Comparative Biochemistry and Physiology, Part D 4 (2009) 268–289
Contents lists available at ScienceDirect
Comparative Biochemistry and Physiology, Part D
j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / c b p d
Chemical transmission in the sea anemone Nematostella vectensis:
A genomic perspective
Michel Anctil ⁎
Département de sciences biologiques and Centre de recherches en sciences neurologiques, Université de Montréal, Case postale 6128, Succursale Centre-Ville, Montréal, Québec,
Canada H3C 3J7
a r t i c l e
i n f o
Article history:
Received 26 March 2009
Received in revised form 30 June 2009
Accepted 7 July 2009
Available online 16 July 2009
Keywords:
Anthozoa
Cnidaria
Genomic analysis
Hormone signalling
Nematostella
Neuropeptides
Neurotransmitters
Starlet sea anemone
a b s t r a c t
The sequencing of the starlet sea anemone (Nematostella vectensis) genome provides opportunities to
investigate the function and evolution of genes associated with chemical neurotransmission and hormonal
signaling. This is of particular interest because sea anemones are anthozoans, the phylogenetically basal
cnidarians least changed from the common ancestors of cnidarians and bilaterian animals, and because
cnidarians are considered the most basal metazoans possessing a nervous system. This analysis of the
genome has yielded 20 orthologues of enzymes and nicotinic receptors associated with cholinergic function,
an even larger number of genes encoding enzymes, receptors and transporters for glutamatergic (28) and
GABAergic (34) transmission, and two orthologues of purinergic receptors. Numerous genes encoding
enzymes (14), receptors (60) and transporters (5) for aminergic transmission were identified, along with
four adenosine-like receptors and one nitric oxide synthase. Diverse neuropeptide and hormone families are
also represented, mostly with genes encoding prepropeptides and receptors related to varying closeness to
RFamide (17) and tachykinin (14), but also galanin (8), gonadotropin-releasing hormones and vasopressin/
oxytocin (5), melanocortins (11), insulin-like peptides (5), glycoprotein hormones (7), and uniquely
cnidarian peptide families (44). Surprisingly, no muscarinic acetylcholine receptors were identified and a
large number of melatonin-related, but not serotonin, orthologues were found. Phylogenetic tree
construction and inspection of multiple sequence alignments reveal how evolutionarily and functionally
distant chemical transmitter-related proteins are from those of higher metazoans.
© 2009 Elsevier Inc. All rights reserved.
1. Introduction
Cnidarians are regarded as the most basal metazoans possessing
nervous systems. Consequently, they have attracted considerable
interest among neurobiologists as the evolutionary implications of
studying their nervous systems became increasingly apparent.
Cnidarian neurons are largely organized into planar nerve nets of
varying density that are distributed in the ectoderm and, especially
within the anthozoan class, in the endoderm (Pantin, 1952; Batham,
1965). As the neuronal processes cross over each other in the nerve
net, they form more or less specialized synaptic junctions in which
synaptic vesicles were identified (Anderson and Grünert, 1988;
Westfall and Grimmelikhuijzen, 1993; Westfall et al., 1995). A large
body of electrophysiological investigations showed that cnidarian
neurons fundamentally work like neurons of physiologically more
complex animals and that mechanisms of chemical neurotransmis-
⁎ Tel.: +1 514 343 7691; fax: +1 514 343 2293.
E-mail address: [email protected].
1744-117X/$ – see front matter © 2009 Elsevier Inc. All rights reserved.
doi:10.1016/j.cbd.2009.07.001
sion in cnidarians are similar to those of higher invertebrates and of
vertebrates (Anderson and Spencer, 1989; Mackie, 1990).
Although many forms of neuronal activity were documented in
cnidarian nervous systems, the lack of strong evidence for classical
transmitters (Martin and Spencer, 1983) coupled with evidence of
widespread use of epithelial conduction and of electrical synapses
between neurons in hydrozoan experimental models (Mackie, 2004),
led to skepticism about the nature and extent of transmitter use in these
animals. However, the last 25 years have witnessed the emergence of a
large body of biochemical, immunohistochemical, molecular and
physiological investigations demonstrating the presence in neurons
and the biological activity of neuropeptides, biogenic amines and fastacting small transmitters such as glutamic acid and GABA (Grimmelikhuijzen et al., 1996, 2002; Kass-Simon and Pierobon, 2007 for reviews).
Yet, while the evidence for the role of both neuropeptides and classical
transmitters in effector activities of all cnidarian classes is persuasive,
the body of existing data is too incomplete and fragmented among
several species to gain a satisfactory picture of the set of transmitter
systems available to cnidarians.
The recent sequencing of the genome of the starlet sea anemone
Nematostella vectensis (Putnam et al., 2007) provides a unique opportunity to explore the full repertoire of putative gene products known to be
M. Anctil / Comparative Biochemistry and Physiology, Part D 4 (2009) 268–289
involved in transmitter biosynthesis, transport and receptors, all within a
single cnidarian species. Recently, the morphological organization of the
nervous system of this anemone was described (Marlow et al., 2009). The
starlet sea anemone is a representative of the Anthozoa, the most basal
cnidarian class which includes also corals and sea pens. Representatives
of this class are considered to be closer to the ancestor of bilaterian
animals than are other cnidarians (Bridge et al., 1992, 1995). Therefore,
any analysis of protein families from the genome of N. vectensis is likely
to provide insights on the evolution of protein structure and function
in the context of the eumetazoan ancestry of cnidarians and of their
predating the emergence of bilaterian animals. In addition, it is the
intention of this genomic approach to provide a resource for investigators
to explore new avenues and hypotheses in their efforts to understand
various aspects of transmitter function in cnidarians.
Transmitter-associated proteins belong to different classes of
proteins (enzymes, transporters, receptors), all of which contributing
to transmitter function while potentially exhibiting distinctive
features that reflect their evolutionary history. The following analysis
of the repertoire of putative transmitter-related genes in the genome
of N. vectensis aims at assessing for the first time the range, functional
capability and evolutionary implications of transmitter systems in a
cnidarian. For the purpose of this analysis, the word «transmitter» is
used in a broad sense to include all substances that may be released
from neurons and that act as bona fide neuroactive substances
(triggering a post-synaptic response), modulators (modulating a
pre- or post-synaptic event) or hormones (triggering a response
significantly away from the release site).
2. Methods
Protein sequences were searched from the US Department of
Energy Joint Genome Institute website for N. vectensis (http://
genome.jgi-psf.org/Nemve1/Nemve1.home.html). Searches were conducted using annotation keywords or search engines such as KOG and
BLAST available on the site. PHI-BLAST was also used to improve hit
returns on queries of neuropeptide precursor proteins, using PHI
patterns for the various neuropeptide families. All hits with an e-value
below e− 10 were selected. The selected sequences were downloaded
through the MEGA v.4 software (Tamura et al., 2007) and directed to
the RPS-BLAST alignment tool for inspection. Sequences of interest
that were deemed too short or that included incomplete expected
conserved domains were discarded. Duplicates and splice variants
were identified by manually inspecting all aligned sequences and
were removed from the pool of analysed sequences.
For phylogenetic analyses each protein transcript of interest was
aligned with BLAST against the entire nr database. Sequences among
the first 100 hits were selected and used to construct phylogenetic
trees. The hits and related N. vectensis sequences were next aligned
using ClustalW with MegAlign (Lasergene, DNASTAR) and trees were
constructed with the MEGA implementation of distance neighborjoining with complete deletion of gaps. Manual deletions were also
performed to emphasize conserved domains. For sequences of
membrane proteins, the vast majority of which are G protein-coupled
receptors (GPCR), the N- and C-tails were removed. Consensus trees
were obtained by bootstrapping the data (1000 replicates). To further
validate some of the phylogenies, maximum likelihood analyses were
conducted with PUZZLE (Strimmer and von Haeseler, 1996).
