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First published online March 27, 2009
Journal of Experimental Biology 212, 1067-1077 (2009)
Published by The Company of Biologists 2009
doi: 10.1242/jeb.027599
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NGFFFamide and echinotocin: structurally unrelated myoactive neuropeptides derived from neurophysin-containing precursors in sea urchins

Maurice R. Elphick* and Matthew L. Rowe

School of Biological and Chemical Sciences, Queen Mary University of London, Mile End Road, London E1 4NS, UK


Figure 1
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  		Fig. 1. The Strongylocentrotus purpuratus NGFFFamide precursor. The sequence of a cDNA (lowercase, 1302 bases) encoding the NGFFFamide precursor protein (bold uppercase, 266 amino acid residues) is shown. The DNA sequence was derived from genomic sequence data but EST data were used to determine the length of 5' and 3' non-coding regions and the positions of introns. The positions of introns in the gene encoding the NGFFFamide precursor are shown by highlighting the pairs of bases (bold and underline) in the cDNA sequence that are interrupted by an intron in the corresponding genomic sequence. The predicted signal peptide is shown in blue and the two copies of the NGFFFG sequence are shown in red, interrupted and flanked by putative dibasic cleavage sites (KR) shown in green. The C-terminal neurophysin-like region of the precursor is shown in purple with the 14 cysteine residues underlined. The asterisk shows the position of the stop codon.

 

Figure 2
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  		Fig. 2. The S. purpuratus echinotocin precursor. The nucleotide sequence (lowercase) encoding the echinotocin precursor is shown, as predicted by the GLEAN3 gene prediction algorithm, with the corresponding protein sequence (165 residues) shown in bold uppercase. The positions of introns in the gene encoding the echinotocin precursor are shown by highlighting the pairs of bases (bold and underline) in the sequence that are interrupted by an intron. The predicted signal peptide is shown in blue, the echinotocin sequence (CFISNCPKGG) is shown in red followed by a putative dibasic cleavage site (KR) shown in green. The neurophysin-like region of the precursor is shown in purple with the 14 cysteine residues underlined. The asterisk shows the position of the stop codon.

 

Figure 3
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  		Fig. 3. ClustalX multiple alignment of the sequences of the S. purpuratus NGFFFamide precursor, the S. purpuratus echinotocin precursor and precursors of vasopressin/oxytocin-like peptides in other species. Signal peptides are shown in blue, neuropeptides are shown in red, dibasic cleavage sites are shown in green and neurophysin-like domains are shown in purple. The conserved cysteine residues in the neurophysin-like domains are underlined and numbered 1–14. The precursors of vasopressin/oxytocin-like peptides from other species include precursors of human vasopressin (Mohr et al., 1985Go), human oxytocin (Mohr et al., 1985Go), vasotocin from the lamprey Lethenteron japonicum (Suzuki et al., 1995Go), an oxytocin-like peptide (SOP) from the urochordate Styela plicata (Ukena et al., 2008Go), Lys-conopressin from the mollusc Lymnaea stagnalis (Van Kesteren et al., 1992Go), cephalotocin from the mollusc Octopus vulgaris (Reich, 1992Go), annetocin from the annelid Eisenia foetida (Oumi et al., 1994Go) and inotocin from the arthropod (insect) Tribolium castaneum (Aikins et al., 2008Go; Stafflinger et al., 2008Go). Also included is a precursor from the nematode Caenorhabditis elegans (GenBank: NP_001033548, GI:86564869) that has not been reported previously in the literature; this precursor contains an unusual putative vasopressin/oxytocin-like peptide comprising just eight residues (CFLNSCPY), which we have named `nematocin'.

 

Figure 4
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  		Fig. 4. Neighbour-joining tree (with bootstrap values) based on a ClustalX multiple alignment of neurophysin (NP) sequences, incorporating residues from the first to the fourteenth conserved cysteines. The tree shows that the neurophysin domain of the S. purpuratus NGFFFamide precursor does not have a higher level of overall sequence similarity with the neurophysin domain of the S. purpuratus echinotocin precursor than with the neurophysins from other species.

 

Figure 5
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  		Fig. 5. NGFFFamide and echinotocin cause contraction of sea urchin tube foot and oesophagus preparations. Representative traces show that application (arrows) of NGFFFamide (3x10–6 mol l–1; A,B) and echinotocin (3x10–6 mol l–1; C,D) causes contraction of oesophagus (A,C) and of tube foot (B,D) preparations from the sea urchin Echinus esculentus. (E) Graph showing the dose-dependent effect of NGFFFamide on tube foot (filled squares) and oesophagus (open squares) preparations. Data points are mean values (N=4) with bars showing s.e.m. (F) Graph showing the dose-dependent effect of echinotocin on tube foot (filled squares) and oesophagus (open squares) preparations. Data points are mean values (N=4) with bars showing s.e.m.

 

Figure 6
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  		Fig. 6. Comparative alignment of the amino acid sequence of echinotocin with the sequences of vasopressin, oxytocin, vasotocin and vasopressin/oxytocin-like peptides identified in other invertebrate species. Cysteine residues, which are conserved in all of the peptides, are shown in bold. References: 1present study; 2(Light and Du Vigneaud, 1958Go); 3(Suzuki et al., 1995Go); 4(Kawada et al., 2008Go); 5(Ukena et al., 2008Go); 6(Van Kesteren et al., 1992Go); 7(Reich, 1992Go); 8(Oumi et al., 1994Go); 9(Proux et al., 1987Go); 10(Aikins et al., 2008Go); 11(Stafflinger et al., 2008Go); 12GenBank NP_001033548, GI:86564869.

 

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© The Company of Biologists Ltd 2009