (LomTK-1 and LemTrp-1) (Siviter et al., 2002a). This ability to break down amidated peptides might be of special significance since the presence of an amidated C-terminal amino acid is essential for full biological activity of many insect peptides. ACER can also be classed as both a peptidyl dipeptidase and an endopeptidase, but it has a much more restricted range of substrates compared with ANCE (Houard et al., 1998; Siviter et al., 2002a). ANCE has a vital role in insect development. D. melanogaster that lack the Ance gene die during late embryogenesis and an hypomorphic allele, originally classified as another lethal, results in infertile males because of a failure in spermatid differentiation (A.D. Shirras and R.E. Isaac, unpublished data; Hurst et al., 2003). ACE has also been detected in the testes of several other insect species and is often localised to germ cells (Isaac et al., 1999; Loeb et al., 1998; Schoofs et al., 1998). In the haematophagous fly, Haematobia irritans exigua, testicular ACE is induced after a blood meal indicating a specific role for the peptidase in the maturation of spermatozoa (Wijffels et al., 1996). The failure of spermatids to undergo individualisation in Ance mutants has led to the suggestion that ANCE is required for the processing of peptides regulating spermatogenesis in D. melanogaster (Hurst et al., 2003). ACE-like peptidases have also been implicated in several other aspects of insect reproduction. The demonstration that a trypsin modulating oostatic peptide is an in vivo substrate for haemolymph ACE in the adult fleshfly, Neobellieria bullata, suggested that the enzyme might have a role in regulating the synthesis of yolk proteins after a protein meal (Zhu et al., 2001). In the female mosquito, Anopheles stephensi, ACE is induced after a blood meal and appears to have a role in egg-laying (Ekbote et al., 1999, 2003). ANCE may also have important physiological roles during metamorphosis since ACE activity increases three-fold during early pupal development in D. melanogaster (Siviter et al., 2002b). This increase has been attributed to the strong induction of Ance expression in imaginal cells by 20-hydroxyecdysone. Increased expression of an ACE-like gene in imaginal cells (wing disc), under the influence of 20-hydroxyecdysone, also

occurs in the silkworm, Bombyx mori, suggesting that high levels of ACE expression might be characteristic of this stage of development in holometabolous insects (Quan et al., 2001). However, there have been no biochemical studies to support the suggestion that lepidopteran insects also display an increase in ACE activity during the transition from larva to pupa. We now report the existence of ACE activity in the larval, pupal and adult stages of the tomato moth, Lacanobia oleracea, which resembles biochemically D. melanogaster ANCE. The ACE specific activity increases around four-fold during the last larval instar and early pupal stages and high levels of enzyme activity are found in the male and female reproductive tissues of L. oleracea.

2. Materials and methods 2.1. Chemicals Hippuryl-Histidyl-Leucine (Hip-His-Leu), captopril, amastatin, hippuric acid, protein assay kit (BCA), peptides and all immunochemicals were purchased from Sigma-Aldrich Co., Poole, Dorset, UK. RXP 407, lisinopril and fosinoprilat were generously provided by Vincent Dive, Commissariat a` l’Energie Atomique, CESaclay, 91191 France, Dr. H. Bull, Merck Sharpe and Dohme Laboratories, Rahway, NJ, USA and BristolMyers Squibb, Pharmacological Research Institute, Princton, NJ, USA, respectively. p-Nitro blue tetrazolium chloride and 5-bromo-4-chloro-3-indolyl phosphate were purchased from Promega UK Ltd., Southampton, UK. HPLC-grade solvents were obtained from Rathburn Chemicals, Walkerburn, Scotland, UK. HPLC-grade orthophosphoric acid was purchased from Fisons Laboratory Chemicals, Loughborough, UK. 2.2. Insects L. oleracea were cultured at 20 °C and 65% relative humidity, under a 16 h light:8 h dark cycle, as described previously (Corbitt et al., 1996). Larvae were reared on an artificial diet (Bio-Serv, Frenchtown, NJ, USA) and adults were fed on 50% aqueous honey solution, smeared on a cotton pad soaked previously with adult diet solution. Sixth instar larvae were used for determining larval ACE activity. One batch (3–4 days old) were taken from the diet during the feeding phase and were starved for 4–6 h before being frozen in liquid N2 and stored at ⫺80 °C until required. A second batch of sixth instar larvae, that had reached the wandering stage (6– 7 days old), were also taken and frozen as described above. The mean pupal period in the non-diapausing condition lasts for 25 days. Male and female pupae were collected in three groups, early (3–4 days post pupa for-