In addition, sequences were selected from the constructed trees of
each protein class to create alignments designed to assess the extent
to which the sea anemone proteins retained aa residues important for
functional features of the corresponding protein class. For this
purpose multiple alignments of proteins and their putative sea
anemone orthologues were generated with MegAlign using the
ClustalW algorithm and were displayed with the GeneDoc editor
program (version 2.7; http://www.psc.edu/biomed/genedoc). The
Statistics report function of GeneDoc was also used to evaluate
269
percentages of residue similarity and identity in pairwise alignments.
For this purpose the N- and C-tails of all membrane protein sequences
were removed. SignalP was used to detect signal peptides of
neuropeptide precursor proteins and NeuroPred for prediction of
cleavage sites.
3. Results and discussion
3.1. An overview of the repertoire
Nearly 280 N. vectensis sequences of appropriate length and/or
including integral functional domains were retained for analysis.
Receptors represent nearly 70% of these sequences. The remaining
sequences are distributed among biosynthetic or inactivating
enzymes, cell membrane or vesicular membrane transporters and
neuropeptide precursors. Although every effort was made to cull from
the genome all transcripts relevant to transmitters, it is likely that
some were missed due to oversight or to their belonging to hitherto
unknown transmitter categories.
For convenience candidate genes are classified in three categories:
small transmitters acting through both ionotropic and metabotropic
receptors such as acetylcholine, amino acid and purinergic transmitters (Table 1), aminergic and other small transmitters such as
adenosine and nitric oxide (Table 2) and neuropeptides/hormones
Table 1
Genes of N. vectensis predicted to code for proteins associated with acetylcholine, amino
acids and ATP.
Transmitter type
Acetylcholine
Choline
acetyltransferase
Acetylcholinesterase
Nicotinic receptors
Amino acids
Glutamate
AMPA receptors
Kainate receptors
NMDA receptors
Metabotropic
receptors
Glutamate
transporters
Vesicular
transporters
GABA
Glutamate
decarboxylase
GABAa receptors
Glycine receptor
GABAb receptors
Protein ID number
EST ID number
Nv_416, Nv_95805, Nv_203043
Nv_163430
Nv_31599, Nv_87444, Nv_119959,
Nv_209664 Nv_211382
Nv_40919, Nv_85091, Nv_91696,
Nv_91941 Nv_110265, Nv_198343,
Nv_198927, v_199721 Nv_200917,
Nv_205808, Nv_205855,
Nv_214990
Nv_160761, Nv_171912
Nv_239659, Nv_244109
Nv_240779, Nv_247410
Nv_13877, Nv_24412, Nv_50912,
Nv_104623 Nv_117160, Nv_132356
Nv_141731
Nv_11315, Nv_31895, Nv_51517,
Nv_211456
Nv_31331, Nv_40374, Nv_105783,
Nv_197524 Nv_198894, Nv_201378,
Nv_218792, Nv_229374
Nv_110362, Nv_210965, Nv_230013
Nv_241281, Nv_244506
Nv_173595
Nv_10128, Nv_11440, Nv_81701,
Nv_123866 Nv_138860, Nv_231086
Nv_60452, Nv_60834, Nv_70014
Nv_40863, Nv_60804, Nv_93322,
Nv_103931 Nv_111552, Nv_114921,
Nv_201724, Nv_204447 Nv_211643,
Nv_215047, Nv_230093
Nv_22284
Nv_86565, Nv_87697, Nv_206093,
Nv_223171
GABA transporters Nv_60521, Nv_79255, Nv_79785,
Nv_81637 Nv_109800, Nv_228010
Vesicular
Nv_1064, Nv_22306, Nv_33294,
transporters
Nv_60758 Nv_96724, Nv_142801,
Nv_206710, Nv_214632 Nv_222356
ATP
Purinergic
receptors (P2X)
Nv_239847
Nv_171792
Nv_102596, Nv_104653
Nv_158857, Nv_176489
Nv_239821, Nv_243252
Nv_244104
Nv_161287, Nv_187148
Nv_236066, Nv_247614
Nv_241391, Nv_241392
Nv_247861
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Table 2
Genes of N. vectensis predicted to code for proteins associated with biogenic amines and other non-peptidergic transmitters.
Transmitter type
Protein ID Number
EST ID Number
Monoamines
Enzymes
Tyrosine/tryptophan hydroxylase
Nv_203775, Nv_216737
Nv_237576
Nv_240046
Nv_245974
Nv_164399
Nv_164601,
Nv_179511
Nv_191334
Tyrosinase
DOPA decarboxylase
Dopamine-β-hydroxylase
PNMT-like (adrenaline)
HIOMT (melatonin)
Monoamine oxidase
Receptors
Monoamine-like (DA/5-HT)
Monoamine-like (adrenergic)
Monoamine-like (histamine)
Melatoninergic
Transporters
Monoamine transporters
Vesicular amine transporters
Adenosine
Adenosine-like receptors
Nitric oxide
Nitric oxide synthase (NOS)
NOS binding protein
Nv_98001, Nv_204120
Nv_79900, Nv_93717
Nv_209258
Nv_34681, Nv_91623, Nv_123672
Nv_94865, Nv_136792, Nv_196827, Nv_229539
Nv_13440, Nv_14501 Nv_24241, Nv_41866
Nv_164564,
Nv_239384
Nv_247298
194295
Nv_175230
Nv_85851, Nv_117699, Nv_118705, Nv_122716, Nv_207168, Nv_214347, Nv_222805,
Nv_222806, Nv_223523, Nv_1848, Nv_1944, Nv_82546, Nv_85943
Nv_85995, Nv_97538, Nv_119248, Nv_131304, Nv_148199, Nv_212680, Nv_197923,
Nv_1897, Nv_23357, Nv_97861, Nv_109382
Nv_123316, Nv_136356, Nv_199298, Nv_200221, Nv_204979, Nv_212860, Nv_1499,
Nv_1642, Nv_2167, Nv_6197
Nv_13917, Nv_33192, Nv_34809, Nv_52597, Nv_105626, Nv_113440, Nv_113494,
Nv_196476, Nv_197968, Nv_198502, Nv_198600, Nv_202553, Nv_203795, Nv_208482,
Nv_209463, Nv_209464 Nv_209465 Nv_211467, Nv_211963, Nv_212429
Nv_209450, Nv_209454, Nv_229612
Nv_213661, Nv_214544
Nv_172825,
Nv_189286
Nv_241669,
Nv_244057
Nv_171569
Nv_1683, Nv_82495, Nv_169811, Nv_216997
Nv_110559
Nv_44036
Table 3
Genes of N. vectensis predicted to code for proteins associated with neuropeptides.
