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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
NGFFFamide and echinotocin: structurally unrelated myoactive neuropeptides derived from neurophysin-containing precursors in sea urchins
School of Biological and Chemical Sciences, Queen Mary University of London, Mile End Road, London E1 4NS, UK
* Author for correspondence (e-mail: m.r.elphick{at}qmul.ac.uk)
Accepted 28 January 2009
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Summary |
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 TOP Summary INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONReferences |
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Key words: NGFFFamide, echinotocin, vasopressin, oxytocin, vasotocin, neurophysin, NGIWYamide, echinoderm, Strongylocentrotus purpuratus, Echinus esculentus, neuropeptide, nematocin, Caenorhabditis elegans
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INTRODUCTION |
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TOPSummaryINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONReferences |
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; Grimmelikhuijzen et al.,
1999; Hoyle, 1999;
O'Shea and Schaffer, 1985;
Strand, 1999). However,
relatively little is known about neuropeptide structure and function in the
phylum Echinodermata (e.g. sea urchins, starfish, sea cucumbers). New
opportunities to identify and characterize echinoderm neuropeptides have
emerged recently with the sequencing of the genome of the sea urchin
Strongylocentrotus purpuratus (Order Echinoida; Family
Strongylocentrotidae) (Burke et al.,
2006; Sodergren et al.,
2006). Moreover, there are a number of reasons why analysis of
neuropeptides in echinoderms is of interest.
Adult echinoderms are unique in the animal kingdom in having a pentaradial
morphological organization, which is both evolutionarily and developmentally
derived from bilateral symmetry (Burke et
al., 2006). It is of particular interest, therefore, to determine
how neuropeptides participate in the neural coordination of physiology and
behaviour in the context of a pentaradial bauplan. Furthermore, analysis of
neuropeptide expression provides a useful approach for investigation of the
changes in neuroarchitecture that accompany the transition from bilaterally
symmetrical larvae to radially symmetrical adult echinoderms
(Byrne and Cisternas,
2002).
As deuterostomian invertebrates, echinoderms occupy an interesting
phylogenetic position in the animal kingdom because, together with
hemichordates and xenoturbellids, they form a sister clade to the chordates
(Bourlat et al., 2006;
Bromham and Degnan, 1999;
Dunn et al., 2008).
Comparative analysis of echinoderms and chordates therefore provides a basis
for identifying synapomorphies shared within the deuterostome clade as well as
characters that differentiate echinoderms from chordates.
Echinoderms have many unusual biological properties, which include
remarkable powers of autotomy and regeneration and the ability to rapidly and
reversibly change the mechanical state of their body wall and/or body
wall-associated appendages (Byrne,
2001; Patruno et al.,
2001; Thorndyke et al.,
2001; Wilkie,
2001; Wilkie,
2005). Neuropeptides have been implicated as potential regulators
of these processes (Birenheide et al.,
1998; Mladenov et al.,
1989; Tamori et al.,
2007) but more detailed investigation of the role of neuropeptides
in these and other aspects of echinoderm biology is needed.
The first neuropeptides to be identified in echinoderms were a family of
peptides known as SALMFamides, which have a characteristic C-terminal motif,
Sx(L/F)xFamide (where x is variable). The
prototypes for this family, S1 (GFNSALMFamide) and S2 (SGPYSFNSGLTFamide),
were both isolated from the starfish Asterias rubens and Asterias
forbesi (Elphick et al.,
1991a; Elphick et al.,
1991b). Subsequently, members of the SALMFamide family have been
identified in sea cucumbers, including GFSKLYFamide and SGYSVLYFamide from
Holothuria glaberrima
(Díaz-Miranda et al.,
1992). Pharmacological studies have revealed that SALMFamide
neuropeptides cause relaxation of muscle preparations in starfish and sea
cucumbers (Díaz-Miranda and
García-Arrarás, 1995;
Elphick et al., 1995;
Elphick et al., 1991a;
Melarange and Elphick, 2003;
Melarange et al., 1999) and
SALMFamides may have a general role as muscle relaxants throughout the phylum
Echinodermata (Elphick and Melarange,
2001). Furthermore, evidence of other physiological roles of
SALMFamides in echinoderms has been reported, including modulation of
luminescence in brittle stars (De
Bremaeker et al., 1999) and regulation of neurohormone
(gonad-stimulating substance) secretion in starfish
(Mita et al., 2004).
Sequencing of the genome of the sea urchin S. purpuratus
facilitated identification of a gene encoding SALMFamides, the first
neuropeptide precursor gene to be characterized in an echinoderm
(Elphick and Thorndyke, 2005).
The S. purpuratus SALMFamide gene comprises two protein-coding exons:
the first exon encodes an N-terminal signal peptide and the second exon
encodes seven putative SALMFamide neuropeptides known as SpurS1–SpurS7
(Elphick and Thorndyke, 2005).
Discovery of this gene is of interest because it has revealed an unprecedented
diversity of SALMFamides in an echinoderm species. Moreover, identification of
the SALMFamide gene in S. purpuratus has paved the way for the
identification of other neuropeptide genes in this species.