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mation, PPF), middle (10–12 days PPF) and late pupae (21–22 days PPF). They were immediately frozen in liquid N2 and stored at ⫺80 °C. Virgin and mated L. oleracea adults were collected 1–2 days post-emergence. The wings and appendages were removed from the bodies, which were frozen in liquid N2 and stored at ⫺80 °C. 2.3. Preparation of larval and pupal homogenates Insects were homogenised for 2 min in ice-cold 50 mM Tris/HCl, 0.1% (w/v) Triton X-100, pH 7.4 (6 ml) with a motor driven homogeniser (Ultra-Turrax, Janke and Kunkel, Mannheim, Germany). The homogenate was filtered to remove any cuticular material and then centrifuged at 13,000 g for 7 min at 4 °C in a Jouan BR 3.11 centrifuge. The supernatant (5 ml) was collected and stored at ⫺80 °C until required. 2.4. Preparation of tissue homogenates Tissues (foregut, midgut, hindgut, Malpighian tubules, nervous system, fatbody and integument) were dissected from sixth instar larvae in Ringer’s saline (Ephrussi and Beadle, 1936), after collection of the haemolymph from an incision in a proleg. The entire gut, Malpighian tubules, fatbody, and flight muscle were taken from adult female insects, whereas whole reproductive tissues were taken from both male and female adults. Adult tissues were also dissected in insect Ringer’s saline. In a separate experiment, the reproductive tissues from virgin male insects were separated into testes, vesicula seminalis (including the ampulla ductus deferentis), accessory glands with duplex, primary simplex, cuticular simplex and the phallus (Fig. 1). Female reproductive tissues from virgin insects were separated into ovary-a, ovaryb, ovary-c, accessory glands, spermatheca and bursa copulatrix (Fig. 1). All tissues were immediately frozen in liquid N2 and stored at ⫺80 °C until required. Tissue homogenates were prepared in ice-cold 50 mM Tris/HCl, 0.1% (w/v) Triton X-100, pH 7.4, with a glass homogeniser and were centrifuged at 13,000 g (Micro centaur, MSE) for 7 min at 4 °C. The supernatant was collected and stored at ⫺80 °C.

Fig. 1. Diagrammatic representations of the male (A) and female (B) reproductive tissues of L. oleracea. (A) The male genital tract: a, testis; b, seminal vesicle and vas deferens; c, accessory glands and duplex; d, primary simplex; e, cuticular simplex. (B) The female reproductive tissues: a, ovary-section a; b, ovary-section b; c, ovary-section c; d, spermatheca; e, bursa copulatrix; f, accessory gland. Broken lines delineate the sub-divisions of the genital tract (based on an original diagram of Barbosa, 1974).

2.6. Hydrolysis of peptides by bursa copulatrix peptidases

2.5. Assay for ACE activity The supernatant prepared from whole animal or tissue homogenates was used for the determination of ACE activity using Hip-His-Leu, according to the method previously described (Lamango and Isaac, 1994). The method relies upon HPLC to quantify the release of hippuric acid from the substrate by insect ACE. Protein concentrations were estimated using the bicinchoninic acid (BCA) assay with bovine serum albumin as a protein standard (Smith et al., 1985).

Peptides (250 µM of YGGFL, YGGFLR, YGGFLRR and 1 mM of FL) were incubated at 35 °C with cleared homogenate of a bursa copulatrix dissected from a female moth 2 h after mating (2.5 µg of protein) for 15 min in 0.1 M Tris/HCl, pH 7.5, in the presence and absence of either 10 µM captopril or 10 µM amastatin (final incubation volume, 20 µl). Peptides were resolved and quantified by reversed-phase HPLC with the UVdetector set at 214 nm and 0.2 full-scale absorbance units, as described previously (Wilson et al., 2002).

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Metabolites were identified by co-chromatography with authentic enkephalin fragments purchased from SigmaAldrich Co., Poole, Dorset, UK.

Table 1 Changes in the specific activity of ACE during the sixth stadium and the development of male (M) and female (F) pupae Stage

Specific activity (unit of activity/mg of protein)

Data were analysed using Minitab (Windows version 9.1 and 10). Data were tested for the basic statistical function such as mean, standard deviation and standard error of the mean and two way t-test for significant values.

