Defective processing of neuropeptide precursors in Caenorhabditis elegans lacking proprotein convertase 2 (KPC‐2/EGL‐3): mutant analysis by mass spectrometry

Abstract

Biologically active peptides are synthesized as larger inactive proprotein peptide precursors which are processed by the concerted action of a cascade of enzymes. Among the proprotein convertases, PC2 is widely expressed in neuroendocrine tissues and has been proposed to be the major convertase involved in the biosynthesis of neuropeptides. In this study, we have examined the role of the Caenorhabditis elegans orthologue PC2/EGL-3 in the processing of proprotein peptide precursors. We recently isolated and identified 60 endogenous peptides in the nematode C. elegans by twodimensional nanoscale liquid chromatography -quadrupole time-of-flight tandem mass spectrometry. In the present study, we compare the peptide profile of different C. elegans strains, including PC2/EGL-3 mutants. For this purpose, we used an offline approach in which HPLC fractions are analysed by a matrix-assisted laser desorption ionisation -time of flight mass spectrometer. This differential peptidomic approach unambiguously provides evidence for the role of PC2/EGL-3 in the processing of FMRFamide-like peptide (FLP) precursors and neuropeptide-like protein (NLP) precursors in nematodes. Keywords: EGL-3, FMRFamide-like peptide gene, mass spectrometry, neuropeptide, neuropeptide-like protein gene, proprotein convertase. J. Neurochem. (2006Neurochem. ( ) 98, 1999Neurochem. ( -2012 Neuropeptides are derived from large proprotein peptide precursor genes, which encode for a single or multiple peptides. Several highly regulated and specific post-translational events are required for the formation of biologically active peptides from their larger inactive precursor proteins. A typical proprotein peptide precursor contains an amino terminal signal peptide, which is cleaved off upon entrance into the secretory pathway by a signal peptidase. Subsequently, proprotein convertases (PCs) cleave the remaining part of the precursor at specific cleavage sites. KR and RR constitute a large majority of all cleavage places, while RK and KK are found with much lower frequency (Veenstra 2000;Fricker 2005). In addition to these sites, some cleavages occur at sites containing pairs of basic amino acids separated by two, four, six or eight other residues. These sites were earlier described as 'monobasic' (Veenstra 2000). After cleavage from the proprotein peptide precursor, the peptide is further processed by a specific carboxypeptidase that removes the carboxyterminal basic amino acids. Next, the carboxyterminal glycine residue is transformed into an amide by a-amidating enzymes. This post-translational modification (conversion of a glycine into an amide) is a common feature of secretory peptides.In mammals, seven members of the kex2/subtilisin-like PCs have been described in detail (Rouille et al. 1995;Beinfeld 1998;Canaff et al. 1999). These are designated furin, PC1/3, PC2, PC4, PC5/6, PC7/8/LPC and PACE4 and they all share a characteristic overall similarity to the prototype member of the family, Kex2p, that was initially identified in yeast (Julius et a...

Intestinal signaling to GABAergic neurons regulates a rhythmic behavior in Caenorhabditis elegans

“…aex-5 encodes a pro-protein convertase and was identified in the same screen that isolated aex-4 (1,12,13). We confirmed that the pro-protein convertase gene is mutated in aex-5(sa23) by sequencing (lesion is C443W) and by genomic fosmid rescue (Fig.…”

“…aex-3 and aex-6 regulate synaptic transmission, probably by regulating exocytosis of neurotransmitter: aex-3 is a guanine nucleotide exchange factor that regulates Rab small guanosine triphosphatase function, and aex-6 (also known as rab-27) is a Rab small guanosine triphosphatase that regulates secretory vesicle exocytosis (10,11). aex-5 encodes a pro-protein convertase, an enzyme that is copackaged with propeptides and processes them to make mature secretory molecules (12,13). Lastly, Doi and Iwasaki (12) demonstrated that aex-1 is a distant homologue of the synaptic gene unc-13 (or Munc13), which acts in the intestine to regulate the DMP.…”

FMRFamide‐like peptides encoded on the flp‐18 precursor gene activate two isoforms of the orphan Caenorhabditis elegans G‐protein‐coupled receptor Y58G8A.4 heterologously expressed in mammalian cells

“…3,[26][27][28] Recently, SYFDEKKSVPGVLRF-NH 2 was isolated 3,27 and EMPGVLRF-NH 2 was both predicted and isolated 3,27 ; neither was included in the current study. When we tested KSVPGVLRF-NH 2 (a predicted sequence 25 ) and SVPGVLRF-NH 2 (the isolated peptide [26][27][28] ), both peptides were equally potent to activate FLP-18R1b but SVPGVLRF-NH 2 was two fold more potent at the FLP-18R1a receptor (Table I). It is interesting to note that the shorter peptides were more potent than longer peptides and that the most potent was not a C. elegans but an A. suum peptide, AVPGVLRF-NH 2 (EC 50 7.6-13.1 nM).…”

FMRFamide-Like Neuropeptides and Mechanosensory Touch Receptor Neurons Regulate Male Sexual Turning Behavior inCaenorhabditis elegans

“…Each neuropeptide processing and secretion component controls multiple behavioral programs. Although immunostaining and mass spectrometry studies have shown that egl-3 and egl-21 mutants have significantly reduced levels of mature FMRFamide-related peptides (Jacob and Kaplan, 2003;Husson et al, 2006), there have been no functional studies linking EGL-3, EGL-21, IDA-1, or UNC-31 to any of the C. elegans neuropeptides. Our newly identified male repetitive turning phenotype of the flp mutants provided an opportunity to genetically address the question.…”