SALMFamide neuropeptides were originally isolated from starfish and sea
cucumbers because of their cross-reactivity with antibodies to molluscan
FMRFamide-related peptides
(Díaz-Miranda et al.,
1992; Elphick et al.,
1991a). Subsequently, Iwakoshi and colleagues used a different
strategy for the isolation and identification of echinoderm neuropeptides
(Iwakoshi et al., 1995).
Radial longitudinal muscle and intestinal preparations from the sea cucumber
Apostichopus japonicus were used to test for the presence of
myoactive peptides in body wall extracts of the same species
(Iwakoshi et al., 1995;
Ohtani et al., 1999). Amongst
the peptides identified were two members of the SALMFamide family
(GYSPFMFamide and FKSPFMFamide) and, consistent with previous pharmacological
tests with SALMFamides, both peptides caused relaxation of muscle preparations
(Ohtani et al., 1999). Many of
the other peptides identified had indirect effects on muscle contractility,
either potentiating or inhibiting electrically evoked contractions. However,
one of the peptides identified (NGIWYamide) was found to cause contraction of
the muscle preparations tested (Iwakoshi
et al., 1995; Ohtani et al.,
1999).
The physiological roles of NGIWYamide in holothurians have been
investigated in detail by testing the effects of NGIWYamide on longitudinal
body wall muscle, tentacles and intestine from Apostichopus japonicus
(Inoue et al., 1999).
NGIWYamide caused contraction of body wall muscle and tentacle preparations,
consistent with the effects of NGIWYamide originally observed by Iwakoshi and
colleagues (Iwakoshi et al.,
1995). However, NGIWYamide also caused inhibition of the
spontaneous rhythmic contractile activity of intestine preparations. Using
antibodies to NGIWYamide to analyse the distribution of this peptide in
Apostichopus japonicus, abundant NGIWYamide immunoreactivity was
observed in the radial nerve cords and circumoral nerve ring, localized in
neuronal cell bodies and their processes. In addition, and consistent with the
pharmacological effects of NGIWYamide, NGIWYamide immunoreactivity was
detected in the innervation of body wall dermis, intestine, tentacles and tube
feet (Inoue et al., 1999).
More recently, Saha and colleagues tested the effects of NGIWYamide on tube
foot preparations from the starfish species Asterina pectinifera and
found that the peptide causes contraction
(Saha et al., 2006).
Furthermore, antibodies to NGIWYamide revealed the presence of NGIWYamide-like
immunoreactivity in the radial nerve cords and tube foot innervation in
Asterina pectinifera. Collectively, these data indicate that
NGIWYamide-related peptides may occur throughout the phylum Echinodermata and
may have a general role in neural regulation of muscle contraction in
echinoderms. However, to test these hypotheses it will be necessary to
identify NGIWYamide-related peptides in other echinoderms apart from sea
cucumbers. Therefore, building on a successful strategy that led to the
identification of a SALMFamide gene in the sea urchin S. purpuratus,
here we have investigated the occurrence of a gene encoding an
NGIWYamide-related peptide in this species.
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MATERIALS AND METHODS |
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TOPSummaryINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONReferences |
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)]
facility available on the Baylor College of Medicine Human Genome Sequencing
Center website
(http://www.hgsc.bcm.tmc.edu/blast/blast.cgi?organism=Spurpuratus).
The strategy used was similar to that employed previously to identify a gene
encoding novel SALMFamide neuropeptides in S. purpuratus
(Elphick and Thorndyke, 2005).
Thus, the query sequence comprised three copies of the sequence NGIWY
separated by the sequence GKR, with the glycine (G) residues putative
substrates for C-terminal amidation and the lysine–arginine (KR)
dipeptide sequences putative cleavage sites for endopeptidases (i.e.
NGIWYGKRNGIWYGKRNGIWYG). Using this approach, a contig containing a DNA
sequence encoding two copies of a putative NGIWYamide-like peptide
(NGFFFamide) was identified. The full-length sequence of the putative NGFFFamide precursor protein was determined by analysis of S. purpuratus genome and cDNA sequence data using resources available on the Baylor College of Medicine Human Genome Sequencing Center Sea Urchin Genome Project website (http://www.hgsc.bcm.tmc.edu/projects/seaurchin) and the NCBI Sea Urchin Genome Resources website (http://www.ncbi.nlm.nih.gov/genome/guide/sea_urchin/). As described in detail in the Results section, determination of the full-length sequence of the putative NGFFFamide precursor revealed that it shares sequence similarity with the precursor of a vasopressin/oxytocin-like peptide in S. purpuratus, which we have named `echinotocin'.
Comparison of the sequences of the NGFFFamide precursor, the echinotocin
precursor and precursors of vasopressin/oxytocin-like peptides in other
species was performed using ClustalX for multiple sequence alignment and NJ
plot for construction of trees with bootstrap analysis
(Saitou and Nei, 1987;
Thompson et al., 1997).
In vitro pharmacology
The pharmacological activity of NGFFFamide and echinotocin was investigated
by testing the effects of these peptides on in vitro preparations of
tube feet and oesophagus from specimens of the sea urchin Echinus
esculentus L. (Order Echinoida; Family Echinidae), which were collected
off the coast of Ayrshire in Scotland, transported to QMUL and maintained in a
seawater aquarium at about 11°C. NGFFFamide and echinotocin were custom
synthesized by the Advanced Biotechnology Centre at Imperial College London.