Sixth instar (feeding) Sixth instar (wandering) Early pupa Middle-stage pupa Late pupa

6.6 ± 1.2 15.4 ± 2.5

– –

23.6 ± 1 (M) 19 ± 1.8 (M) 18.3 ± 0.9 (M)

34 ± 6 (F) 24 ± 2 (F) 23 ± 6 (F)

2.8. SDS-PAGE and immunodetection of insect ACE

One unit of activity = 1 nmol of Hip - His - Leu hydrolysed / min. Values are the means of replicate experiments ± s.e.m., n = 5.

2.7. Statistical analysis

SDS-PAGE analysis was performed using a 10% polyacrylamide gel and a Bio-Rad Mini-Protean II Gel Electrophoresis Cell run at a constant voltage of 200 V for 45 min (Laemmli, 1970). The electrophoresed proteins were then transferred to a 0.3 µm pore size nitrocellulose paper (Hybond, Amersham) at 100 V for 1 h on Bio-Rad Mini Trans-Blot Electrophoretic Transfer Cell (Bio-Rad, U.K.). The membrane was blocked 17 h at 4 °C with 5% (w/v) dried milk in Tris–buffered saline (0.05% w/v Tween 20, 10 mM Tris/HCl, pH 7.5, 150 mM NaCl; TBST). The next day the membrane was washed three times in TBST at intervals of 6 min, before incubating for 1 h with antiserum to D. melanogaster ANCE (provided by Dr. P.M. Dani, University of Leeds), diluted 1:5000 in 5% (w/v) dried milk in TBST. The membrane was washed three times in TBST before incubating for a further 1 h with rabbit anti-rat IgG coupled to alkaline phosphatase (1:5000 in 5% (w/v) dried milk in TBST). The secondary antibody was detected using the substrates, p-nitro blue tetrazolium chloride and 5-bromo-4-chloro-3-indolyl phosphate. Recombinant ANCE protein was generously provided by Dr. R.J. Siviter, University of Leeds.

3. Results

level in female pupae that was almost five-fold greater than in insects 3–4 days into the sixth stadium (Table 1). ACE specific activity dropped slightly in later stages of pupal development. In addition to inhibition by captopril, both larval and pupal ACE activities were inhibited by two other ACE inhibitors, lisinopril and fosinoprilat, but not by RXP 407, a selective inhibitor of D. melanogaster ACER and the N-domain of human ACE (Table 2). 3.2. Tissue distribution of ACE in the sixth stadium larva ACE activity was found in all tissues examined, but the highest specific activity was found in the fat body, followed by the midgut and the integument (Table 3). However, by far the greatest source of ACE activity was not in the tissues, but in the haemolymph. The ACE specific activity of the haemolymph was 15.2 ± 1.0 nmol / min / mg of protein. The total haemolymph activity is around seven times greater than that found in the fat body, which is itself 8–10 times greater than the amount present in the foregut, hindgut, Malpighian tubules and nervous system.

3.1. Changes in the specific activity of ACE during the last larval stage and in pupae

Table 2 Effect of mammalian ACE inhibitors on ACE activity of L. oleracea

ACE activity was measured in the cleared homogenates of whole insects from five stages of development. The stages selected were, day 3–4 of the sixth stadium (feeding larvae), day 6–7 of the sixth stadium (wandering larvae), 3–4 days post-pupation (early pupae), 10–12 days post-pupation (mid-pupae), and 21– 22 days post-pupation (late pupae). The specificity of the enzyme assay was routinely checked by ensuring that ⬎90% of the enzyme activity was inhibited by 30 µM captopril. ACE specific activity increased by over 100% as the larvae progressed from feeding to the wandering phase and increased further after pupation, reaching a

Enzyme source % Inhibition

Sixth instar larva (wandering) Early Pupa Adult male reproductive tissue

Captopril

Lisinopril

Fosinoprilat

RXP 407

96

83

94

0

98 94

84 64

92 85

7 2

Data are the means of triplicate assays. The standard error of the mean for all data was less than ±10%.