Transmitter type
Transcript ID number
EST ID number
RFamide-related
Precursors
Antho-RFamide
Other RFaPs
Amidating enzymes
Receptors
RFa/NPFF/GnIH/NPY
Nv_1374
Nv_9531, Nv_16904/97952
Nv_101809, Nv_230646
Nv_172604
Nv_13858, Nv_24147, Nv_33569, Nv_34324, Nv_34835, Nv_61644, Nv_81082, Nv_84747, Nv_99552,
Nv_113178, Nv_210664
Nv_238927, Nv_241688
Nv_244873, Nv_245559
Other cnidarian peptides
Antho-RIamide-like precursor
Antho-RNamide-like precursor
Antho-RPamide-like precursors
Antho-RWamide-like precursor
LWamide precursor
Unique cnidarian neuropeptide
receptors (unclassifiable)
Galanin-related
Galanin-like precursors
Galanin-like receptors
Tachykinin-related
Putative precursors
Receptors
Tachykinin/SIFamide receptors
GnRH/vasopressin-related
GnRH-like precursor
Vasopressin-like
precursor
GnRH/vasopressin-like
receptors
Melanocortin-related
α-MSH-like precursor
Melanocortin-like receptors
Insulin-like peptides
Peptides
Receptors
Glycoprotein hormone
receptors (LGR type A)
Nv_65111
Nv_244953⁎
Nv_200817
Nv_37852
Nv_141747
Nv_126270
Nv_1668, Nv_1720, Nv_1919, Nv_1951, Nv_1990, Nv_13425, Nv_13739, Nv_21366, Nv_24914, Nv_33886,
Nv_237937
Nv_34191, Nv_34333, Nv_34364, Nv_34518, Nv_41807, Nv_52016, Nv_79851, Nv_83657, Nv_86966, Nv_87779
Nv_90581, Nv_91081, Nv_98771, Nv_100423, Nv_101072, Nv_105690, Nv_107948, Nv_111898, Nv_112480,
Nv_119626, Nv_119692, Nv_127852, Nv_157068, Nv_196320, Nv_197583, Nv_198963, Nv_200814 Nv_209920,
Nv_212697
Nv_96465, Nv_128410, Nv_142194
Nv_108333, Nv_133576, Nv_206354, Nv_206604, Nv_211853
Nv_88765, Nv_94714
Nv_13531, Nv_22729, Nv_34692, Nv_41719, Nv_61697, Nv_87682, Nv_103257, Nv_106757, Nv_122333,
Nv_133598, Nv_196253, Nv_207006
Nv_244378
Nv_216820
Nv_65450, Nv_206388
Nv_241190
Nv_201906, Nv_209131
Nv_8156, Nv_38399
Nv_199985, Nv_204373, Nv_205839, Nv_207495, Nv_208724, Nv_210358, Nv_211188, Nv_219411, Nv_244403 Nv_246236
Nv_199266, Nv_207484
Nv_50070, Nv_85808, Nv_198971
Nv_80429, Nv_204412, Nv_212618, Nv_197977, Nv_200697, Nv_217594, Nv_221138
Nv_238729
M. Anctil / Comparative Biochemistry and Physiology, Part D 4 (2009) 268–289
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Fig. 1. Relationships of the ACh-like enzymes of N. vectensis. (A) Rooted neighbor-joining (NJ) tree showing that the choline acetyltransferase (ChAT)-like sea anemone transcripts
(shaded box) cluster with carnithine acetyltransferases (CrAT) of higher metazoans rather than with ChATs. Numbers on the nodes in this and subsequent tree figures represent the
percentage of bootstrap replicates supporting this topology. Scale for branch lengths at bottom represents the number of substitutions per site in this and subsequent tree figures.
(B) Alignment of ChAT β strands 8 (upper panel) and 12 (lower panel) showing residues important for catalytic activity (⁎) and substrate interaction (+). (C) Unrooted tree in which
acetylcholinesterase (AChase)-like sea anemone sequences (shaded box) stand as an outgroup to various cholinesterases. (D) Alignment of Chase β strand 9/helix 10 (upper panel)
and of segment between β strand 10 and helix 14 (lower panel) showing conserved active site residues (⁎) and conserved residues in active site gorge (+).
Fig. 2. Relationship of the nicotinic-like receptors of N. vectensis. (A) Rooted NJ tree showing the sea anemone subclades (shaded) forming an outgroup to invertebrate and vertebrate
counterparts. Note that the GABA-A and glycine receptors are not nested with the nicotinic and sea anemone sequences. (B) Alignment of β strands 4 and 6 (upper panel) and of sequence
fragments flanking β strand 10 (lower panel) showing residues important for ligand binding (o), for binding pocket boundary formation (+) and for contacting docked ACh and nicotine (⁎).
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(Table 3). In addition to the candidate genes, Tables 1–3 also provide a
list of available EST transcripts in order to gain an appreciation for the
distribution of expressed proteins among the transmitter categories.
3.2. Acetylcholine
Evidence so far for acetylcholine as transmitter in cnidarians is weak
and controversial (Scemes, 1989; Kass-Simon and Pierobon, 2007).
Cholinergic agonists were reported to induce muscle contractions and
increase bioelectric activity in hydrozoans and sea anemones (Romanes,
1885; Mendes and Freitas, 1984; Kass-Simon and Passano, 1978).
Although early histochemical studies reported acetylcholinesterase
(AChE) activity in Hydra (Lentz and Barnett, 1961), later attempts failed
to detect AChE or choline acetyltransferase (ChAT) activity in other
hydrozoans and in sea anemones (Scemes, 1989; Van Marle, 1977).
In view of this background, it may seem surprising that 20 sequences
apparently related to cholinergic function were found among the
transcripts of N. vectensis: 3 ChATs, 5 AChEs and 12 nicotinic receptors
(Table 1). Phylogenetic analysis suggests that the putative ChAT
sequences appear closer to the carnithine-AT (CrAT) clade than to the
ChAT clade (Fig. 1A). This is consistent with the higher residue identity
score of transcripts against CrAT (40%) than against ChAT (35%). In
Fig. 3. Relationships of the glutamate-like receptors of N. vectensis. (A) Unrooted NJ tree showing some sea anemone sequences clustering with AMPA/kainate receptors and others
with NMDA receptors. (B) Alignment of sea anemone sequences with corresponding AMPA receptor (upper panel) and with NMDA receptor sequences (lower panel). The
represented sequence segment is in the N-terminal part and just precedes the first transmembrane region. The conserved motif PLTxxxxR is important for glutamate interaction at
the binding site. (C) Unrooted NJ tree showing some sea anemone sequences clustering with metabotropic glutamate receptors and others with calcium sensors. (D) Alignment of sea
anemone sequences with corresponding metabotropic glutamate receptor sequences in the N-terminus. Consensus residue for agonist binding (⁎) and residues important for
binding pocket formation (+) in groups I (red), II (green) and III (violet) receptors are shown. (For interpretation of the references to colour in this figure legend, the reader is
referred to the web version of this article.)
M. Anctil / Comparative Biochemistry and Physiology, Part D 4 (2009) 268–289
contrast, multiple alignment shows retention of conserved residues for
catalytic activity and substrate interaction consistent with both ChAT
and CrAT function (Govindasamy et al., 2004; Cai et al., 2004) (Fig. 1B).
An arginine residue critical for electrostatic interaction with carnithine
in helix 18 is conserved by the sea anemone enzymes (not shown).
Tree analysis shows that sea anemone transcripts form outgroups
to AChEs and butyrylChEs (BChE) from both vertebrates and
invertebrates (Fig. 1C). Residue identity scores of transcripts against
AChEs or BChEs range from 32 to 37%. Multiple alignment of these
sequences revealed that some of the residues important for catalytic
activity or for the formation of the substrate interaction gorge (Bourne
et al., 2003; Lazari et al., 2004) are preserved in the sea anemone
orthologues (Fig. 1D). That these enzymes are expressed and
potentially functional in N. vectensis is hinted by the presence of
several EST sequences related to AChE and ChAT (Table 1).
The transcripts annotated as nicotinic receptor subunits showed
a range of 44–59% similarity and 24–37% identity with several
vertebrate and invertebrate counterparts. NJ analyses suggest that
the putative sea anemone nicotinic receptor subunits cluster sep-
273
arately from those of bilateral animals (Fig. 2A). The tree also shows
that the sea anemone sequences are orthologues of nicotinic receptor
subunits and not of GABA-A and glycine receptor subunits to which
they are related. Most of the residues considered important for ligand
binding and for the formation of the ligand binding pocket (Le Novère
et al., 1999; Schapira et al., 2002) are preserved in the predicted
sea anemone nicotinic receptors (Fig. 2B). While all the proteins
necessary for nicotinic neurotransmission are apparently present
in the sea anemone, no muscarinic receptor was detected. All 10
transcripts assigned to the muscarinic class in the JGI Genome
Assembly of N. vectensis turned out to belong to other classes of
metabotropic GPCRs. Data supporting effects of muscarinic agents on
muscle contraction and bioelectric activity exist for cnidarians (see
Kass-Simon and Pierobon, 2007 for review), but these are insufficient
to establish the existence of muscarinic receptors. A blast search
apparently yielded Hydra genes for muscarinic receptors (Watanabe
et al., 2009), but none is identified and there is no evidence that the
gene sequences were subjected to scrutiny for functional domains.