Echinotocin (CFISNCPKGamide) was synthesized with a disulphide bridge between
the cysteine residues, consistent with the occurrence of a disulphide bridge
in other members of the vasopressin/oxytocin neuropeptide family
(De Bree and Burbach, 1998;
Light and Du Vigneaud,
1958).
Tube foot preparations were obtained from specimens of E. esculentus by severing extended tube feet. Silk ligatures were tied around each end of the severed tube foot and one of the ligatures was attached to a glass rod. The preparation was then suspended in a 20 ml bath containing aerated seawater at 11°C and the second ligature was attached to an isometric force transducer (Harvard Apparatus, Edenbridge, Kent, UK). Likewise, oesophageal preparations were set up using approximately 1.5 cm sections of oesophagus. Once set up, tube foot and oesophageal preparations were allowed to equilibrate until a stable resting tension was obtained. The effects of NGFFFamide and echinotocin on tube foot and oesophageal preparations were examined by applying the peptides to achieve bath concentrations within the range 10–11 to 10–6 mol l–1. Additionally, NGFFFamide and echinotocin at a concentration of 3x10–6 mol l–1 were tested consecutively on tube foot and oesophagus preparations to enable direct comparison of their efficacy.
After dissection of tube foot and oesophagus preparations, sea urchins were anaesthetized in seawater containing 0.1 mol l–1 magnesium chloride.
|
RESULTS |
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TOPSummaryINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONReferences |
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-amidating
monooxygenase could give rise to two copies of the NGIWYamide-like peptide
Asn-Gly-Phe-Phe-Phe-(NH2) or NGFFFamide.
|
)]
predicts an N-terminal signal peptide (Fig.
1). Following the predicted 26 amino acid residue signal peptide
there is a 114 amino acid residue sequence, which is then followed by the 18
residue sequence KRNGFFFGKRNGFFFGKR, comprising two copies of the putative
NGFFFamide neuropeptide separated and flanked by potential dibasic cleavage
sites (KR). On the C-terminal side of the NGFFFamide-encoding region of the
precursor is a 108 amino acid residue sequence that contains 14 cysteine
residues. Moreover, submission of the putative NGFFFamide precursor as a
BLASTp query against the GenBank non-redundant database revealed that the
C-terminal region of the protein shares a high level of sequence identity with
neurophysins, proteins that form the C-terminal region of precursors for the
neuropeptide hormones vasopressin, oxytocin and vasotocin. For example,
residues 181–260 of the NGFFFamide precursor share 46% sequence identity
with residues 37–116 of the chicken vasotocin precursor. Comparison of the RNSP-5L15 cDNA sequence with the S. purpuratus genome sequence revealed that the 266 amino acid NGFFFamide precursor is encoded by a gene comprising four exons (Fig. 1). Exon 1 is a 206 base 5' non-coding sequence, which is separated from exon 2 by an intron comprising 37,141 bases. Exon 2 (150 bases) consists of a 5' non-coding region (42 bases) followed by 118 bases that encode the N-terminal signal peptide and the first 10 amino acid residues of the 114 residue polypeptide that separates the signal peptide from the NGFFFamide-encoding region. A short second intron (715 bases) is followed by exon 3, which comprises 444 bases encoding the remaining 104 residues of the 114 residue polypeptide, two copies of the NGFFFG sequence separated and flanked by putative dibasic cleavage sites (Lys–Arg) and then a 26 residue sequence. The third intron comprises 16,420 bases and is followed by exon 4 (502 bases), which comprises 246 bases encoding a neurophysin-like sequence followed by a stop codon and a 253 base 3' non-coding sequence.
In the sea urchin genome project, annotation of S. purpuratus
genome sequence data was facilitated by production of a list of genes
predicted by the GLEAN3 gene prediction algorithm
(Elsik et al., 2007;
Sodergren et al., 2006).
Interestingly, the NGFFFamide precursor gene was one of a number of genes that
were not predicted by the GLEAN3 algorithm. Therefore, we manually annotated
this gene as part of the sea urchin genome project annotation process and the
NGFFFamide gene has been assigned the official ID number SPU_030074 (see
http://www.spbase.org/SpBase/search/viewAnnoGeneInfo.php?spu_id=SPU_030074
for further details).
Identification of a gene encoding a vasopressin/oxytocin-like peptide (`echinotocin') in S. purpuratus
Our discovery that the C-terminal region of the putative NGFFFamide
precursor contains a polypeptide sequence similar to neurophysins that occur
in precursors of the peptide hormones vasopressin and oxytocin prompted us to
investigate the occurrence of a gene or genes encoding
vasopressin/oxytocin-like peptides in S. purpuratus. To do this the
human vasopressin precursor sequence was submitted as a BLASTp query against
putative S. purpuratus proteins predicted by the gene prediction
algorithm GLEAN3 (Elsik et al.,
2007; Sodergren et al.,
2006). The protein with the highest level of sequence identity
with the query sequence was a putative 225 amino acid residue protein
(GLEAN3_06899). Analysis of the sequence of this protein revealed that
residues 87–98 comprised a vasopressin/oxytocin-like peptide sequence
(CFISNCPKG) followed by a potential substrate for C-terminal amidation (G) and
a putative dibasic cleavage site (KR). Moreover, the C-terminal region of the
protein contained a neurophysin-like sequence. However, the N-terminal part of
the protein sequence (residues 1–86) did not share sequence similarity
with vasopressin and oxytocin precursors. Furthermore, analysis of the protein
sequence using SignalP 3.0 revealed that a predicted N-terminal signal peptide
was located between residues 61 and 86 of the putative 225 residue protein.