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Table 3 Tissue distribution of ACE Stage

Tissue

Table 4 Distribution of ACE activity in the reproductive tract of male moths ACE activity (unit of activity/tissue)

Sixth instar larva Haemolymph Foregut Midgut Hindgut Malpighian tubules Nervous tissue Fat body Integument

14.10 ± 0.95 (0.094 ± 0.006a) 0.11 ± 0.03 0.76 ± 0.19 0.24 ± 0.06 0.23 ± 0.1

Adult

13.99 ± 2.77a 0.39 ± 0.12 1.30 ± 0.29

Haemolymph Gut Malpighian tubules Fat body Flight muscle Reproductive tissue (F) Reproductive tissue (M)

993

0.15 ± 0.03 2.03 ± 0.50 0.50 ± 0.11

1.49 ± 0.43 0.55 ± 0.04 462 ± 141 351 ± 49

One unit of activity = 1 nmol of Hip - His - Leu hydrolysed /min. Values are the means of replicate experiments ± s.e.m., n = 5. a nmoles of Hip/min/µl of haemolymph.

3.3. Tissue distribution of ACE in adult moths ACE activity was determined for the haemolymph and in homogenates of the gut, Malpighian tubules, fat body and flight muscle, all of which were taken from the adult female, and in the complete male and female reproductive tissues (Table 3). ACE activity of the male and female reproductive tissues far exceeded the activity found in the gut, Malpighian tubules and flight muscle. Although it was not possible to estimate the total volume of haemolymph in an adult insect, it is clear that the ACE activity in the haemolymph was also high. 3.4. The distribution of ACE activity in the reproductive tissues of virgin adult moths The six regions that constitute the male reproductive system (Fig. 1A) were dissected from virgin insects and were used to determine the source of the high ACE activity found in adult male L. oleracea. Over 99% of the total ACE activity in the reproductive tissues of virgin male moths was located in the accessory glands (633 ± 141 nmol / min / tissue, Table 4). Levels of ACE activity also varied within the regions of the reproductive system of adult virgin females (Fig. 1B; Table 5). The spermatheca (220 ± 25.9 nmol / min / tissue) and bursa copulatrix (267 ± 101 nmol / min / tissue) accounted for 87% of the total ACE activity found in adult female moths.

Tissue

Testis Vas deferens, Seminal vesicle Accessory gland Primary simplex Cuticular simplex Phallus

ACE (unit of activity/tissue) Virgin

Mated

1.42 ± 0.25 0.06 ± 0.02

1.32 ± 0.05 0.17 ± 0.15

633 ± 141 ∗ 1.49 ± 0.68 1.26 ± 0.31 1.06 ± 0.45

200 ± 91∗ 1.19 ± 0.99 1.82 ± 0.34 1.02 ± 0.25

One unit of activity = 1 nmol of Hip-His-Leu hydrolysed/min. Values are the means of replicate experiments ± s.e.m., n = 5. The significance of the difference between the specific activities for tissues from virgin and mated animals was performed using Student’s t-test. ∗ P = 0.015.

Table 5 Distribution of ACE activity in the reproductive tract of female moths Tissue

Ovary-a Ovary-b Ovary-c Accessory gland Spermatheca∗ Bursa copulatrix∗∗

ACE (unit of activity/tissue) Virgin

Mated

3.04 ± 0.57 37.05 ± 3.7 25 ± 2.55 5.58 ± 4.7 220 ± 25.9 267 ± 101

4.32 ± 1.01 39.9 ± 5.56 55.5 ± 7 1.83 ± 0.8 347 ± 86.6 533 ± 83.5

One unit of activity = 1 nmol of Hip-His-Leu hydrolysed/min. The values presented are the means of replicate experiments ± s.e.m., n = 5. The significance of the difference between the specific activities for tissues from virgin and mated animals was performed using Student’s t-test. ∗ P = 0.065. ∗∗ P = 0.06.

3.5. Changes in the levels of ACE activity in the reproductive tissues of male and female L. oleracea during mating A comparison of the levels of ACE activity in the reproductive tissues of virgin and mated moths, suggested that copulation resulted in a reduction in the level of male accessory gland ACE, accompanied by an increase in ACE levels in the female spermatheca and bursa copulatrix (Tables 4 and 5). The changes in ACE levels occurring during mating were studied in a larger cohort of insects. The results confirmed that the specific activity of male accessory gland ACE declined during mating, by around 40%, whereas the specific activity of the peptidase in the female bursa copulatrix and spermatheca increased by around 160 and 184%, respectively

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Table 6 Transfer of ACE from the male accessory gland to the female bursa copulatrix and spermatheca Tissue

ACE (unit of activity/mg of protein) Virgin

Male accessory gland∗ 682 ± 54 Female bursa copulatrix∗∗ 245 ± 43 582 ± 49 Female spermatheca∗∗∗

Mated 432 ± 72 397 ± 29 1072 ± 111

One unit of activity = 1 nmol of Hip-His-Leu hydrolysed/min. Values are the means of replicate experiments ± s.e.m., n = 15. The significance of the difference between the specific activities for tissues from virgin and mated animals was performed using Student’s t-test. ∗ P = 0.0054. ∗∗ P = 0.0037. ∗∗∗ P = 0.0004.

