An endothelin‐converting enzyme homologue in the locust, Locusta migratoria: functional activity, molecular cloning and tissue distribution

Abstract

Endothelin-converting enzyme is the key enzyme in the process of endothelin production. Endothelin is a peptide that plays an important role in vasoconstriction and the development of neural crest-derived cells in vertebrates. Activity assays performed on membrane extracts from Locusta migratoria brain revealed the existence of a protease activity responsible for the formation of mature endothelin-1 from its precursor, big endothelin. Cloning experiments led to a cDNA sequence (Lom ECE) with an open reading frame of 727 amino acid residues displaying all the characteristic ECE features. A comparison of ECE activity levels among different tissues of the locust showed a high enzyme activity in the gonads and midgut. RT-PCR experiments showed a wide tissue distribution of Lom ECE mRNA, with transcription being most abundant in brain tissue.

Zinc-metalloproteases in insects: ACE and ECE

“…This activity was found less susceptible to thiorphan inhibition when compared to phosphoramidon (Macours et al, 2003c). Because vertebrate NEP is inhibited by phosphoramidon and thiorphan, while vertebrate ECE is only inhibited by phosphoramidon, this points towards an ECE-like activity.…”

“…It constitutes the catalytic site and contains 10 potential glycosylation signals (Asn-X-Ser/thr). Ten cysteine residues, presumably involved in proper folding and in maintenance of the protein's active conformation, corresponding to those conserved among all known NEP-ECE-Kell protein sequences, are also conserved in LomECE (Macours et al, 2003c).…”

Differential gene expression profiles in the venom gland/sac of Orancistrocerus drewseni (Hymenoptera: Eumenidae)

“…NEP is a cell surface metallopeptidase that degrades various biologically active peptides, and ECE functions as a key enzyme in the final processing step of endothelin (ET) in mammals (Roques et al, 1993;Xu et al, 1994). High levels of ECE activity in the gonads and midgut, as well as a wide distribution of ECE transcripts in various tissues, including the brain and fat body of desert locust, Locusta migratoria, imply that ECE may have multiple functions in insects (Macours et al, 2003). ECE/NEP also have been known to hydrolyze various small peptides such as neurotensin, substance P, bradykinin, oxidized insulin B chain, and angiotensin in vitro, as efficiently as big ET, a precursor of ET (Hoang and Turner, 1997;Johnson et al, 1999;Turner and Nalivaeva, 2007).…”

“…However, only three contigs (4 ESTs) actually retained the common characters of ECE and NEP, such as the active site residues (HexxH, sequences not shown) and the third zinc-binding ligand (E) (Hooper et al, 1997;Shimada et al, 1995). The rest of them lacked amino acids crucial for proper enzyme maturation and enzyme activity of ECE and NEP (MacLeod et al, 2001;Macours et al, 2003), but contained an overall sequence resemblance to M13 family members.…”

Bioinformatic analysis of the neprilysin (M13) family of peptidases reveals complex evolutionary and functional relationships

“…Neprilysin-like activity is enriched in the brain neuropil and in isolated synaptic membranes of relatively basal insects, the locusts, Schistocerca gregaria and Locusta migratoria and the cockroach Leucophea maderae , indicating an evolutionarily conserved role for M13 peptidases in the functioning of nervous systems that use neuropeptides extensively as neurotransmitters/modulators [2,24]. Recently an ECE-like gene was identified in L. migratoria and was shown to be highly expressed in the central nervous system and the midgut [25]. Insect M13 peptidases are associated with metamorphosis [26,27] and immunity to bacterial, fungal and protozoan infections [28,29].…”