This suggested that inclusion of the N-terminal 60 residues of the 225 residue
protein, as predicted by GLEAN3, is likely to be erroneous. Thus, it appears
that in S. purpuratus there is a 165 residue
vasopressin/oxytocin-like precursor protein, which comprises a 26 residue
N-terminal signal peptide, a putative vasopressin/oxytocin-like peptide
(CFISNCPKGamide), which we have named `echinotocin', and a neurophysin-like
protein (Fig. 2).
|
A large number of S. purpuratus ESTs have been deposited in the GenBank database but cDNA/EST data have as yet not been obtained for the echinotocin precursor. Therefore, the sequence shown in Fig. 2 is derived from the GLEAN3 prediction (06899). The predicted echinotocin precursor gene comprises 3 exons, with the first exon (138 bases) encoding the N-terminal signal peptide, echinotocin and the N-terminal region of neurophysin. Exons 1 and 2 are separated by an intron comprising 24,141 bases. The second exon (208 bases) encodes the core of the neurophysin protein and is followed by an intron comprising 2379 bases. The third exon (152 bases) encodes the C-terminal region of the neurophysin protein and is followed by a stop codon (Fig. 2). This gene has been assigned the official gene ID number SPU_006899 (see http://www.spbase.org/SpBase/search/viewAnnoGeneInfo.php?spu_id=SPU_006899).
Comparison of the NGFFFamide precursor, the echinotocin precursor and precursors of vasopressin/oxytocin-like peptides in other species
To facilitate comparison of the sequences of the NGFFFamide precursor and
the echinotocin precursor and comparison of these sea urchin precursors with
precursors of vasopressin/oxytocin-like peptides in other species, ClustalX
was used to generate a multiple sequence alignment
(Fig. 3). This revealed that
whilst the 14 cysteine residues that are characteristic of neurophysins are
conserved in both the echinotocin precursor and the NGFFFamide precursor,
there is variation in the length of the peptide sequences between some of the
conserved cysteine residues. For example, between cysteines 7 and 8 in the
NGFFFamide-associated neurophysin there are seven residues, whereas in the
neurophysins associated with echinotocin and with vasopressin/oxytocin-like
peptides in other species there are nine residues. Conversely, there are six
residues between cysteines 12 and 13 in the NGFFFamide-associated neurophysin,
whereas in the neurophysins associated with echinotocin and with
vasopressin/oxytocin-like peptides in other species there are only four
residues.
|
|
NGFFFamide and echinotocin cause contraction of sea urchin tube foot and oesophagus preparations
Both NGFFFamide (Fig. 5A,B)
and echinotocin (Fig. 5C,D)
caused contraction of tube foot and oesophagus preparations from the sea
urchin E. esculentus. Comparison of the effects of NGFFFamide and
echinotocin suggested that the magnitude of the NGFFFamide-induced contraction
was larger than the echinotocin-induced contraction of both tube foot and
oesophagus preparations. Thus, the mean force of contraction induced by
3x10–6 mol l–1 NGFFFamide on tube feet
was 1.35±0.21 mN (±s.e.m.; N=3), whereas the mean force
of contraction induced by 3x10–6 mol
l–1 echinotocin on tube feet was 0.81±0.42 mN
(±s.e.m.; N=3). Similarly, the mean force of contraction
induced by 3x10–6 mol l–1 NGFFFamide
on oesophagus was 1.47±0.23 mN (±s.e.m.; N=3), whereas
the mean force of contraction induced by 3x10–6 mol
l–1 echinotocin on oesophagus 0.71±0.01 mN
(±s.e.m.; N=3). However, statistical analysis of these data
using a t-test did not reveal significant differences in the
magnitudes of contraction induced by NGFFFamide and echinotocin.
|
NGFFFamide caused dose-dependent contraction of oesophagus preparations at concentrations ranging from 10–11 to 10–6 mol l–1 (Fig. 5E). With tube foot preparations, dose-dependent contractile effects were only observed with higher concentrations of NGFFFamide within the range 10–8 to 10–6 mol l–1 (Fig. 5E). These data indicate that NGFFFamide is more potent as a contractant of oesophagus than as a contractant of tube feet.
Echinotocin caused dose-dependent contraction of tube foot (10–8 to 10–6 mol l–1) and oesophagus (10–9 to 10–7 mol l–1) preparations (Fig. 5F).
|
DISCUSSION |
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TOPSummaryINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONReferences |
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; Ohtani et al.,
1999). NGIWYamide-like immunoreactive peptides also occur in
starfish (Saha et al., 2006)
and therefore NGFFFamide and NGIWYamide may be members of a family of
neuropeptides that occur throughout the phylum Echinodermata. A cDNA encoding
the NGFFFamide precursor protein was identified in a cDNA library generated
from S. purpuratus radial nerve tissue, demonstrating that the
NGFFFamide gene is expressed in the sea urchin nervous system and indicating
that NGFFFamide may function as a neuropeptide.
