Premetazoan Origin of Neuropeptide Signaling

Abstract

Neuropeptides are a diverse class of signaling molecules in metazoans. They occur in all animals with a nervous system and also in neuron-less placozoans. However, their origin has remained unclear because no neuropeptide shows deep homology across lineages, and none have been found in sponges. Here, we identify two neuropeptide precursors, phoenixin (PNX) and nesfatin, with broad evolutionary conservation. By database searches, sequence alignments, and gene-structure comparisons, we show that both precursors are present in bilaterians, cnidarians, ctenophores, and sponges. We also found PNX and a secreted nesfatin precursor homolog in the choanoflagellate Salpingoeca rosetta. PNX, in particular, is highly conserved, including its cleavage sites, suggesting that prohormone processing occurs also in choanoflagellates. In addition, based on phyletic patterns and negative pharmacological assays, we question the originally proposed GPR-173 (SREB3) as a PNX receptor. Our findings revealed that secreted neuropeptide homologs derived from longer precursors have premetazoan origins and thus evolved before neurons.

Keywords: choanoflagellate; ctenophore; nesfatin; neuropeptide; phoenixin; sponge.

Fig. 1.

Sequence alignment and genomic structure of PNX precursors. (A) Alignment of the PNX precursors containing the PNX peptides. Predicted signal peptides and mature peptides are indicated with lines. Residues that are conserved in more than 50% of the sequences are shown in black, and conservative substitutions are shown in gray. Amidation sites are highlighted in red. (B) Exon–intron structure of PNX precursor genes. The regions encoding the signal peptides are in blue with their length indicated. Amino acids encoded at the exon–intron junctions are shown above the exon boxes. Introns are shown as lines, and their length in base pairs is indicated below. The intron phase is shown above the introns.

Fig. 2.

Sequence alignment of nesfatin-1 and genomic structure of NUCB precursors. (A) Alignment of the N-terminal NUCB precursor region containing the nesfatin-1 peptides. The conserved residues are highlighted, with conservation in more than 50% of sequences shown in black, and conservative substitutions shown in gray. (B) The genomic exon–intron structure of NUCB precursors. The regions encoding the signal peptides are shown in blue. The nesfatin-1-peptide coding region is indicated in dark red. Introns are shown as lines, with the phase of the introns shown above the lines. An empty/white box indicates a missing part in the mRNA-genome alignment.

Fig. 3.

Presence and absence of PNX and nesfatin precursors and SREB receptors in the investigated species. Phylogenomic tree of the investigated species, annotated with the presence/absence of PNX precursor, NUCB and its nesfatin-1 peptide, and SREB receptors. The PNX neuropeptide precursor is conserved across metazoans and in the choanoflagellate S. rosetta, whereas GPR173 (proposed as a potential receptor for this peptide) is only present in Bilateria. The NUCB precursor gene is conserved across metazoans and present in choanoflagellates and Tunicaraptor, whereas the nesfatin-1 peptide is only encoded in metazoans. A question mark indicates that the NUCB sequence was only partially recovered with the N-terminal part that encodes the nesfatin-1 peptide missing in the transcriptome. An unfilled box indicates that the corresponding gene was not identified. Dashed lines in main bilaterian branches indicate generally contradicting results in different phylogenomic analyses.