Without more reliable data, it can only be assumed that muscarinic
Fig. 4. Relationships of the GABAergic-like receptors of N. vectensis. (A) Unrooted NJ tree showing that sea anemone sequences form outgroups in relation with GABA-A receptors
whereas one sequence clusters clearly with a glycine receptor. (B) Alignment of sea anemone sequences with corresponding GABA-A receptors. The represented sequence segments are on
the C-terminal side of loop E (upper panel) and in loops B and C (lower panel) of the ligand-binding domain. Residues important for GABA binding in α1 (orange) and β2 subunits (red), and
residues important for benzodiazepine binding in γ subunit (green) are shown. A cysteine residue involved in a sulfhydryl bridge is also shown. (C) Unrooted NJ tree showing that a few sea
anemone sequences are nested with GABA-B2-like receptors but not with GABA-B1 receptors. (D) Alignment of sea anemone sequences with corresponding GABA-B receptors. The
represented sequence segments are in the first extracellular loop (upper panel) and in the area of the fifth transmembrane region (lower panel). Residues important for agonist binding (⁎)
and affinity (+) are shown. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
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M. Anctil / Comparative Biochemistry and Physiology, Part D 4 (2009) 268–289
receptors may have evolved in the common ancestors of protostomes
and deuterostomes, representatives from both of which possess muscarinic receptor genes.
3.3. Amino acids
Excitatory (glutamate/aspartate) and inhibitory (GABA/glycine)
transmitters have long been considered of paramount importance in
the nervous system of both vertebrates and invertebrates (Hall et al.,
1979; Bowery and Smart, 2006). In cnidarians these transmitters were
reported to be involved in activities as diverse as nematocyst discharge
(Kass-Simon and Scappaticci, 2004), pacemaker networks associated
with motor activity (Kass-Simon et al., 2003; Ruggieri et al., 2004) and in
the feeding response (Pierobon et al., 1995, 2001, 2004). Predicted
proteins for amino acid transmitters are well represented in N. vectensis.
A large number of ionotropic glutamate receptors were identified
(Table 1). Several AMPA-like and NMDA-like receptors are present, but
only one kainate-like. While AMPA/kainate-like sea anemone receptors
do not cluster readily with specific vertebrate subtypes, the NMDA-like
sea anemone proteins appear closer to the NMDA2 than to the NMDA1
subtype (Fig. 3A). The AMPA orthologues show higher similarity/
identity with their bilaterian counterparts (up to 61/40%) than the
NMDA orthologues (up to 40/22%). Only some of the residues
considered important for glutamate binding in mammalian AMPAs
and NMDAs (Lampinen et al., 1998; Furukawa and Gouaux, 2003; Chen
and Wyllie, 2006) are present in the sea anemone orthologues (Fig. 3B).
Fig. 5. Relationships of the amino acid-like transporters of N. vectensis. (A) Unrooted NJ tree showing that sea anemone sequences form an outgroup in relation to various excitatory amino
acid transporters (EAAT). (B) Alignment of sea anemone sequences with corresponding EAATs. The represented sequence segments are in the third extracellular loop (upper panel),
seventh (middle panel) and tenth (lower panel) transmembrane regions. Consensus residues for the EAAT family (⁎) and residues implicated in glutamate transport (+) are shown.
(C) Unrooted NJ tree showing that some sea anemone sequences form an outgroup in relation with various GABA (GAT) and glycine (GlyT) transporters whereas others are nested with
GATs. (D) Alignment of sea anemone sequences with corresponding GATs, taurine (TauT) and creatine (CreaT) transporters. The represented sequence segments are in the first (upper
panel) and sixth (lower panel) transmembrane regions. Consensus residues for the SLC6 family of transporters (⁎) and a residue involved in sodium ion interaction (o) are shown.
M. Anctil / Comparative Biochemistry and Physiology, Part D 4 (2009) 268–289
In contrast to the cholinergic suite of receptors, many metabotropic glutamate receptors (mGlutR), members of the GPCR superfamily, were clearly identified among the sea anemone transcripts
(Table 1). In addition, calcium-sensing receptors (CaSR), which are
closely related to mGlutR but are not involved in chemical transmission, were detected. Phylogenetic analyses show a clear cleavage
between CaSR and mGlutR, although the relationship of the sea
anemone CaSR clade with that of vertebrates is poorly resolved
(Fig. 3C). The position of the sea anemone mGlutR clade outside of the
vertebrate mGlutR classification is strongly supported by bootstrap
validation and by the level of transcript residue identity with
vertebrate orthologues (27–38%). It is also supported by the poor
conservation of the signature residues for the various vertebrate
mGlutR subtypes even though residues important for glutamate
binding by the sea anemone sequences are conserved (Fig. 3D;
Rosemond et al., 2002; Kuang et al., 2006; Muto et al., 2007). The large
number of ionotropic and metabotropic receptors suggests an
important role of glutamic acid as transmitter in sea anemones and
hints at the diversity of responses possible. The few identified ESTs
displaying homology with these receptors indicate that the latter are
expressed and functional in N. vectensis.
275
Glutamate dehydrogenase (GAD) is involved in the synthesis of
gamma-aminobutyric acid (GABA). GABA and GAD immunoreactivities were reported in neurons of the anthozoan Eunicella cavolini
(Girosi et al., 2007) and GABA immunoreactivity in the ectodermal
nerve net of the starlet sea anemone (Marlow et al., 2009). There are
at least two apparent splice variants of GAD in the sea anemone
genome (Table 1). However, they exhibit more conserved residues
with prokaryote (32% residue identity) than with eukaryote GADs
(13%).
There are twice as many GABAa as GABAb receptors in the sea
anemone genome and one unambiguous glycine receptor was also
found (Table 1). The sea anemone GABAa receptors appear to be
distantly related to their various vertebrate and cephalopod orthologues whereas one sea anemone transcript appeared to nest with a
glycine receptor (Fig. 4A). This distant relationship to GABAa receptors
is also reflected in the shared residue conservation scores (up to 52%
similarity, 32% identity) and in the several residues important for
GABA binding (Galvez et al., 1999; Ci et al., 2008) that are not present
among sea anemone transcripts (Fig. 4B). The sea anemone metabotropic GABAb receptors, in contrast, appear to form sister clades
with vertebrate and invertebrate type 2 GABAb orthologues, but not
Fig. 6. Relationships of the vesicular amino acid-like transporters of N. vectensis. (A) Rooted NJ tree showing that sea anemone sequences form a clade with a mammalian vesicular
glutamate transporter (vGluT1) but not with other vertebrate (vGlut2-6) and invertebrate glutamate transporters. (B) Alignment of sea anemone sequences with corresponding
vGluTs. The represented sequence segments are in helices 5 (upper panel) and 10 (lower panel). Residues facing the center of the pore separating the N- and C-tail domains are
shown (⁎). (C) Rooted NJ tree showing that sea anemone sequences form outgroups in relation to various vesicular inhibitory amino acid transporters (VIAAT). (D) Alignment of sea
anemone sequences with corresponding VIAATs. The domains in the represented sequence segments are identified above the panels and include highly conserved residues.
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with type 1 receptors (Fig. 4C). However, examination of residues
important for ligand binding or affinity shows that the sea anemone
transcripts have conserved both type 1 and type 2 specific residues
(Fig. 4D).
