Structure, evolutionary conservation, and functions of angiotensin- and endothelin-converting enzymes

Angiotensin-converting enzyme, a member of the M2 metalloprotease family, and endothelin-converting enzyme, a member of the M13 family, are key components in the regulation of blood pressure and electrolyte balance in mammals. From this point of view, they serve as important drug targets. Recently, the involvement of these enzymes in the development of Alzheimer's disease was discovered. The existence of homologs of these enzymes in invertebrates indicates that these enzyme systems are highly conserved during evolution. Most invertebrates lack a closed circulatory system, which excludes the need for blood pressure regulators. Therefore, these organisms represent excellent targets for gaining new insights and revealing additional physiological roles of these important enzymes. This chapter reviews the structural and functional aspects of ACE and ECE and will particularly focus on these enzyme homologues in invertebrates.status: publishe

Cyclorraphan yolk proteins and lepidopteran minor yolk proteins originate from two unrelated lipase families

Vitellogenins, cyclorraphan yolk proteins and lepidopteran minor yolk proteins are three classes of female-specific proteins that serve as an embryonic nutritional store. Similarity to vertebrate lipid-binding proteins was established for vitellogenins and yolk proteins, vitellogenins being related to apolipoprotein B and yolk proteins to lipases. Recently, similarity between yolk proteins and minor yolk proteins was reported and it was suggested that yolk proteins are more related to minor yolk proteins than to vertebrate lipases. In this study, we cloned five additional yolk proteins from the grey fleshfly Neobellieria bullata, formerly known as Sarcophaga bullata. We used this sequence data, combined with sequence data retrieved from the NCBI protein database to evaluate the yolk protein-lipase and the yolk protein-minor yolk protein relationship. We found no similarity between yolk proteins and minor yolk proteins, but we showed that yolk proteins are related to a family of lipases containing vertebrate hepatic and pancreatic lipases while minor yolk proteins are related to a family of lipases containing vertebrate gastric and lingual lipases. The fact that three different classes of yolk storage proteins show similarity to three different classes of vertebrate lipid-binding proteins strongly suggests that this lipid-binding feature is important for insect yolk storage proteins.status: publishe

Cloning and expression of the yolk protein of the tsetse fly Glossina morsitans morsitans

Two major families of nutritional proteins exist in insects, namely the vitellogenins and the yolk proteins. While in other insects only vitellogenins are found, cyclorraphan flies only contain yolk proteins. Possible sites of yolk protein synthesis are the fat body and the follicle cells surrounding the oocyte. We report the cloning of the yolk protein of the tsetse fly Glossina morsitans morsitans, a species with adenotrophic viviparity. The tsetse fly yolk protein could be aligned with other dipteran yolk proteins and with some vertebrate lipases. In contrast to the situation in most fly species, only a single yolk protein gene was found in the tsetse fly. Northern blot analysis showed that only the ovarian follicle cells, and not the fat body represents the site of yolk protein synthesis. (C) 2004 Published by Elsevier Ltd.status: publishe

Cloning and tissue distribution of the cyclic AMP generating peptide of the grey flesh fly Neobellieria bullata (Diptera : Sarcophagidae)

Originally, two forms of cyclic AMP Generating Peptide (cGP) were identified in whole body extracts of the flesh fly Neobellieria bullata. The long form, a 48-mer, stimulated cAMP production by Malpighian tubules of Manduca sexta. The short form, which had been discovered previously with an ovarian bioassay, turned out to be the 1-15 aa sequence of the 48-mer. We here report on the cloning, sequencing and expression profile of the cGP-gene in N. bullata. The full-length cDNA sequence, obtained by RT-PCR in combination with 5' and 3' RACE, encodes a single copy of the cGP precursor. No signal peptide is present in this precursor, which, compared to the mature peptide, is only extended at the N-terminus with an extra methionine residue. RT-PCR revealed that Neb-cGP is expressed in both larval and adult brain, testis and ovaries, flight muscles, Malpighian tubules, midgut, and fat body.status: publishe

Isolation of angiotensin converting enzyme from testes of Locusta migratoria (Orthoptera)

By means of a tracer assay using a labeled synthetic angiotensin converting enzyme (ACE) substrate hippurylglycylglycine, we have detected high ACE activity in the testes of the African migratory locust, Locusta migratoria. Lower, but significant, ACE activity was observed in midgut and hemolymph. In a two-step purification procedure involving anion exchange and gel permeation chromatography, we have purified LomACE from the locust testes. The enzyme of approximately 80 kDa shows substantial amino-acid sequence homology with ACE from both vertebrate and invertebrate origin. The ACE identity of the purified enzyme was further confirmed by cDNA cloning of the Locusta ACE fragment, which, after in silico translation, revealed a mature protein of 623 amino acids with a large structural similarity to other known ACE proteins.status: publishe

Isolation and characterization of an angiotensin converting enzyme substrate from vitellogenic ovaries of Neobellieria bullata