To investigate the physiological roles of NGFFFamide, the pharmacological
effects of synthetic NGFFFamide on in vitro preparations of tube feet
and oesophagus from the sea urchin E. esculentus were examined.
NGFFFamide caused contraction of Echinus tube foot and oesophagus
preparations, consistent with the contracting action of NGIWYamide on sea
cucumber body wall muscle and tentacle preparations and starfish tube foot
preparations (Inoue et al.,
1999; Saha et al.,
2006). Thus, it appears that members of the NGIWYamide/NGFFFamide
neuropeptide family typically cause muscle contraction in echinoderms. Further
studies are now required to investigate the mechanisms by which NGIWYamide and
NGFFFamide affect muscle activity in sea cucumbers and sea urchins,
respectively. One scenario would be direct interaction with receptor proteins
expressed by muscle cells; an alternative possibility is that these peptides
act indirectly by stimulating the release of myoactive factors from nerves or
other cell types.
The NGFFFamide precursor contains a neurophysin domain
The discovery of a new family of myoactive neuropeptides in echinoderms is
of interest with respect to the neurobiology and physiology of these animals.
However, perhaps of more general interest is our discovery that the NGFFFamide
precursor, in addition to encoding two copies of the NGFFFamide peptide, also
comprises a polypeptide that shares a high level of sequence identity with
neurophysins, a family of proteins that are derived from the precursors of
vasopressin/oxytocin-type neuropeptides. Neurophysins act as carrier proteins,
which are important for packaging, processing and protection of
vasopressin/oxytocin-type neuropeptides
(De Bree, 2000;
De Bree and Burbach, 1998;
Legros and Geenen, 1996).
Hitherto neurophysins have been uniquely associated with
vasopressin/oxytocin-type neuropeptides and to the best of our knowledge the
NGFFFamide precursor is the first to be discovered comprising neurophysin and
a neuropeptide that is not a member of the vasopressin/oxytocin family of
peptides.
Echinotocin: a vasopressin/oxytocin-like peptide in sea urchins
The vasopressin/oxytocin neuropeptide family has a widespread phylogenetic
distribution indicative of an ancestry that dates back at least as far as the
common ancestor of bilaterian animals. Accordingly, vasopressin/oxytocin-like
peptides have been identified in vertebrates
(Hoyle, 1999;
Urano et al., 1992),
protostomian invertebrates (Cruz et al.,
1987; Oumi et al.,
1994; Proux et al.,
1987; Reich, 1992;
Van Kesteren et al., 1992) and
most recently in two deuterostomian invertebrates, the urochordates Ciona
intestinalis and Styela plicata
(Kawada et al., 2008;
Ukena et al., 2008). However,
vasopressin/oxytocin like peptides have thus far not been identified in any
echinoderm species. Against this background and our discovery that the sea
urchin NGFFFamide precursor contains a neurophysin domain, it was of interest
to investigate the occurrence of a gene encoding a vasopressin/oxytocin-like
peptide in sea urchins. BLAST analysis of S. purpuratus genomic
sequence data using the human vasopressin precursor as a query enabled
identification of a gene encoding a peptide (CFISNCPKGamide) that is a member
of the vasopressin/oxytocin-type neuropeptide family and which we have named
`echinotocin'. Likewise, if vasopressin/oxytocin-like peptides are identified
in other echinoderm species, we suggest that these are collectively known as
`echinotocins'.
Comparison of the sequence of echinotocin with other members of the
vasopressin/oxytocin neuropeptide family reveals that residues Cys1
and Cys6 in echinotocin are conserved throughout the family
(Fig. 6). This is not
surprising because in vasopressin/oxytocin-like peptides these two residues
form a disulphide bridge, conferring a cyclic conformation that is important
for the biological activity of these peptides
(Hruby et al., 1990;
Sawyer, 1977). Other residues
in the echinotocin sequence are shared with some of the known
vasopressin/oxytocin-like peptides. Thus, the C-terminal amidated glycine
residue and residues Asn5 and Pro7 in echinotocin are
also features of most vasopressin/oxytocin-like peptides, with notable
exceptions being two vasopressin/oxytocin-like peptides identified in
urochordates (Kawada et al.,
2008; Ukena et al.,
2008) and a putative neuropeptide (CFLNSCPY or `nematocin') in the
nematode Caenorhabditis elegans (NP_001033548; GI:86564869). Residues
2 and 3 in echinotocin are phenylalanine and isoleucine, respectively, which
is consistent with the occurrence of hydrophobic residues (Phe, Tyr, Leu or
Ile) in these positions in other vasopressin/oxytocin-like peptides. Finally,
the presence of a basic amino acid residue (Lys) at position 8 in echinotocin
confers similarity with vasopressin, which has an arginine residue at this
position, whereas oxytocin has a leucine residue in this position
(Fig. 6).
|
Structure of the echinotocin precursor and organization of the echinotocin gene
The predicted structure of the echinotocin precursor protein is consistent
with precursors of vasopressin/oxytocin-like peptides in vertebrates and in
other invertebrates (De Bree and Burbach,
1998; Hoyle,
1999). Thus, the echinotocin sequence is preceded by an N-terminal
signal peptide and followed by a C-terminal neurophysin-like domain
(Fig. 3). A signal peptide,
vasopressin/oxytocin-like neuropeptide and neurophysin occur in all of the
known precursor proteins for vasopressin/oxytocin-like neuropeptides in both
vertebrates and invertebrates (De Bree and
Burbach, 1998; Hoyle,
1999). However, there is variability in the length of the
C-terminal polypeptide sequence following the highly conserved neurophysin
domain. For example, in the oxytocin, annetocin and nematocin precursors it is
very short (nine residues) or absent (Fig.