Glutamate can be inactivated by reuptake from the synaptic cleft
through excitatory amino acid transporters (EAAT; Palacin et al.,
1998), members of the solute carrier superfamily (SLC). Three
transporters related to this superfamily, showing the typical 10
transmembrane domains, were identified in the sea anemone genome
(Table 1). They show significant similarity with vertebrate glial and
neuronal EAAT, but also with other EAATs, sharing 59–66% residue
similarity and 42–46% identity with various vertebrate EAATs. Tree
analysis shows that the sea anemone sequences form an outgroup in
relation to a host of vertebrate EAATs (Fig. 5A). Therefore, it is unclear
if any of these sequences are related to EAATs involved in transport at
neuronal membranes as opposed to less selective, epithelial amino
acid transports. Signature residues and those involved in glutamate
binding (Yernool et al., 2004; Pedretti et al., 2008) are largely shared
by the sea anemone EAAT (Fig. 5B). Glutamate is also stored in
synaptic vesicles through vesicular transporters (SLC17 family). Five
transcripts from the sea anemone genome (Table 1) are nested with a
rat vesicular glutamate transporter (vGlut1) which is remote from
other types of vertebrate and invertebrate vGlut orthologues (Fig. 6A).
However, the consensus residues shared by the rat vGlut1 and sea
anemone transcripts are within the score range of those shared with
other vGluts (48–64% similarity, 27–43% identity). There are divergences in the extent to which the various sea anemone transcripts
show conservation of the residues important for binding pocket
formation and for glutamate binding (Almqvist et al., 2008) (Fig. 6B).
A surprisingly large number of inhibitory amino acid transporters
were found (Table 1). Among them, five are plasma membrane
transporters (SLC6 family) and nine are vesicular inhibitory amino
acid transporters (SLC32, VIAAT). Immunoreactivity to GABA vesicular
transporters was previously reported in the sea fan (Girosi et al., 2007).
Some of the SLC6-like sea anemone sequences cluster with vertebrate
GABA GAT1 proteins while others cluster with unclassified vertebrate
and invertebrate SLC6 members (Fig. 5C). Their sequences are highly
conserved, with up to 70% residue similarity and 49% identity with
various GAT1 orthologues. None clustered with glycine transporters.
Signature residues of the SLC6 family and residues important for
substrate binding (Palacin et al., 1998) are highly conserved in most
transcripts (Fig. 5D). The VIAAT transcripts are distributed in two clades,
one of which clusters with A1 members of the vertebrate SLC32 family
and with a few invertebrate VIAATs whereas the other forms a separate,
more distantly related clade (Fig. 6C). The sea anemone transcripts
share a large number of conserved residues with the A1 type of VIAAT
(Fig. 6D).
3.4. ATP
P2X receptors are cation channels gated by ATP released from
purinergic neurons (see Burnstock, 2007 for review). ATP was shown to
trigger circular muscle contraction more potently than other nucleotides
in the sea anemone Actinia equina (Hoyle et al., 1989). In addition,
evidence for a role of ATP, presumably released from sensory neurons, in
sea anemone sensory hair cell repair was presented (Watson et al.,
1999). The case for a transmitter role for ATP is bolstered by the
identification of two P2X receptors in the N. vectensis genome (Table 1)
sharing strong sequence homology with vertebrate P2X4 (54% residue
identity) and, to a lesser extent, with a Hydra P2X-like receptor (48%
identity). Phylogenetic analysis provides strong support for the sea
anemone receptors forming a clade with invertebrate (plathelminth and
Aplysia) P2X receptors that is separate from a variety of vertebrate P2X
receptors (Fig. 7A). Interestingly, inclusion of the Hydra P2X in the sea
anemone clade was not validated by bootstrapping. Examination of
alignments shows that the residues important for ligand binding and ion
channel activities (Silberberg et al., 2005) are largely preserved in the
sea anemone transcript (Fig. 7B).
3.5. Biogenic amines
Next to neuropeptides the most compelling case for the physiological involvement of neurotransmitters in cnidarians comes from
studies on monoamines (Anctil and Bouchard, 2004; Anderson,
2004). Although not all criteria for neurotransmitter identification
have been met, studies on the sea pansy Renilla koellikeri, an
anthozoan like N. vectensis, have converged to provide evidence of
the presence in neurons (Umbriaco et al., 1990; Pani et al., 1995;
Mechawar and Anctil, 1997; Anctil et al., 2002), biosynthesis (Pani and
Anctil, 1994), exocytotic release (Gillis and Anctil, 2001), receptor
binding (Awad and Anctil, 1993a,b; Hajj-Ali and Anctil, 1997) and
inactivation (Anctil et al., 1984; Dergham and Anctil, 1998) of various
monoamines. Dopaminergic transmission was also investigated in sea
anemones and hydrozoans (Carlberg et al., 1984; Carlberg, 1992;
Chung et al., 1989; Chung and Spencer, 1991a,b).
As will become apparent below, proteins related to aminergic
transmitters are heavily represented in the genome of N. vectensis
(Table 2). The most striking feature of the suite of proteins (enzymes
of the aminergic pathway, receptors, transporters) is that they belong
to classical vertebrate types of biogenic amine proteins. For example,
Fig. 7. Relationship of the ATP-like receptor of N. vectensis. (A) Unrooted NJ tree showing that the sea anemone P2X-like sequence forms with plathelminth and mollusk orthologues a
separate clade from vertebrate P2X receptors. (B) Alignment of sea anemone sequences with corresponding P2X receptors. The represented sequence segments are in the first (upper
panel) and second (lower panel) transmembrane regions. Residues important for ligand binding affinity are shown (+). Red letters indicate residues which when substituted by
tryptophan lead to non-conducting channels. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
M. Anctil / Comparative Biochemistry and Physiology, Part D 4 (2009) 268–289
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Fig. 8. Relationships of biogenic amine-related enzymes of N. vectensis. (A) Unrooted NJ tree showing that a sea anemone EST clusters with various vertebrate phenylalanine
hydroxylases (PaH) but not with tyrosine (TH) or tryptophan (TPH) hydroxylases. (B) Rooted NJ tree showing two sea anemone sequences clustering with monooxygenase X (moxd)
sequences from various vertebrates instead of with related dopamine β hydroxylases (DBH). (C) Unrooted NJ tree in which several genomic and EST sea anemone sequences form an
outgroup in relation to various vertebrate hydroxyindole-O-methyltransferases (HIOMT) and to related enzymes from microorganisms. (D) Rooted NJ tree showing two sea anemone
sequences clustering with vertebrate and invertebrate monoamine oxidases (MAO), but not with amine oxidases from unicellular organisms.
neither enzymes of the octopamine biosynthetic pathway nor
octopaminergic receptors were identifiable in the sea anemone
genome, which is consistent with failure to detect octopamine by
HPLC in the sea pansy (Pani and Anctil, 1993).
Tyrosine (TH) and tryptophan (TPH) hydroxylases are key ratelimiting enzymes in the catecholaminergic and indolaminergic pathways. No transcript was found in the sea anemone genome that shares
homology with these enzymes except one EST (Table 2). This
sequence clustered robustly with phenylalanine hydroxylases rather
than with TPHs or THs (Fig. 8A), which is consistent with a higher
residue identity score with the zebrafish PaH (63%) than with the
honeybee TH or human TPH (54%). This may account for the weak sea
pansy TH-like and TPH-like activities relative to the vertebrate
enzymes (Pani and Anctil, 1994). In contrast, a tyrosinase was
extracted from the sea anemone Metridium senile that catalysed the
hydroxylation of tyrosine (Carlberg et al., 1984). In this regard, the two
tyrosinases present in the N. vectensis genome (Table 2) could be
predicted to participate in the catecholaminergic pathway. The
presence of a tyrosinase EST (Table 2) suggests that tyrosinases are
expressed and functional in sea anemones.
