Abstract
The recent identification of catalytically active peptidylglycine α-amidating monooxygenase (PAM) in Chlamydomonas reinhardtii, a unicellular green alga, suggested the presence of a PAM-like gene and peptidergic signaling in the last eukaryotic common ancestor (LECA). We identified prototypical neuropeptide precursors and essential peptide processing enzymes (subtilisin-like prohormone convertases and carboxypeptidase B-like enzymes) in the C. reinhardtii genome. Reasoning that sexual reproduction by C. reinhardtii requires extensive communication between cells, we used mass spectrometry to identify proteins recovered from the soluble secretome of mating gametes, and searched for evidence that the putative peptidergic processing enzymes were functional. After fractionation by SDS-PAGE, signal peptide-containing proteins that remained intact, and those that had been subjected to cleavage, were identified. The C. reinhardtii mating secretome contained multiple matrix metalloproteinases, cysteine endopeptidases, and serine carboxypeptidases, along with one subtilisin-like proteinase. Published transcriptomic studies support a role for these proteases in sexual reproduction. Multiple extracellular matrix proteins (ECM) were identified in the secretome. Several pherophorins, ECM glycoproteins homologous to the Volvox sex-inducing pheromone, were present; most contained typical peptide processing sites, and many had been cleaved, generating stable N- or C-terminal fragments. Our data suggest that subtilisin endoproteases and matrix metalloproteinases similar to those important in vertebrate peptidergic and growth factor signaling play an important role in stage transitions during the life cycle of C. reinhardtii.
Highlights
Identification of the enkephalins as endogenous ligands for opioid receptors led to the successful description of hundreds of additional bioactive peptides in the nervous systems of species as diverse as Drosophila, Caenorhabditis elegans, and Hydra [1,2,3]
To search for any neuropeptide-like precursors encoded by the C. reinhardtii genome, we identified primary transcripts (Phytozome v5.5; a total of 17,741) encoding proteins with a predicted signal peptide (SignalP v4.1) and no transmembrane helices (TMHMM v2.0), resulting in a list of 771 proteins
We limited our search to the 771 soluble proteins predicted to contain an N-terminal signal peptide (Figure 2A)
Summary
Identification of the enkephalins as endogenous ligands for opioid receptors led to the successful description of hundreds of additional bioactive peptides in the nervous systems of species as diverse as Drosophila, Caenorhabditis elegans, and Hydra [1,2,3]. The precursors to these neuropeptides have N-terminal signal sequences, multiple potential paired. Proteomes 2018, 6, 36 basic amino acid endoproteolytic cleavage sites, potential amidation sites, generally lack recognized domains, and often contain multiple copies of similar peptides (Figure 1). The reactions catalyzed by subtilisin-like prohormone convertases, carboxypeptidase B (CPB)-like enzymes and peptidylglycine α-amidating monooxygenase (PAM) are shown. Endoplasmic reticulum (ER) entry requires an N-terminal signal peptide, which is quickly removed. A family of subtilisin-like endoproteases, referred to as prohormone convertases (PCs), catalyze a series of ordered endoproteolytic cleavages, with furin (PCSK3), PC1 (PCSK1), and PC2 (PCSK2) playing especially important roles in many neurons and endocrine cells. CPB-like enzymes (CPE and CPD) remove the C-terminal Lys and Arg residues produced by furin, PC1, and PC2. The amidating enzyme, PAM, requires only a C-terminal Gly residue to amidate the penultimate residue (–X–amide); in the presence of adequate copper, ascorbate, and molecular oxygen, PAM can function throughout the biosynthetic pathway [6]
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