Vitellogenic ovaries of the gray fleshfly Neobellieria bullata contain a variety of unidentified substances that interact, either as a substrate or as an inhibitor, with angiotensin converting enzyme (ACE). We here report the isolation and characterization of the first ACE interactive compound hereof. This 1312.7 Da peptide with the sequence NKLKPSQWISL, is substrate to both insect and human ACE. It is a novel peptide that shows high sequence similarity to a sequence at the N-terminal part of dipteran yolk polypeptides (YPs). We propose to call it N. bullata ovary-derived ACE interactive factor or Neb-ODAIF Both insect and human ACE hydrolyze Neb-ODAIF by sequentially cleaving off two C-terminal dipeptides. K-m values of Neb-ODAIF and Neb-ODAIFI-9 (NKLKPSQWI) for human somatic ACE (sACE) are 17 and 81 muM, respectively. Additionally, Neb-ODAIF(1-7) (NKLKPSQ) also interacts with sACE (K-m/i = 90 muM). These affinity-constants are in range with those of the physiological ACE substrates and suggest the importance of Neb-ODAIF and its cleavage products in the elucidation of the physiological role of insect ACE. Alternatively, they can serve as lead compounds in the development of new drugs against ACE-related diseases in humans. (C) 2002 Elsevier Science Inc. All rights reserved.status: publishe

Characterization of four substrates emphasizes kinetic similarity between insect and human C-domain angiotensin-converting enzyme

Angiotensin converting enzyme (ACE) was already discovered 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-separated 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.status: publishe

Captopril, a specific inhibitor of angiotensin converting enzyme, enhances both trypsin and vitellogenin titers in the grey fleshfly Neobellieria bullata

A strong and constitutive angiotensin converting enzyme- or ACE-like activity was demonstrated in the hemolymph of the adult grey fleshfly Neobellieria bullata, In a competition assay, the N. bullata trypsin modulating oostatic factor (Neb-TMOF) was confirmed to be an in vitro substrate for this circulating Neb-ACE, Oral uptake of captopril, a selective and specific inhibitor of ACE, resulted in a complete phenotypic knockout of circulating ACE activity. When compared with control animals, captopril-fed female flies showed an increase in the liver meal-induced trypsin peak in the midgut and elevated levels of protein meal-induced yolk polypeptides in the hemolymph, The latter effect was not due to a slower vitellogenin uptake by the ovaries, because oocyte growth was not affected by the captopril treatment. The apparent synergism between the demonstrated ACE functionality and the previously reported effects of the oostatic peptide Neb-TMOF are discussed in the context of our recent finding that Neb-TMOF represents a prime candidate for being the first known in vivo substrate for circulating insect ACE. (C) 2001 Wiley-Liss, Inc.status: publishe

Immunocytochemical distribution of angiotensin-I converting enzyme in the central nervous system of insects and speculations about its possible function

Insect peptidyl-dipeptidase A [angiotensin I - converting enzyme (ACE)] is a soluble single-domain peptidyl-dipeptidase that has many properties in common with the C-domain of mammalian somatic ACE and with the single-domain mammalian ACE. In agreement with a variety of insects, immunocytochemical studies reveal the presence of an ACE-like protein in Locusta migratoria. ACE-like immunoreactivity is present in neurosecretory cells of the pars intercerebralis. These cells have axons projecting into the nervus corporis cardiaci I and into the storage part of the corpus cardiacum, a neuroendocrine organ directly releasing into the aorta. The localisation of ACE in neurosecretory cells is consistent with its proposed role as a processing enzyme that is involved in the generation of active peptide hormones.status: publishe

Gene expression profiling defines ATP as a key regulator of human dendritic cell functions.

Extracellular ATP and PGE2 are two cAMP-elevating agents inducing semimaturation of human monocyte-derived dendritic cells (MoDCs). We have extensively compared the gene expression profiles induced by adenosine 5'-O-(3-thiotriphosphate) (ATPgammaS) and PGE2 in human MoDCs using microarray technology. At 6 h of stimulation, ATPgammaS initiated an impressive expression profile compared with that of PGE2 (1125 genes compared with 133 genes, respectively) but after 24 h the number of genes regulated by ATPgammaS or PGE2 was more comparable. Many target genes involved in inflammation have been identified and validated by quantitative RT-PCR experiments. We have then focused on novel ATPgammaS and PGE2 target genes in MoDCs including CSF-1, MCP-4/CCL13 chemokine, vascular endothelial growth factor-A, and neuropilin-1. ATPgammaS strongly down-regulated CSF-1 receptor mRNA and CSF-1 secretion, which are involved in monocyte and dendritic cell (DC) differentiation. Additionally, ATPgammaS down-regulated several chemokines involved in monocyte and DC migration including CCL2/MCP-1, CCL3/MIP-1alpha, CCL4/MIP-1beta, CCL8/MCP-2, and CCL13/MCP-4. Interestingly, vascular endothelial growth factor A, a major angiogenic factor displaying immunosuppressive properties, was secreted by MoDCs in response to ATPgammaS, ATP, or PGE2, alone or in synergy with LPS. Finally, flow cytometry experiments have demonstrated that ATPgammaS, ATP, and PGE2 down-regulate neuropilin-1, a receptor playing inter alia an important role in the activation of T lymphocytes by DCs. Our data give an extensive overview of the genes regulated by ATPgammaS and PGE2 in MoDCs and an important insight into the therapeutic potential of ATP- and PGE2-treated human DCs.Comparative StudyJournal ArticleResearch Support, Non-U.S. Gov'tValidation Studiesinfo:eu-repo/semantics/publishe