Two transcripts each of dopa decarboxylases (DopaDC), which
catalyse the conversion of Dopa into dopamine, and dopamine beta
decarboxylases (DBH), which catalyse the formation of norepinephrine,
were found (Table 2). Recently, Marlow et al. (2009) have demonstrated
DBH expression by in situ hybridization in presumptive oral ectodermal
neurons and pharyngeal ectodermal cells. While the DopaDC transcripts
formed an outgroup clade to the vertebrate and invertebrate DopaDCs
and to the histidine or tyramine counterparts (not shown), the DBH
transcripts clustered with the moxd subfamily of monooxygenases
(Fig. 8B). In support of this, the sea anemone transcripts possess in their
copper-binding domain a motif (LxF; Xin et al., 2004) shared with moxd
but not with DBH-related enzymes (DBHR) or DBHs. However, the
residue identity score between transcripts and DBHRs was similar to
that with moxd (35–37%). In contrast, the DopaDC transcripts share
strong residue identity with vertebrate DopaDC counterparts (up to
50%) and possess nearly all the residues considered important for
substrate binding and catalytic activity (not shown). No specific
phenylethanolamine N-methyltransferase (PNMT) transcript was identified in N. vectensis. An N-methyltransferase transcript and a related EST
sequence (Table 2) shared substantial residue identity with human
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PNMT (36%), but tree analysis placed them as a sister clade to a clade
encompassing PNMTS, nicotinamide (NNMT) and indolethylamine Nmethyltransferases (INMT) (not shown). In addition, the sea anemone
transcripts lack the PNMT residues critical for norepinephrine binding
(Martin et al., 2001), thus making it unlikely that efficient synthesis of
epinephrine may occur. Consistent with this, PNMT-like activity in the
sea pansy showed little substrate specificity and yielded only minute
amounts of epinephrine (Pani and Anctil, 1994).
Hydroxyindolamine-O-methyltransferase (HIOMT) converts Nacetylserotonin to melatonin, the vertebrate pineal hormone. Melatonin immunoreactivity levels were reported to peak during the
reproductive season in the sea pansy and immunoreactive melatonin
was found in sea pansy neurons (Mechawar and Anctil, 1997). In
addition, melatonin was found to have opposite actions to serotonin
on sea pansy peristaltic activity (Anctil et al., 1991). Thus, although the
presence of a HIOMT was expected, it is surprising that as many as
three HIOMT gene transcripts and three related EST sequences were
found in N. vectensis (Table 2). Multiple alignments show that the sea
anemone transcripts share significant homology with vertebrate
HIOMTs (28–40% residue identity). However, tree analysis suggests
Fig. 9. Relationships of biogenic amine-like receptors of N. vectensis to catecholaminergic and histaminergic receptors. (A) Rooted NJ tree showing that the sea anemone
sequences form a sister clade of histaminergic H2 receptors while dopaminergic and
adrenergic receptors fall outside the two sister clades. Note that two sea anemone
sequences form a subclade with a receptor from another anthozoan, the sea pansy.
(B) Alignment of sea anemone sequences with various catecholaminergic and
histaminergic receptors. The represented sequence segments are in the fifth transmembrane region. Consensus residues for receptor interaction with biogenic amines
(+) and for GPCRs (⁎) are shown.
that the transcripts form a clade separate from both vertebrate
HIOMTs and O-methyltransferases of microorganisms (Fig. 8C).
Monoamine oxidase (MAO) inactivates catecholamines and indoleamines by converting them into acid forms. Evidence of such
conversions were reported in the sea pansy (Pani and Anctil, 1994).
Four MAO transcripts were identified in the sea anemone genome
(Table 2), two of which were shown by tree analysis to cluster with
invertebrate and vertebrate MAOs, but not with amine oxidases from
microorganisms (Fig. 8D). Residue identity in paired alignments with
either vertebrate or invertebrate orthologues ranged from 23 to 48%.
Although catecholaminergic-like and serotonergic-like receptor
binding and monoaminergic modulation of behaviour are documented in cnidarians (Anctil and Bouchard, 2004; Kass-Simon and
Pierobon, 2007), only two aminergic-like receptors from the sea
pansy have been cloned and despite successful attempts to express
them no ligand has been identified (Bouchard et al., 2003, 2004). It is
bewildering, then, that as many as 54 GPCR sequences in the sea
anemone genome appear to be orthologues of biogenic amine
receptors (Table 2). Another surprise is that of these, twenty-four
are melatonin-like receptors, which however is consistent with the
presence of the HIOMT transcripts mentioned above.
BLAST queries yielded a mixture of hits from different classes of
aminergic receptors for all non-melatonergic sea anemone transcripts,
in keeping with difficulties in classifying physiologically investigated
and cloned sea pansy receptors. The classification arrived at in Table 2
Fig. 10. Relationship of indoleaminergic-like receptors of N. vestensis. (A) Rooted NJ tree
showing that the sea anemone sequences form a sister clade with various vertebrate
melatonin receptors, but not with serotonin receptors. (B) Alignment of sea anemone
sequences with melatonergic and serotonergic receptors. The represented sequence
segments overlap the third transmembrane region and second intracellular loop.
Consensus residues (+) and motif (CxxCH) for melatonin receptors, and a conserved
GPCR residue (⁎) are shown. Note that D in the DRY motif is substituted by N in both the
sea anemone and melatonergic sequences.
M. Anctil / Comparative Biochemistry and Physiology, Part D 4 (2009) 268–289
is tentative only and is based on the top hits of each transcript and on
the clustering of the various transcripts in phylogenetic trees. Thus
some of the transcripts present more similarity to dopaminergic or
serotonergic receptors than to other aminergic receptor classes, others
to adrenergic receptors, and still others to histaminergic receptors
(Table 2). Vertebrate and protochordate orthologues largely dominate
the hit lists, although occasional sequences from invertebrates such as
the sea urchin are among the hits. Fig. 9A shows that many of the sea
anemone transcripts, along with Ren 1 from the sea pansy (Bouchard
et al., 2003), tend to cluster with histamine H2 receptors rather than
with dopamine and adrenergic receptors. The best scores for residue
identity are in the dopamine/serotonin group (40% for Nv_24241
against sea urchin D1B), followed by the histamine group (34%
Fig. 11. Relationships of biogenic amine-like transporters of N. vectensis. (A) Unrooted
NJ tree showing that the two sea anemone sequences shown form a sister clade with
various aminergic nerve terminal transporters, but not with GABA, taurine or creatine
transporters. The panel below shows an alignment of sea anemone sequences with
various aminergic transporters. The represented sequence segments overlap the sixth
transmembrane region with residues considered to be involved in interaction with
monoamines (⁎) and the fourth intracellular loop containing many residues where
conformational changes occur during substrate binding and translocation (+).
(B) Rooted NJ tree showing that the sea anemone sequences form an outgroup relative
to aminergic vesicular transporters (VMAT) and members of the major facilitator
superfamily (MSF) and related transporters. The tree is rooted with a VIAAT. The panel
below shows an alignment of sea anemone sequences with aminergic vesicular
transporters and a MFS. The represented sequence segments are in the second (left
segment) and fourth (right segment) transmembrane regions. Two conserved motifs of
VMATs are highlighted.
279
for Nv_136356 against Amphioxus H2). Inspection of multiple alignments indicates that the sea anemone transcripts largely share with
vertebrate orthologues the signature residues of GPCRs of the rhodopsin family. Residues important for histamine binding are conserved in some sea anemone transcripts (Fig. 9B) whereas other
transcripts tend to conserve residues important for binding of amines
other than histamine (Shi and Javitch, 2002).
