The protein precursors of peptides that affect the mechanics of connective tissue and/or muscle in the echinoderm Apostichopus japonicus

Abstract

Peptides that cause muscle relaxation or contraction or that modulate electrically-induced muscle contraction have been discovered in the sea cucumber Apostichopus japonicus (Phylum Echinodermata; Class Holothuroidea). By analysing transcriptome sequence data, here the protein precursors of six of these myoactive peptides (the SALMFamides Sticho-MFamide-1 and -2, NGIWYamide, stichopin, GN-19 and GLRFA) have been identified, providing novel insights on neuropeptide and endocrine-type signalling systems in echinoderms. The A. japonicus SALMFamide precursor comprises eight putative neuropeptides including both L-type and F-type SALMFamides, which contrasts with previous findings from the sea urchin Strongylocentrotus purpuratus where L-type and F-type SALMFamides are encoded by different genes. The NGIWYamide precursor contains five copies of NGIWYamide but, unlike other NG peptide-type neuropeptide precursors in deuterostomian invertebrates, the NGIWYamide precursor does not have a C-terminal neurophysin domain, indicating loss of this character in holothurians. NGIWYamide was originally discovered as a muscle contractant, but it also causes stiffening of mutable connective tissue in the body wall of A. japonicus, whilst holokinins (PLGYMFR and derivative peptides) cause softening of the body wall. However, the mechanisms by which these peptides affect the stiffness of body wall connective tissue are unknown. Interestingly, analysis of the A. japonicus transcriptome reveals that the only protein containing the holokinin sequence PLGYMFR is an alpha-5 type collagen. This suggests that proteolysis of collagen may generate peptides (holokinins) that affect body wall stiffness in sea cucumbers, providing a novel perspective on mechanisms of mutable connective tissue in echinoderms.

Figure 1. The Apostichopus japonicus SALMFamide precursor.

The cDNA sequence (lowercase, 1054 bases) encoding the SALMFamide precursor protein (bold uppercase, 290 amino acid residues) is shown. The predicted signal peptide is shown in blue and the eight putative SALMFamide neuropeptides are shown in red, flanked by putative dibasic cleavage sites (KR or RR) shown in green. The asterisk shows the position of the stop codon.

Figure 2. The Apostichopus japonicus NGIWYamide precursor.

The cDNA sequence (lowercase, 1226 bases) encoding the NGIWYamide precursor protein (bold uppercase, 238 amino acid residues) is shown. The predicted signal peptide is shown in blue and the five copies of NGIWYamide are shown in red, flanked by putative dibasic cleavage sites (KR) shown in green. The asterisk shows the position of the stop codon.

Figure 3. The Apostichopus japonicus stichopin precursor.

The cDNA sequence (lowercase, 449 bases) encoding the stichopin precursor protein (bold uppercase, 39 amino acid residues) is shown. The predicted signal peptide is shown in blue and the stichopin peptide sequence is shown in red. The asterisk shows the position of the stop codon.

Figure 4. The Apostichopus japonicus GN-19 precursor.

The cDNA sequence (lowercase, 582 bases) encoding the GN-19 precursor protein (bold uppercase, 82 amino acid residues) is shown. The predicted signal peptide is shown in blue and the GN-19 peptide sequence is shown in red, flanked by putative dibasic cleavage sites (KR, RR) shown in green. The asterisk shows the position of the stop codon.

Figure 5. The Apostichopus japonicus GLRFA precursor.

The cDNA sequence (lowercase, 429 bases) encoding the GLRFA precursor protein (bold uppercase, 62 amino acid residues) is shown. The predicted signal peptide is shown in blue and the single copy of the GLRFA peptide sequence is shown in red, flanked N-terminally by a putative dibasic cleavage site (KR) and C-terminally by a putative monobasic cleavage site (R), which are shown in green. The asterisk shows the position of the stop codon.

Figure 6. The holokinin peptide sequence PLGYMFR is present in Apostichopus japonicus alpha-5 type collagen.

A. The sequence of isotig 14428 is shown (lowercase, 1582 bases) with the partial sequence of the protein that it encodes shown underneath in bold uppercase (298 amino acid residues). The holokinin sequence PLGYMFR is shown in red and the asterisk shows the position of the stop codon. B. BLAST alignment showing that the protein encoded by isotig 14428 (Query) shares a high level of sequence similarity with the C-terminal region of alpha-5 type collagen from the sea urchin Strongylocentrotus purpuratus (Subjct; XP_780851.3, GI:390347052).