[
International Worm Meeting,
2003]
In all metazoa the regulation of cell-cell-signaling is essential for development and behaviour. Neprilysins (NEP, neutral endopeptidases), transmembrane proteins belonging to the big family of zinc-metalloproteases, play a central regulatory role in these processes. In mammals, their main function is the hydrolysis of small neuropeptides at the cell surface leading to a termination of signals between neurons. In C. elegans more than 20 putative neprilysin genes are known but until now the physiological function of their corresponding proteins is unclear. Five of these neprilysins (F18A12.8, F26G1.6, T05A8.4, T16A9.4 and ZK20.6) show more than 30 % amino acid sequence identity to the mammalian neprilysin EC3.4.24.11. NEP-knockout mice show a decreased -opioid receptor density in the brain, increased aggression behaviour and altered locomotion activity. We are interested in effects on C. elegans behaviour and development caused by knockout of neprilysins. RNAi experiments were performed without detecting a change of the wild type phenotype. In parallel, a NEP deletion mutant was isolated by screening EMS-mutagenesis libraries. The knockout strain shows decreased, uncoordinated locomotion when compared to wild type. A temperature shift (from 20 to 26C) leads to a high embryonal lethality in these NEP deletion animals. We are continuing the characterisation of the deletion mutant strain and screen for additional NEP deletion mutants.
[
European Worm Meeting,
1998]
Neprilysins (NEPs) and endothelin-converting enzymes (ECEs) are structurally related zinc metallo-proteinases of the type II class of integral membrane proteins that are widely distributed in many tissues and cell types. They are involved in the processing and inactivation of peptides involved in cell-cell signalling during development and in a variety of other biological processes, such as the immune response, control of blood pressure and peptidergic neurotransmission. NEPs have a broad substrate specificity towards oligopeptides, cleaving peptide bonds on the amino side of hydrophobic residues. In contrast, only the pro-peptide (big endothelin) of the vasoconstrictor endothelin, has been identified as a substrate for mammalian ECE. The aim of this project is to improve our understanding of the roles of NEPs and ECEs in the processing and inactivation of peptides involved in animal development and behaviour using the nematode, Caenorhabditis elegans, as a model. The sequencing of the entire genome of C. elegans provides a unique opportunity to study the function of a complete family of related proteins in a metazoan. We have partially characterised NEP/ECE activity present in membranes prepared from a mixed-stage culture of C. elegans. The enzyme activity is inhibited by chelators of divalent metal ions, has a neutral pH optimum and hydrolyses internal peptide bonds of a C. elegans neuropeptide (PF1, Ser-Asp-Pro-Asn-Phe-Leu-Arg-Pheamide) adjacent to Phe5, Leu6 and Phe8. This activity is inhibited by phosphoramidon and thiorphan, both of which are site-directed inhibitors of mammalian NEP and ECE. Sequence and text searches of ACeDB identify 6 NEP/ECEs (F12A10.4, F26G1.6, T16A9.4, T25B6.2, ZK20.6, ZK970.1), all of which show conserved active site motifs for zinc co-ordination, when aligned with mammalian NEP/ECEs. Hydrophobicity plots of the predicted amino acid sequence of these genes revealed putative secretion signal peptides at the N-terminus. In order to obtain expression patterns for these genes, promoter::LacZ constructs have been generated by PCR with fusions prior to the N-terminal transmembrane domain and are being transformed into C. elegans. cDNAs for the C. elegans NEP/ECEs, lacking coding sequence of the N-terminal transmembrane domain anchor and secretion signal sequence, have been cloned into the yeast expression vector pPIC9 (Invitrogen). Fusion with the prepro alpha-factor signal sequence ensures efficient secretion of the recombinant protein into the medium when expression is induced by methanol on the AOX promoter. Recombinant strains are undergoing small scale culturing to assess levels of expression by SDS-PAGE. High yielding strains will be cultured under optimised conditions on a large scale to produce sufficiently pure (>95%) recombinant protein. This will be used for biochemical analysis of enzymatic activity and for antiserum production for immunocytochemical localisation of C. elegans NEP/ECEs.