In contrast to non-melatonin aminergic receptors, all BLAST
alignments of any melatonin receptor transcript of the sea anemone
yielded first numerous hits for other sea anemone transcripts,
followed by hits of much lower e-values for vertebrate melatonin
receptors. No other aminergic or non-aminergic receptors were
represented in the hits. This suggests that these transcripts form a
tight assemblage of interrelated sea anemone receptors with some
resemblance to vertebrate melatonin receptors. This is supported by
the relatively low residue identity scores of the transcripts with a
range of melatonin receptors (21–29%). Tree analysis shows that the
sea anemone transcripts cluster robustly with vertebrate melatonin
receptors but not with serotonin receptors (Fig. 10A). Alignment
inspection revealed that while rhodopsin GPCR signature residues are
largely shared by the sea anemone transcripts, only half the residues
important for melatonin binding (Barrett et al., 2003) are retained
(Fig. 10B). Melatonin receptor-specific motifs such as NRY/F at the exit
Fig. 12. Relationships of MECA receptors and nitric oxide synthase (NOS) of N. vectensis.
(A) Rooted NJ tree showing that some sea anemone sequences cluster with adenosine
receptors whereas others, together with coral sequences, cluster with melanocortin
receptors. (B) Unrooted NJ tree showing that one sea anemone sequence forms with a
coral NOS an outgroup to a broad variety of invertebrate and vertebrate NOSs.
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of TM3 and NaxxY in TM7 are largely preserved (not shown), but
CxxCH in the second intracellular loop is absent (Fig. 10B).
Pharmacological uptake experiments in the sea pansy (Anctil et al.,
1984; Dergham and Anctil, 1998) suggested the existence of plasma
membrane transporters for catecholamines and indoleamines in
cnidarians. Three such transporters were identified in the sea anemone
genome (Table 2). They cluster with aminergic rather than with amino
acid transporters in tree analysis (Fig. 11A). Alignment inspection
revealed that they show a high level of residue conservation with
aminergic transporters (43–45% residue identity), including a residue
involved in the interaction with monoamines in TM1 and leucine repeats
in TM2 (not shown). A glycophorin-like motif in TM6 and residues
involved in conformational change during substrate binding and
translocation in the third intracellular loop (Torres et al., 2003) are
also shared by the sea anemone transcripts (Fig. 11A). In addition, two
vesicular monoamine transporters (VMAT) were found (Table 2). They
formed a clade separate from vertebrate and invertebrate VMATs and
from vertebrate members of the major facilitator superfamily (MFS)
(Fig. 11B). Alignment inspection showed residue identity scores against
vertebrate VMATs were low (11–15%) compared with those against
MSFs and related transporters (22–30%). In addition, the sea anemone
sequences lacked all conserved motifs and substrate-relevant aspartate
residues of vertebrate VMATs (Fig. 11B). In contrast, they shared more
conserved residues with insect VMATs than with MFSs and related
transporters (not shown).
3.6. Adenosine
So far there is no report of adenosine acting as transmitter in
Cnidaria. Adenosine receptors are members of the melanocortinendoglin-cannabidoid-adenosine (MECA) family of GPCRs (Fredriksson et al., 2003; Schiöth and Fredriksson, 2005). Four sea anemone
transcripts were identified that showed some homology with
adenosine receptors (Table 2), two of which clustered with a
vertebrate/invertebrate mix of adenosine receptors in tree analysis,
whereas others clustered with melanocortin GPCRs (Fig. 12A).
However, the residue identity scores against adenosine receptors are
low (23–26%) and none of these transcripts share with adenosine
receptors any of the consensus residues important for ligand binding
(Fredholm et al., 2001). A similarly weak relationship to adenosine
receptors was reported in four MECA-like GPCRs cloned from the coral
Acropora millipora (Anctil et al., 2007). Thus the case for adenosine as
a cnidarian transmitter is inconclusive at this point.
3.7. Nitric oxide
The gaseous transmitter nitric oxide (NO) is a well-known
modulator of blood vessel tone and gut muscles and is widespread
across animal phyla (Moroz, 2001). Its involvement as neurotransmitter in cnidarians was bolstered by evidence of the involvement of NO in
the feeding response of Hydra (Colasanti et al., 1997) and the
subsequent localization of NADPH-diaphorase activity in Hydra
neurons (Cristino et al., 2008). Similar findings in the jellyfish
Aglantha digitale (Moroz et al., 2004) and the sea pansy (Anctil et al.,
2005) were reported, in which NO was found to modulate swimming
and fluid circulation, respectively. One NO synthase (NOS) transcript is
present in N. vectensis (Table 2) and it forms with another cnidarian
NOS an outgroup in relation to vertebrate neuronal and inducible NOS
clades and to an invertebrate NOS clade (Fig. 12B). The transcript
shares 55% residue identity with a previously cloned cnidarian NOS
(mushroom coral) and 46–48% with vertebrate and invertebrate
orthologues. All functional domains and residues important for iron
binding are preserved in the sea anemone sequence. Residues
important for substrate binding and pterin interaction in nNOS
(Crane et al., 1998; Li and Poulos, 2005) are also preserved (not
shown). The sea anemone sequence shares many consensus residues
Table 4
Neuropeptides predicted from putative preprohormones identified in the genome of
N. vectensis.
Peptide name
Transcript
Predicted peptide sequence
Antho-RFamide
Nv-RFamide I
Nv-RFamide II
Nv-NPY
Antho-RIamide II
Nv-RNamide I
Nv-RNamide II
Nv-RPamide I
Nv-RPamide II
Nv-RPamide III
Nv_1374
Nv_16904
Nv_9531
Nv_9531
Nv_65111
Nv_200817
Nv_200817
Nv_37852
Nv_37852
Nv_244953
Nv-RPamide IV
Nv_244953
Nv-RWamide I
Nv-RWamide II
Nv-LWamide I
Nv-LWamide II
Nv-LWamide III
Nv-LWamide IV
Nv-LWamide V
Nv-Galanin I
Nv-Galanin II
Nv-Tachykinin I
Nv-Tachykinin II
Nv-GnRH I
Nv-GnRH II
Nv-Vasopressin I
Nv-Vasopressin II
Nv_141747
Nv_141747
Nv_126270
Nv_126270
Nv_126270
Nv_126270
Nv_126270
Nv_128410
Nv_96465
Nv_94714
Nv_88765
Nv_216820
Nv_216820
Nv_65450
Nv_206388
Nv-Vasopressin III
Nv_241190
Nv-α-MSH I
Nv-α-MSH II
Nv-α-MSH III
Nv_38399
Nv_38399
Nv_8156
b Glu-Gly-Arg-Phe-NH2
b Glu-Ile-Thr-Arg-Phe-NH2
Val-Val-Pro-Arg-Arg-Phe-NH2
Val-Val-Leu-Arg-Arg-Tyr-NH2
Tyr-Arg-Ile-NH2
Gly-Met-Asp-Gly-Arg-Asn-NH2
Gly-Met-Tyr-Arg-Arg-Asn-NH2
Trp-Ser-Cys-Ser-Leu-Arg-Pro-NH2
Trp-Ser-Cys-Cys-Leu-Arg-Pro-NH2
b Glu-Asp-Ala-Phe-Leu-Pro-LysPro-Arg-Pro-NH2
b Glu-Asp-Ser-Ser-Asn-Tyr-Glu-PhePro-Pro-Gly-Phe-His-Arg-Pro-NH2
Leu-Val-Gly-Arg-Trp-NH2
Asp-Arg-Trp-NH2
b Glu-Ala-Gly-Ala-Pro-Gly-Leu-Trp-NH2
b Glu-Ala-Gly-Pro-Pro-Gly-Leu-Trp-NH2
Gly-Pro-Pro-Gly-Leu-Trp-NH2
Gly-Ala-Pro-Gly-Leu-Trp-NH2
Asn-Ala-Pro-Gly-Leu-Trp-NH2
Gly-Asp-Thr-Gly-Ile-Thr-NH2
b Glu-Gly-Met-Thr-NH2
Tyr-Gln-Val-Ile-Phe-Glu-Gly-Val-Arg-NH2
Thr-Leu-Gln-Val-Gly-Arg-Arg-NH2
Gly-Ser-Ser-Ile-Pro-Arg-Pro-Gly-NH2
Asn-Tyr-Ser-Leu-Arg-Arg-Pro-Gly-NH2
Ala-Asn-Asp-Gly-Pro-Arg-Gly-NH2
b Glu-Glu-Glu-Gly-Val-Pro-Leu-ProArg-Gly-NH2
Pro-Gln-Pro-Gln-Arg-Ser-Met-Pro-ArgGly-NH2
Gly-Ala-Val-Pro-Val-NH2
Ser-Ala-Val-Pro-Val-NH2
b Glu-Tyr-Asn-Ile-His-Leu-Ala-Leu-Val-NH2
with iNOS as well, which is consistent with the dual nNOS/iNOS profile
of NO action in the sea pansy (Anctil et al., 2005). An orthologue of
NOS-binding protein was also found (Table 2).