3), whereas in the vasopressin precursor there is a 39 amino acid
residue peptide, which is known as copeptin. Moreover, following cleavage at a
monobasic site separating it from neurophysin, copeptin is co-secreted with
vasopressin. Three notable characteristics of copeptin are that it is preceded
by a monobasic cleavage site, it is glycosylated at a site
(N6-X-T8) located near its N-terminus and it has a
conserved hydrophobic LLLRLV sequence comprising residues 17–22
(De Bree and Burbach, 1998).
Interestingly, the C-terminal region of the echinotocin precursor has some of
these features; it has a putative glycosylation site (NGS) that aligns with
the glycosylation site in the vasopressin precursor (NAT) and it has a
hydrophobic sequence (LLDLLL) that aligns with the LLLRLV sequence in the
vasopressin precursor (Fig. 3).
There is also a potential dibasic cleavage site (KR) at residues 134 and 135
in the echinotocin precursor, which if utilized in vivo would
liberate a 30 amino acid residue copeptin-like molecule.
Interestingly, glycosylation of copeptin is a characteristic hitherto
uniquely associated with vasopressin precursors
(De Bree and Burbach, 1998).
Therefore, the presence of a putative glycosylation site in the C-terminal
region of the echinotocin precursor is intriguing and worthy of further
investigation to assess whether it is glycosylated in vivo in sea
urchins. Measurement of serum levels of copeptin can be used as a biomarker
for several clinical conditions in humans
(Katan et al., 2008) but
little is known about the physiological relevance of this molecule. It has
been postulated that copeptin may act as a modulator of excitatory
neurotransmission in the brain (Van den
Hooff et al., 1990) and as a prolactin-releasing factor
(Nagy et al., 1988), but
further studies are required (Hyde et al.,
1989). Comparative studies on the echinotocin-associated
copeptin-like peptide in sea urchins may provide new insights on this
issue.
The predicted echinotocin precursor protein is encoded by three exons,
which is consistent with the structural organization of genes encoding
vasopressin/oxytocin-like peptides in other animals
(De Bree and Burbach, 1998;
Hoyle, 1999). The first and
third exons of genes encoding precursors of vasopressin/oxytocin-like peptides
also have 5' and 3' non-coding sequences, respectively
(Ivell and Richter, 1984), and
it is likely, therefore, that the echinotocin gene is similar in this respect.
The positions of introns interrupting the coding sequence are conserved
between the echinotocin gene and other genes encoding
vasopressin/oxytocin-like peptides. Thus, the first intron is located between
the codons for residues Gln46 and Cys47 in the
echinotocin precursor and the second intron interrupts the codon for residue
Asn116 (Fig. 2);
introns are located at equivalent positions in genes encoding precursors of
vasopressin/oxytocin-like peptides in mammals
(Ivell and Richter, 1984) and
in the gastropod mollusc Lymnaea stagnalis
(Van Kesteren et al., 1995).
Thus, the conserved positioning of the two introns in genes encoding
vasopressin/oxytocin-like precursors presumably dates back to the common
ancestor of all bilaterian animals. However, this feature appears to have been
secondarily lost in some lineages because, for example, genes encoding
vasopressin/oxytocin-like peptides (cephalotocin and octopressin) in the
mollusc Octopus vulgaris lack introns
(Kanda et al., 2003).
Physiological roles of echinotocin in sea urchins
Analysis of the in vitro pharmacological effects of synthetic
echinotocin on Echinus tube feet and oesophagus revealed that, like
NGFFFamide, it causes contraction. However, as with NGFFFamide, the mechanisms
by which echinotocin causes muscle contraction in sea urchins remain to be
determined. We can, however, speculate on the molecular identity of a receptor
that may mediate the effects of echinotocin because, as part of a genome-wide
annotation of genes associated with nervous system function, we have
identified a gene (SPU_021290) encoding a G-protein-coupled receptor in S.
purpuratus that is an orthologue of vasopressin/oxytocin receptors
(Burke et al., 2006) (see also
http://www.spbase.org/SpBase/search/viewAnnoGeneInfo.php?spu_id=SPU_021290).
The myoactivity of echinotocin is consistent with the effects of
vasopressin/oxytocin-like peptides in other animals. For example, in mammals
vasopressin regulates blood pressure by causing vasoconstriction. However,
perhaps the most well known physiological role of vasopressin is in
osmoregulation, acting as an anti-diuretic hormone
(Sawyer, 1977). Interestingly,
a gene encoding a vasopressin/oxytocin-like peptide (Styela
oxytocin-related peptide or SOP) was recently identified in an invertebrate
chordate, the sea-squirt Styela plicata
(Ukena et al., 2008). Analysis
of the expression of the SOP gene in the cerebral ganglion of Styela
revealed that it is upregulated when animals are exposed to dilute (60%)
seawater, which also causes closure of their inhalant and exhalant siphons.