(Table 6). The ACE content of the accessory gland from virgin and mated male moths was also subjected to immunoelectrophoretic analysis using antibodies raised to D. melanogaster ANCE (Fig. 2). The results indicate a reduction in the level of ACE protein after mating, supporting the conclusions derived from the enzymatic analysis. 3.6. ACE and other peptidase activities of the spermatophore/bursa copulatrix of mated female moths Several peptidases are thought to be responsible for protein and peptide metabolism in the spermatophore of mated female lepidopteran insects (Kasuga et al., 1987; Shepherd, 1974). We therefore sought information about the type of peptide-degrading enzymes present in the

Fig. 2. Immunoelectrophoretic detection of an ACE-like protein in the male accessory gland of L. oleracea. Equal quantities of protein (30 µg per lane) from the accessory gland of a virgin (A) and mated (B) adult male moth and pre-stained broad-range molecular weight marker proteins (C) were loaded on to the SDS/PAGE gel.

spermatophore/bursa copulatrix of L. oleracea and whether ACE was a relatively important peptidase activity when short peptides were employed as substrates. Since we did not know the identity of any natural peptide substrates for these enzymes, we used three readily available peptides ([Leu5]enkephalin (YGGFL), YGGFLR and YGGFLRR), whose degradation products could be identified by HPLC analysis. Five enkephalin fragments were generated after incubation of [Leu5]enkephalin with a supernatant fraction prepared from a cleared homogenate of the spermatophore/bursa copulatrix and separated by HPLC. Captopril partially protected YGGFL from degradation and totally inhibited the formation of the direct ACE cleavage products, YGG and FL (Fig. 3A and B). The low amount of detectable FL relative to YGG, was probably due to the efficient hydrolysis of FL to F by a dipeptidase, since captopril greatly reduced the F peak of the HPLC chromatogram (Fig. 3B). This reaction was shown to be rapid by incubating FL with the spermatophore/bursa copulatrix enzymes (Fig. 4). Two of the enkephalin fragments formed in the presence of captopril were identified as GGFL and Y, indicating the presence of an aminopeptidase. This conclusion was supported by the observation that the aminopeptidase inhibitor, amastatin, totally inhibited the production of GGFL and reduced the amount of Y formed by around 40% (Fig. 3C). The generation of Y was also strongly inhibited by captopril, indicating that a large proportion of Y probably resulted from the hydrolysis of the Y-G peptide bond of YGG by ACE activity (Fig. 3A and B). YGGFLRR was rapidly cleaved to YGGFL (enkephalin) by the captopril-sensitive ACE activity of the spermatophore/bursa copulatrix (Fig. 3D). The failure to form YGGFL in the presence of captopril was accompanied by the disappearance of YGG and massive reduction in the amount of Y and F formed (Fig. 3E). The small amount of Y remaining probably resulted from aminopeptidase cleavage of the parent peptide, YGGFLRR. Blocking ACE activity led to the identification of YGGFLR as a major metabolite produced as a result of carboxypeptidase activity of the spermatophore/bursa copulatrix (Fig. 3E). The presence of a peptide-degrading carboxypeptidase in the spermatophore/bursa copulatrix was confirmed by showing that YGGFLR was converted to YGGFL (Fig. 3F). However, a more prominent reaction was that catalysed by the ACE activity, since YGGFLR was strongly protected by the presence of captopril (Fig. 3G). The expected product of this reaction, YGGF, is probably the unknown peptide peak (Unk) seen in the HPLC chromatogram (Fig. 3F), whose appearance is inhibited by captopril. The relatively low levels of YGG and the large amount of free Y detected in Fig. 3F can be explained by the dominance of ACE, which by the sequential removal of C-terminal dipeptides from YGGFLR will

