Drosophila uses two distinct neuropeptide amidating enzymes, dPAL1 and dPAL2

Abstract

Neuropeptide a-amidation is a common C-terminal modification of secretory peptides, frequently required for biological activity. In mammals, amidation is catalyzed by the sequential actions of two enzymes [peptidylglycine-a-hydroxylating monooxygenase (PHM) and peptidyl-a-hydroxyglycine a-amidating lyase (PAL)] that are co-synthesized within a single bifunctional precursor. The Drosophila genome predicts expression of one monofunctional PHM gene and two monofunctional PAL genes. Drosophila PHM encodes an active enzyme that is required for peptide amidation in vivo.Here we initiate studies of the two Drosophila PAL genes. dPAL1 has two predicted transmembrane domains, whereas dPAL2 is predicted to be soluble and secreted. dPAL2 expressed in heterologous cells is secreted readily and co-localized with hormone. In contrast, dPAL1 is secreted poorly, even when expressed with a cleaved signal replacing the predicted transmembrane domains; the majority of dPAL1 stays in the endoplasmic reticulum. Both proteins display PAL enzymatic activity. Compared to the catalytic core of rat PAL, the two Drosophila lyases have higher K m values, higher pH optima and similarly broad divalent metal ion requirements. Antibodies to dPAL1 and dPAL2 reveal co-expression in many identified neuroendocrine neurons. Although dPAL1 is broadly expressed, dPAL2 is found in only a limited subset of neurons. dPAL1 expression is highly correlated with the non-amidated peptide proctolin. Tissue immunostaining demonstrates that dPAL1 is largely localized to the cell soma, whereas dPAL2 is distributed throughout neuronal processes.

Genome-wide features of neuroendocrine regulation in Drosophila by the basic helix-loop-helix transcription factor DIMMED

“…These enzymes are LDCV proteins (both luminal and membrane components) that help process neuropeptide precursors transiting through the trans -Golgi to specific secretory granules (22). DIMM targets many of the genes that encode major enzymes in this pathway, including Phm (50), Pal1 and Pal2 (76), amon (dPC2; (57)) and silver (CPD; (58)). Some of these Drosophila genes encode multiple isoforms, only some of which operate in the context of neuropeptide biosynthesis.…”

Role for an Essential Tyrosine in Peptide Amidation

“…Drosophila, Cnidaria, and Planaria), PHM and PAL are encoded by separate genes (9 -11). The Drosophila genome encodes two active PAL proteins (12); whereas Drosophila PAL2 is a soluble protein localized to secretory granules, Drosophila PAL1 is an integral membrane protein localized to the endoplasmic reticulum. Although yeast and humans use similar subtilisin-like endoproteases to produce bioactive peptides from inactive precursors (13,14), yeast cells do not produce amidated peptides, and the yeast genome does not contain sequences homologous to PHM or PAL.…”

A "Neural" Enzyme in Nonbilaterian Animals and Algae: Preneural Origins for Peptidylglycine -Amidating Monooxygenase

“…By contrast, many bilaterian invertebrates (classes Urochordata, Cephalochordata, Echinodermata, Hemichordata, Platyhelminthes, Mollusca, Annelida, and Nematoda) possess loci encoding monofunctional PHM, PAL, or both (sometimes with more than one of each) in addition to a single bifunctional PAM. Among the arthropods, the water flea Daphnia pulex has a single bifunctional PAM, two PHMs, and a PAL, but all insects investigated (representatives of diverse orders) lack PAM and encode the domains separately, as has been reported for D. melanogaster (Kolhekar et al 1997b;Han et al 2004). Although the survey presented here indicates that many invertebrates have discrete loci encoding monofunctional and bifunctional enzymes, there have been few previous reports of this in the literature.…”

“…However, as mentioned by Han et al (2004), C. parasitica does have a PAM sequence (accession AAG44250), distinct from the reported PHM. These data for C. parasitica are broadly consistent with our survey of other cnidarians.…”

“…Some invertebrates, too, possess a gene encoding a bifunctional enzyme: the first to be reported was in the marine mollusc Aplysia californica (Fan et al 2000). Other variations include four different PHM domains upstream of PAL in another mollusc (Spijker et al 1999) and separately encoded PHM and PAL in Drosophila (Kolhekar et al 1997b;Han et al 2004) and the flatworm Schistosoma (Mair et al 2004;Atkinson et al 2010). Stand-alone PHMs were also reported, and a separately encoded PAL posited, for another flatworm (Asada et al 2005) and for a sea anemone (Hauser et al 1997;Williamson et al 2000).…”