Furthermore, SOP causes contraction of in vitro preparations of
inhalant and exhalant siphons from Styela. Ukena and colleagues
(Ukena et al., 2008) conclude
that SOP acts to prevent the influx of dilute seawater in Styela,
suggesting an evolutionarily ancient role for vasopressin/oxytocin-like
peptides in osmoregulation. It is possible, therefore, that the contractile
effect of echinotocin on tube feet in vitro is indicative of a
similar role in sea urchins, with retraction of tube feet reducing water
influx in hypo-osmotic conditions.
Oxytoxin causes uterine contraction and stimulates lactation in mammals
(Sawyer, 1977) and evidence of
an evolutionarily conserved role for vasopressin/oxytocin-like peptides in
reproductive physiology has emerged from studies on invertebrates. For
example, the molluscan peptide conopressin causes contraction of the vas
deferens in the pond snail Lymnaea stagnalis
(Van Kesteren et al., 1992)
and the annelid peptide annetocin induces egg-laying behaviour in earthworms
(Oumi et al., 1996). Moreover,
a recent study suggests that neurons releasing oxytocin/vasopressin-like
peptides are an evolutionarily ancient neuronal population with dual
photosensory–neurosecretory properties coordinating reproduction with
light cycles (Tessmar-Raible et al.,
2007). Consistent with this hypothesis, neurons releasing a
vasopressin/oxytocin-like peptide in the insect Locusta migratoria
are more active in the dark than in the light and this activity is regulated
by extraocular photoreceptors (Thompson
and Bacon, 1991). Interestingly, genes encoding orthologues of
mammalian retinal transcription factors are expressed in sea urchin tube feet,
suggesting that these organs have a photosensory function
(Burke et al., 2006).
Therefore, the contractile effect of echinotocin on tube feet in
vitro may be a manifestation of an in vivo role in mediating
photosensory regulation of physiological processes in sea urchins.
In addition to the peripheral actions of oxytocin and vasopressin in
mammals and other vertebrates, there is growing evidence of roles in the
central nervous system (CNS) associated with reproductive behaviour and social
behaviour/cognition. For example, there is evidence that oxytocin has
important roles in maternal–infant bonding, pair bonding and social
interaction, whilst differences in vasopressin receptor expression in the
brain are associated with monogamy versus polygamy in vole species
(Caldwell et al., 2008;
Donaldson and Young, 2008;
Israel et al., 2008;
Winslow et al., 1993). The
evolutionary origins of these CNS-mediated actions of vasopressin and oxytocin
are unknown; discovery of vasopressin/oxytocin-type peptides in deuterostomian
invertebrates provides new opportunities to address this issue.
The role of neurophysins as carrier proteins for vasopressin/oxytocin-like peptides.
Neurophysins are required to facilitate endopeptidase-mediated cleavage of
vasopressin/oxytocin-like peptides from precursor proteins and for binding and
transport of the biologically active peptides in secretory granules from
neuronal somata to axonal terminals (De
Bree, 2000; De Bree and
Burbach, 1998). There are 14 highly conserved cysteine residues in
neurophysins (see Fig. 3),
which form seven intramolecular disulphide bridges
(De Bree and Burbach, 1998).
Furthermore, neurophysins form dimers and binding of vasopressin/oxytocin-like
peptides favours dimerization (Nicolas et
al., 1978). The interaction of vasopressin/oxytocin-like peptides
with neurophysins was one of the first ligand–protein interactions to be
analysed (Acher et al., 1958)
and more recently it has been investigated in detail using NMR spectroscopy
and X-ray crystallography (Chen et al.,
1991; Sardana and Breslow,
1984; Wu et al.,
2001). The first three amino acids in the N-terminal part of
vasopressin (Cys-Tyr-Phe) and oxytocin (Cys-Tyr-Ile) are the residues that are
most important for binding to neurophysin
(De Bree and Burbach, 1998)
and the corresponding residues in echinotocin are structurally identical or
similar (Cys-Phe-Ile). The strongest interaction is a salt bridge between the
NH +3 group of the N-terminal cysteine residue
and the 
COO– group of Glu47 in the
oxytocin/vasopressin neurophysins and both of these residues are conserved in
the echinotocin precursor. The aromatic side-chain of residue Tyr2
in oxytoxin and vasopressin is located in a pocket formed by the disulphide
bridges Cys10-Cys54 and
Cys21-Cys44, the
Cys21-Phe-Gly-Pro24 backbone, and the side-chains of
Pro24, Glu47 and Asn48. By comparison,
echinotocin has a residue (Phe2) with an aromatic side-chain and
all but one of the residues in neurophysin that form a pocket for the aromatic
side-chain of Tyr2 in oxytoxin and vasopressin are conserved in the
echinotocin precursor sequence, the exception being Phe22, which is
a methionine residue in the sea urchin sequence. Other evolutionarily
conserved characteristics of vasopressin and oxytocin that are important for
binding to neurophysin are the disulphide bridge between Cys1 and
Cys6, the peptide backbone between residues 2 and 3 and the
side-chain of residue 3 (De Bree and
Burbach, 1998). Based on these similarities, it is likely that
echinotocin interacts with the neurophysin domain of the echinotocin
precursor.
Does the neurophysin encoded by the NGFFFamide gene act as a carrier protein for NGFFFamide?