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Fig. 3. Demonstration of peptide-degrading activity in the spermatophore/bursa copulatrix of mated female L. oleracea. YGGFL ([Leu5]enkephalin) (chromatograms A–C), YGGFLRR (chromatograms D and E) and YGGFLR (chromatograms F and G) were incubated for 15 min at 35 °C with a cleared homogenate (2.5 µg of protein) of the spermatophore/bursa copulatrix from a mated adult female in either the absence of inhibitors (A, D and F) or the presence of 10 µM captopril (B, E and G) or 10 µM amastatin (C). Peptide fragments and free amino acids were resolved by using reversed phase-HPLC with UV detection (214 nm, 0.2 full scale absorbance units). The retention times of authentic peptides and amino acids (YGG, Tyr-Gly-Gly; Y, Tyr; F, Phe; FL, Phe-Leu; GGFL, Gly-Gly-Phe-Leu; YGGFL, Tyr-Gly-Gly-Phe-Leu, Enk; Tyr-Gly-Gly-Phe-LeuArg-Arg, EnkRR), all of which were purchased commercially, are indicated. The UV-absorbing peak (Unk) in chromatogram F is probably YGGF, the product of ACE activity towards YGGFLR.

generate YG and not YGG. This dipeptide was probably quickly cleaved by a dipeptidase activity to release the Y detected in Fig. 3F. 4. Discussion There has been much speculation as to the biological role of insect ACE, especially since there is no evidence

for the existence of angiotensin-like peptides in insects and, therefore, it is unlikely that ACE is a component of an invertebrate Renin–Angiotensin System (Isaac et al., 1998a). The widespread occurrence of the enzyme in a range of insect species and the observations that the activity is greatly increased at more than one stage of development suggests that ACE has several important stage-specific roles. The four to five-fold increase in

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Fig. 4. Demonstration of dipeptidase activity in the spermatophore/bursa copulatrix of mated female L. oleracea. FL (1 mM) was incubated for 15 min at 35 °C with a cleared homogenate (2.5 µg of protein) of the spermatophore/bursa copulatrix from a mated adult female. The cleavage of FL was monitored by using reversed phase-HPLC with UV detection (214 nm, 0.2 full scale absorbance units). The retention time of F was confirmed by using the authentic amino acid purchased commercially.

ACE specific activity in L. oleracea during the last larval stadium and pupation is similar to the three-fold increase seen in D. melanogaster at the same stage of development (Siviter et al., 2002b). This increase in the D. melanogaster ACE activity appears to be the result of the induction of Ance expression in imaginal cells by 20hydroxyecdysone. It is possible that the observed increase in L. oleracea ACE activity during the transition between the larval and pupal stage is also the result of ACE synthesis by imaginal cells, under the control of 20-hydroxyecdysone. Supporting evidence for this suggestion comes from the demonstration that 20hydroxyecdysone directly induces expression of an ACE-like gene (BmAcer) in wing discs of another lepidopteran insect, the silkworm, B. mori (Quan et al., 2001). Taken together, this body of evidence is consistent with a fundamental role for ACE peptidases during metamorphosis in holometabolous insects. This role might include converting precursor molecules to biologically active peptides and/or terminating the signalling properties of other peptides. Other peptidases (aminopeptidases, dipeptidase and endopeptidases) in addition to ACE are also induced during metamorphosis in D. melanogaster and it has been suggested that the peptidase expression might be orchestrated to ensure the destruction of larval proteins by converting them into small peptides and the subsequent recycling of amino acids for energy production and the synthesis of new cuticle (Siviter et al., 2002b; Wilson et al., 2002). Since ACE releases dipeptides, a combination of ACE and

