First published online November 4, 2005
Journal of Experimental Biology 208, 4273-4282 (2005)
Published by The Company of Biologists 2005
doi: 10.1242/jeb.01910
Molecular characterisation of SALMFamide neuropeptides in sea urchins
Maurice R. Elphick1,* and
Michael C. Thorndyke2
1 School of Biological and Chemical Sciences, Queen Mary, University of
London, Mile End Road, London, E1 4NS, UK
2 Kristineberg Marine Research Station, Fiskebackskil, S 450 34,
Sweden
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Fig. 1. SALMFamide-like immunoreactivity in whole-body extracts of Echinus
esculentus. The graphs show SALMFamide-like immunoreactivity in a 40%
ACN/TFA Sep-Pak eluate (A,B) and a 60% ACN/TFA eluate (C,D) after HPLC
fractionation using a gradient of ACN/TFA indicated by the line. HPLC
fractions were radioimmunoassayed for both S1-(A,C) and S2-(B,D)
immunoreactivity using the antisera SLII and BGI, respectively. The 40%
Sep-Pak eluate contains two S2-immunoreactive peaks, 1 and 2 (B), which are
weakly immunoreactive with the S1 antiserum SLII. The 60% Sep-Pak eluate also
contains two S2-immunoreactive peaks, 3 and 4 (D), which are weakly
immunoreactive with the S1 antiserum SLII. The elution times of peaks 2 and 3
are the same so they may contain identical immunoreactive peptides. The SLII
antiserum also detects a few minor immunoreactive peaks, which elute before or
after the major peaks 1, 2, 3 and 4.
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Fig. 2. HPLC purification of the S2-like immunoreactive peak 3 from Echinus
esculentus. Seven HPLC steps were required to purify this peak and the
final chromatogram is shown here. The S2-like immunoreactivity elutes in four
fractions corresponding to a single peak of absorbance at 210 nm and amino
acid sequencing of these fractions yielded the partial sequence MRYH.
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Fig. 3. Identification of a SALMFamide precusor gene in Strongylocentrotus
purpuratus. (A) Output data from a tBLASTn analysis of
Strongylocentrotus purpuratus genomic sequence data using a putative
starfish SALMFamide-1 (S1) precursor sequence (GFNSALMFGKR x 6) as the
query. A region of contig 347664 was identified as the sea urchin sequence
with the highest level of sequence identity/similarity with the query sequence
(E=8e-04). The translated sequence of bases 13566-13718 from contig 347664 is
shown aligned with the query sequence and four putative sea urchin SALMFamide
neuropeptides are underlined. (B) Diagram showing data obtained from SignalP
3.0 analysis of a 53 amino acid residue sequence encoded by bases 11827-11986
of contig 347664, using hidden Markov models based on eukaryotic data for
signal peptide prediction. The red line shows cleavage site probability,
indicating that cleavage occurs between residues 25 (S) and 26 (F). The green,
dark blue and light blue lines indicate that the first 25 residues of the
sequence conform with high probability to n-terminal (n), hydrophobic (h) and
c-terminal (c) regions, respectively, that are characteristic of eukaryotic
signal peptides. Collectively, these data provide strong evidence that the
first 25 amino acids of the 53 residue sequence are likely to function as a
signal peptide, which is a characteristic feature of all neuropeptide
precursors.
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Fig. 4. Structure of the SALMFamide precursor gene in Strongylocentrotus
purpuratus. The DNA sequence of the gene is shown in lowercase letters
with base positions in contig 347664 shown in the column on the right
(11762-13920). The predicted precursor sequence is shown in uppercase letters
with amino acid positions shown in bold and in brackets in the column on the
right (1-266). The gene comprises two exons, whose positions are indicated by
the underlying amino acid sequence in uppercase letters, with the start and
end of the intervening intron labelled in bold letters (gt/ag). 24 bases
upstream of the start codon (atg) in the first exon is a putative
TATA-box-like promoter sequence (tttatt), which is shown in bold. The
predicted signal peptide sequence encoded by the first exon is shown in
underlined italics, based on the analysis shown in
Fig. 3B. Amino acid sequences
corresponding to putative SALMFamide neuropeptides encoded by the second exon
are shown underlined, with monobasic and dibasic (K, KR, RR) cleavage sites
shown in bold. In the 3' region of the gene there is consensus
polyadenylation signal sequence (aataaa) located 47 bases downstream from the
stop codon (*).
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Fig. 5. Nomenclature for Strongylocentrotus purpuratus SALMFamide
neuropeptides and comparison with the sequences of other echinoderm SALMFamide
neuropeptides. (A) The predicted amino acid sequence of the
Strongylocentrotus purpuratus SALMFamide precursor showing the
positions of putative SALMFamide neuropeptides (red), which we have named
SpurS1-SpurS7 based on their relative position in the precursor. The signal
peptide is shown in blue and putative monobasic and dibasic (K, KR, RR)
cleavage sites are shown in green. (B). The sequences of starfish and sea
cucumber SALMFamides are shown aligned with the seven putative sea urchin
SALMFamides (SpurS1-SpurS7) identified in this study. Four of the putative
neuropeptides in Strongylocentrotus purpuratus (SpurS1, SpurS2,
SpurS3, SpurS6) have the same consensus sequence as the starfish and sea
cucumber SALMFamides (i.e. SxL/FxFamide, where x is
variable). SpurS4, SpurS5 and SpurS7 have a proline or leucine residue
substituted for the serine residue in the consensus sequence but share the
sequence FxFamide with SpurS1, SpurS2, SpurS3, SpurS6 and the
Stichopus SALMFamides GYSPFMFamide and FKSPFMFamide. References:
1Elphick et al.,
1991a ,b;
2Díaz-Miranda et al.,
1992; 3Ohtani et
al., 1999; 4present study.
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© The Company of Biologists Ltd 2005