By analogy with the role of neurophysins as carrier proteins for
vasopressin/oxytocin-like peptides, the neurophysin domain in the NGFFFamide
precursor may likewise act as a carrier protein for NGFFFamide. Consistent
with this notion, several residues that are involved in binding of
vasopressin/oxytocin-like peptides (see above) are conserved in the NGFFFamide
neurophysin, including the residues corresponding to the cysteines at
positions 10, 21, 44 and 54 and the glutamate at position 47 in the
vasopressin/oyxtocin neurophysins. There are, however, some interesting
differences between the NGFFFamide-associated neurophysin and neurophysins
associated with echinotocin and other vasopressin/oxytocin-like peptides.
Thus, between cysteines 7 and 8 in the NGFFFamide-associated neurophysin there
are seven residues, whereas in the neurophysins associated with echinotocin
and with vasopressin/oxytocin-like peptides in other species there are nine
residues. Furthermore, there are six residues between cysteines 12 and 13 in
the NGFFFamide-associated neurophysin, whereas in the neurophysins associated
with echinotocin and with vasopressin/oxytocin-like peptides in other species
there are only four residues. Unusual structural features such as these may
facilitate binding of NGFFFamide by its associated neurophysin. However,
experimental investigation of an interaction of NGFFFamide with neurophysin,
which was beyond the scope of this study, will be required to address these
issues.
The evolutionary origin of the neurophysin domain in the NGFFFamide precursor
Genes encoding precursors for vasopressin/oxytocin-like peptides with an
associated neurophysin domain have been identified throughout the animal
kingdom (De Bree and Burbach,
1998) and the echinotocin gene reported here is a new member of
this gene family. This widespread phylogenetic distribution indicates that the
evolutionary origin of the vasopressin/oxytocin family of neuropeptide
precursors dates back at least as far as the common ancestor of all bilaterian
animals. The NGFFFamide precursor is the first protein to be identified that
has a neurophysin domain without an associated vasopressin/oxytocin-like
peptide and therefore it is of interest to explore the evolutionary origin of
this novel protein.
The occurrence of the neurophysin domain in the S. purpuratus
NGFFFamide precursor is presumably a consequence of duplication and
transposition of DNA encoding the precursor, or part of the precursor (i.e.
the neurophysin domain), of a vasopressin/oxytocin-like peptide in an ancestor
of S. purpuratus. Consistent with this notion, the NGFFFamide and
echinotocin genes both have an intron preceding the codon encoding the first
cysteine residue of their neurophysin domains. In the echinotocin gene and in
most genes encoding vasopressin/oxytocin-like peptides there is also a second
intron that interrupts the neurophysin-encoding sequence. The NGFFFamide
neurophysin, however, is encoded by a single exon (exon 4). Thus, if the
neurophysin domain of the NGFFFamide precursor originated as a consequence of
complete or partial duplication of a gene encoding a vasopressin/oxytocin-like
peptide, then the second intron that interrupts the neurophysin coding
sequence must have been lost subsequently. There is a precedent for this,
however, because, as discussed above, the two genes encoding
vasopressin/oxytocin-like peptides in Octopus vulgaris both lack
introns (Kanda et al.,
2003).
Based on sequence similarity, NGFFFamide neurophysin is not more closely related to the echinotocin neurophysin than to neurophysins associated with vasopressin/oxytocin-like peptides in other phyla. Moreover, the echinotocin neurophysin shares more similarity with the neurophysin associated with lamprey vasotocin than it does with the NGFFFamide-associated neurophysin (see Fig. 4). This suggests that, with respect to a putative common ancestral sequence, the NGFFFamide-associated neurophysin is more divergent than the neurophysin associated with echinotocin. Furthermore, this feature of the NGFFFamide-associated neurophysin may be related to accommodation of NGFFFamide as a binding partner.
Determination of the timing of the duplication event that gave rise to the
occurrence of a neurophysin domain in the S. purpuratus NGFFFamide
precursor will be facilitated if genes encoding precursors for NGFFFamide-like
peptides with a neurophysin domain are identified in other echinoderms. BLAST
analysis of genome sequence data obtained for the sea urchin Allocentrotus
fragilis
(http://www.hgsc.bcm.tmc.edu/blast.hgsc?organism=15)
reveals the presence of an exon encoding a neurophysin domain that is
identical to residues 185–266 of the S. purpuratus NGFFFamide
precursor. Thus, this feature is not unique to S. purpuratus but also
occurs in other sea urchins. More interesting would be to determine whether
the NGIWYamide precursor protein in the holothurian Apostichopus
japonicus also has a neurophysin domain. If it does not, this would
suggest that the neurophysin domain in the NGFFFamide precursor originated in
an echinoid ancestor of S. purpuratus. If the NGIWYamide precursor
does have a neurophysin domain, this would suggest that it originated prior to
the common ancestor of echinoids and holothurians. It is possible that
precursors comprising NGFFFamide/NGIWYamide-like peptides together with a
neurophysin domain occur throughout the phylum Echinodermata and even in
closely related phyla such as the Hemichordata and the Xenoturbellida (see
Bourlat et al., 2006). Further
investigation of this issue will be possible when genome sequences are
determined for other echinoderm species and for hemichordate and xenoturbellid
species.
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Footnotes |
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References |
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TOPSummaryINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONReferences |
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