Angiotensin-converting enzyme in Spodoptera littoralis: Molecular characterization, expression and activity profile during development

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Abstract

The characterization of the full-length angiotensin-converting enzyme (ACE) cDNA sequence of the lepidopteran Spodoptera littoralis is reported in this study. The predicted open reading frame encodes a 647 amino acids long protein (SlACE) and shows 63.6% identity with the Bombyx mori ACE sequence. A 3D-model, consisting of 26 α-helices and three β-sheets, was predicted for the sequence. SlACE expression was studied in the embryonic, larval and pupal stages of S. littoralis and in different tissues of the last larval stage by reverse-transcribed PCR. This revealed that the gene is expressed throughout the life cycle and especially in brain, gut and fat body tissue of the last stage. These results are in agreement with a role of ACE in the metabolism of neuropeptides and gut hormones. In addition, ACE activity has been studied in more detail during development, making use of a fluorescent assay. High ACE peptidase activity coincides with every transition state, from embryo to larva, from larva to larva and from larva to pupa. A peak value in activity occurs during the early pupal stage. These results indicate the importance of SlACE during metamorphosis and reveal the high correlation of ACE activity with the insect's development, which is regulated by growth and developmental hormones.

Introduction

The angiotensin-converting enzyme (ACE, peptidyl dipeptidase A, EC 3.4.15.1) is a dipeptidyl carboxypeptidase which catalyses the metabolism of bioactive peptides. The enzyme is well studied in vertebrates, where it fulfills an important role in blood pressure regulation and ion balance (Macours et al., 2004). Two distinct forms are present in vertebrates, a single-domain testicular form, referred to as tACE and a double domain somatic form, sACE (Ehlers and Riordan, 1989; Lattion et al., 1989).

The first invertebrate ACE was described by Lamango and Isaac (1994) and was isolated from the housefly, Musca domestica. Since insects lack a vascular system, it was difficult to pinpoint a function to this enzyme. In the first reports about the biological role of invertebrate ACE, processing of peptide hormones and neuropeptides was assigned to it (Lamango et al., 1997; Schoofs et al., 1998). The invertebrate ACEs described until now, all resemble the testicular form of vertebrate ACE, since they possess only one active site, while the somatic vertebrate form (sACE) contains two active site motifs (Ehlers and Riordan, 1989; Lattion et al., 1989). However, where vertebrate tACE and sACE are membrane bound, invertebrate ACE enzymes are in general soluble.

ACE is a zinc metallopeptidase and requires Zn2+ for its activity. The interaction with zinc is coordinated directly by the active site HEXXH. This active site is conserved in fully characterized insect ACE sequences like Drosophila melanogaster Ance and Acer (Q10714 and Q9VLJ6), Haematobia exigua irritans (HeiACE, Q10715), Lutzomyia longipalpis (LlACE, Q5WPT4), Bombyx mori (BmAcer, Q9NDS8), Locusta migratoria (LmACE, Q6RX62) and the Anopheles gambiae proteins AnoACE2, 3 and 9 (A0NFU8, Q7PM24 and Q7Q9W7). The crystal structure of Ance (D. melanogaster, PDB 1J38; Kim et al., 2003) elucidates how the enzyme interacts with its substrates or inhibitors. The structure has a peanut shell shape and shows that the binding site forms a large continuous internal channel. Size limitation of the internal channel prevents binding of longer peptides or globular proteins, while the shape of substrates or inhibitors is of minor importance. Because of these characteristics, ACE can be defined as a relatively non-specific dipeptidyl carboxypeptidase (Kim et al., 2003; Macours and Hens, 2004). However, besides the dipeptidase activity, ACE can also split dipeptideamides and tripeptideamides from the peptide C-terminus. In vitro studies have shown the capability of insect ACE enzymes to convert a broad range of substrates found in vertebrates, although none of these have been detected in insects until now (Cornell et al., 1995; Lamango et al., 1997; Siviter et al., 2002a).

Localization of the enzyme in different stages and tissues has led to the proposal of several functions for invertebrate ACE. Especially a role in vital physiological processes as reproduction and development (both embryogenesis and metamorphosis) has been determined (Macours and Hens, 2004; Isaac et al., 2007). Reproductive tissues are a rich source of ACE. In different insect species, ACE is found in the testes and is often correlated with the germ cells (Loeb et al., 1998; Schoofs et al., 1998; Isaac et al., 1999). In addition, high levels of ACE activity have been found in gut tissues of different larval fly species (Wijffels et al., 1997) where it can function as a processing enzyme for gut hormones, pointing towards a role in digestion. In brain tissue, ACE was localized in the neuropile regions and neurosecretory cells of insects of different orders, having a possible role in the processing of neuropeptides (Schoofs et al., 1998). Insect development is highly correlated with ACE expression and ACE activity. Fruit flies, homozygous for hypomorphic Ance alleles show reduced viability (Tatei et al., 1995; Hurst et al., 2003). The importance of ACE towards a successful metamorphosis is suggested by high ACE activities during last larval stage and early pupal stage in D. melanogaster and Lacanobia oleracea (Siviter et al., 2002b; Ekbote et al., 2003). Evidence from another view, studying the detrimental effect of ACE inhibitors like captopril and fosinopril on growth and development, has been shown for Spodoptera littoralis and Manduca sexta (Vercruysse et al., 2004; Isaac et al., 2007).

Here, we report the cDNA ACE sequence of S. littoralis (Noctuidae family) (SlACE), the first full-length lepidopteran sequence to be characterized after B. mori (BmAcer). The SlACE expression and ACE activity was studied in the embryonic, larval and pupal stages of S. littoralis.

Section snippets

Insects

A continuous colony of the cotton leafworm, S. littoralis, was reared as described (Smagghe et al., 2002). Larvae were fed on a wheat-germ-based artificial diet. Under these conditions, duration of the egg stage tends to be 4 days, first (L1) until fifth (L5) larval stage tend to be 3 days each, the sixth and last larval stage (L6) takes 6 days and the pupal stage 10. Larval stages were selected according to the head capsule width. For the ACE activity assay, the exact age is expressed as time

Molecular characterization

The full-length ACE cDNA sequence of S. littoralis is 2043 bp long. The sequence contains a 5′ UTR of 41 nucleotides and a 3′ UTR of 58 nucleotides, with a putative polyadenylation signal (AATAA). The open reading frame of 1941 bp encodes a protein (SlACE) of 647 amino acids (ABW34729). A signal peptide was predicted with a putative cleavage site between residues Gly23 and Arg24. No other hydrophobic regions were present, indicating that the protein is not trapped in the cellular membrane. In

Discussion

The present study reports the full length sequencing of the S. littoralis ACE cDNA (SlACE). Translation resulted in a mature protein of 624 residues. SlACE has a theoretical molecular weight of 72 kDa and is being secreted. Its structure is in agreement with other functional insect ACE-like enzymes. The sequence homology of SlACE is highest with the ACE-related sequence of the silkmoth B. mori (63.6% identity), the only lepidopteran ACE sequence known until now. In accordance with the D.

Acknowledgement

This research is supported by project 01102703 from the Special Research Fund of the Ghent University.

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