dipeptidase activities would provide a metabolic route for the recycling of amino acids from small peptide fragments generated by endopeptidic cleavage of larval proteins. That haemolymph from L. oleracea larvae and adults displayed relatively high ACE activity was not unexpected as ACE has been found in the circulation of several other insect species, where it is ideally placed for the metabolism of circulating peptide hormones (Lamango and Isaac, 1994; Siviter et al., 2002a). The highest level of ACE activity was found in the reproductive tract of both adult male and female L. oleracea. The enzyme was almost entirely localised to the accessory gland of the male and the spermatheca and bursa copulatrix in the female. Male accessory glands of insects are a rich source of seminal fluid material, which is destined to be transferred to the female during copulation (Boggs and Gilbert, 1979). Accessory gland products include spermatophore proteins, sperm storage proteins, mating plug components, enzymes, nutrients, antibiotics, behaviourmodifying molecules and developmental hormones. Considerable effort has focused on the characterisation of accessory gland peptides that modulate several female reproductive processes, including mating receptivity, egg development and the rate of egg-laying (Chen et al., 1988; Wedell, 1996; Kingan et al., 1995; Heifetz et al., 2001; Jin and Gong, 2001). For example, one of the D. melanogaster accessory gland peptides, known as Acp26Aa, stimulates ovulation (Heifetz et al., 2000). Acp26Aa is generated by limited proteolytic cleavage of a larger precursor polypeptide, which takes place in the female genital tract by male-derived proteases accompanying the prohormone in the seminal fluid (Park and Wolfner, 1995). In lepidopteran species, much of the seminal fluid transferred to the female becomes enclosed together with the motile apyrene and the immotile eupyrene sperm in a spermatophore formed in the female bursa copulatrix. It is only after enclosure in the spermatophore that the eupyrene sperm are released from sperm bundles and become motile (Osanai et al., 1989; Friedlander et al., 2001). Sperm motility requires an energy source, which may be obtained from glycogen and proteins present in the seminal fluid. In B. mori, it has been shown biochemically that a male-specific protein-degradation pathway is activated in the spermatophore to liberate arginine from an arginine-rich male protein (Osanai et al., 1987). High levels of spermatophore arginase converts the arginine to urea and orthinine, which is itself converted to glutamic acid (Osanai et al., 1986). By coupling this pathway to glycolysis, glutamic acid undergoes a transamination reaction to generate oxo-glutarate, which in turn enters the TCA cycle to provide energy for motile eupyrene sperm (Osanai et al., 1987). The proposed proteolytic cascade in the spermatophore of B. mori is initiated by a serine endopeptidase (initiatorin), which generates peptides with C-terminal

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arginine residues (Aigaki et al., 1987). The subsequent release of arginine is achieved through the action of a carboxypeptidase, which is itself activated by initiatorin within the spermatophore (Aigaki et al., 1987, 1988). Although it is not known whether an analogous proteolytic cascade is activated in the spermatophore of L. oleracea, the transfer of large quantities of male ACE to the female during copulation might form a hitherto unknown male component of an energy producing metabolic pathway. In the present study, we have used three mammalian peptides as peptidase substrates to show that, in addition to ACE, there are carboxypeptidase, aminopeptidase and dipeptidase activities in the spermatophore/bursa copulatrix of L. oleracea. The rapid hydrolysis of the Arg-extended enkephalin peptides by a L. oleracea ACE is consistent with the known properties of insect and human ACEs, which show particular preference to peptides with C-terminal basic residues (Isaac et al., 1998b, 1997). We have also provided evidence for the cleavage of FL, and possibly YG, by a dipeptidase present in the spermatophore/bursa copulatrix of L. oleracea. At present the substrate specificity of the dipeptidase is not known, however, a promiscuous dipeptidase working in concert with the ACE activity would provide an effective pathway, complementing the activity of the carboxypeptidase, for the release of amino acids from seminal fluid peptides generated by endopeptidic hydrolysis of male-derived proteins. The high ACE activity seen in the spermatheca and bursa copulatrix of virgin females suggests that female-derived ACE may also be important for peptide metabolism in the female reproductive tract. In mated females this activity might also be required, in addition to the male ACE donated in the spermatophore, to generate dipeptides. It is possible that there is more than one ACE protein contributing to the L. oleracea peptidyl dipeptidase activity assayed in this study. In D. melanogaster there are two isoforms of ACE (ANCE and ACER), which can be distinguished by differences in their substrate specificity and sensitivity to ACE inhibitors (Houard et al., 1998; Coates et al., 2000; Siviter et al., 2002a). For example, ACER does not hydrolyse peptides with pairs of basic residues at the C-terminus and is selectively inhibited by RXP 407, a selective inhibitor of the Ndomain of human ACE. Thus, the observation that the ACE activity of L. oleracea tissues was sensitive to three inhibitors of human ACE, belonging to different chemical classes, but was not inhibited by RXP 407, suggests that the moth ACE is an orthologue of D. melanogaster ANCE and not ACER. The fact that the enkephalin peptide, YGGFLRR, was a good substrate for the spermatophore/bursa copulatrix ACE and the ability of ANCE-specific antibodies to detect a protein from the male accessory gland, provides additional support for this conclusion.

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Acknowledgements We thank Alison Pennington and Fiona Smethurst, CSL, York, for maintaining the insect culture and acknowledge financial support from the school of Biology, University of Leeds.

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