3.8. Neuropeptides
In contrast to classical neurotransmitters, several neuropeptides
were identified and their presence in neurons demonstrated in
cnidarians (Grimmelikhuijzen et al., 2004 for review). While some of
them belong to the RFamide-related peptide superfamily (RFaP),
which has representatives in every investigated metazoan phylum,
others appear so far to be peptide families exclusive to cnidarians.
Mature (secreted) peptides are generated by enzymatic cleavage of
immature copies in larger precursor proteins (preprohormones).
However, it is difficult to identify genes encoding precursors of the
putative neuropeptides because of their peculiar organization and lack
of recognizable functional domains in protein models. In spite of
these hurdles, several precursor proteins were identified (Table 3).
The identification of the putative mature peptides was based
conservatively on the presence of the expected residues for cleavage
at the N- and C-terminals of the immature copies. These include
the conventional basic residues (lysine and/or arginine), but also
acidic residues (aspartic/glutamic acid) or other unconventional pairs
(X-proline, X-alanine) at the N-terminal that were documented in
cnidarians (Grimmelikhuijzen et al., 2004). Other unusual residues
may be involved (Wei et al., 2003), so that the number of predicted
peptides listed in Table 4 is likely underestimated.
Table 3 illustrates the diversity of neuropeptide families predicted to
exist in the starlet sea anemone. Some of these have as yet no
representative identified in cnidarians. Except for a glycoprotein
hormone receptor in another sea anemone (Nothacker and Grimmelikhuijzen, 1993), no gene encoding neuropeptide or hormone receptors
M. Anctil / Comparative Biochemistry and Physiology, Part D 4 (2009) 268–289
281
of its presence in synaptic vesicles (Westfall and Grimmelikhuijzen,
1993). Recent studies showed numerous RFamide-immunoreactive
neurons in the starlet sea anemone (Marlow et al., 2009; Watanabe et al.,
2009).
In the starlet sea anemone a precursor protein predicted to generate 23 copies of Antho-RFamide was found. In addition, two splice
variants of a precursor protein were predicted to produce 27 copies of
pEITRFamide (Nv-RFamide I), a putative new peptide (Tables 3 and 4).
Finally, another transcript was predicted to generate 2 copies of NvRFamide II and of a related sequence of the NPY subfamily (Table 4).
Peptidylglycine α-hydroxylating monooxygenases (PHM) convert a
peptidylglycine into peptidylamide at the C-terminal of immature
peptides and most cnidarian peptides are amidated. An amidating
enzyme orthologue of the CP2 gene product of the sea anemone Calliactis parasitica (Williamson et al., 2000) was also found in N. vectensis
(Table 3), with a residue identity score of 46%.
The diversity of RFa peptides in N. vectensis is matched by the large
number of putative RFa-related receptor orthologues (Table 3). The 11
receptors exhibited strong similarities to Neuropeptide FF (NPFF), RFa,
gonadotropin-inhibiting hormone (GnIH) and Neuropeptide Y (NPY)
receptors from both vertebrates and invertebrates. Paired alignments
showed overall identity scores of 29–35% against human NPFF1 and
pufferfish RFa receptors and of 27–32% against the salmon NPY7
receptor. Multiple alignments revealed that these sequences shared
72% of the conserved consensus residues identified in FMRFamide
receptors (Cazzamali and Grimmelikhuijzen, 2002). Fig. 13A shows
that some sea anemone transcripts cluster with RFaPs but not with
NPY receptors, whereas others appear more or less related with
galanin receptors. An example of conserved RFa-related receptor
residues shared by sea anemone transcripts is shown in Fig. 13B. In
contrast, few of the consensus residues for galanin receptors are
preserved in the corresponding sea anemone transcripts (Fig. 13C)
and the best residue identity score of the transcripts is 31% against the
zebrafish galanin receptor.
Fig. 13. Relationship of RFamide peptide-related and galanin-like receptors of N. vectensis.
(A) Unrooted NJ tree showing that sea anemone sequences cluster with diverse RFamiderelated receptors (RFaR), including neuropeptide FF (NPFF) and gonadotropin-inhibiting
hormone (GnIH) receptors, whereas others cluster with a galanin-like receptor.
(B) Alignment of sea anemone sequences with RFaRs. The represented sequence segment
is in the sixth transmembrane region. The letters represent consensus aa residues of
FMRFamide receptors. (C) Alignment of sea anemone sequences with galanin receptors.
The represented sequence segment overlaps the sixth transmembrane region and third
extracellular loop. Residues highlighted in red are consensus residues for galanin receptors.
(For interpretation of the references to colour in this figure legend, the reader is referred to
the web version of this article.)
has been identified in cnidarians. Over 80 putative neuropeptide
receptors were identified in the genome of N. vectensis that appear to
match the diversity of putative neuropeptides (Table 3).
3.8.1. RFamide-related peptides
The first isolated neuropeptide to be sequenced in cnidarians was
Antho-RFamide in a sea anemone (Grimmelikhuijzen and Graff, 1986)
and in the sea pansy (Grimmelikhuijzen and Groeger,1987). Other RFaPs
were later isolated in hydrozoans (Grimmelikhuijzen et al., 1988, 1992;
Moosler et al., 1996) and scyphozoans (Moosler et al., 1997). In
anthozoans, the role of Antho-RFamide as a neuromuscular transmitter
was supported by its actions on muscle systems (McFarlane et al., 1987;
Anctil and Grimmelikhuijzen,1989) and immunocytochemical evidence
3.8.2. Cnidarian-specific peptides
A variety of new neuropeptides were isolated from the sea
anemone Anthopleura elegantissima that displayed C-terminal signatures unique to cnidarians (Graff and Grimmelikhuijzen, 1988a,b;
Grimmelikhuijzen et al., 1990; Nothacker et al., 1991a,b; Carstensen
et al., 1992, 1993; Leitz et al., 1994). Putative counterparts for each of
these new types of peptides except Antho-KAamide were identified in
predicted precursor proteins of the starlet sea anemone (Table 3).
A precursor was identified that contained 8 copies of Antho-RIamide
II (Table 4), a tripeptide immunolocalized in endodermal sensory
neurons and causing the inhibition of spontaneous sphincter contractions in the sea anemone C. parasitica (Nothacker et al., 1991a,b). The
two N. vectensis members of the RNamide family (Table 4) are present as
one copy each in their precursor protein (Table 3). They possess 3 more
amino acids than Antho-RNamide, a tripeptide with known actions on
antagonistic muscles of sea anemones (McFarlane et al., 1992).
The RPamide family is represented in the starlet sea anemone by
four predicted peptides, two of which (Nv-RPamide I and II) are
heptapeptides encoded in 3 and 2 copies, respectively, in their
precursor protein and differing by only one residue substitution. The
two other members (Nv-RPamide III and IV) are longer sequences
with a N-terminal bGlu residue represented by a copy each in another
precursor protein (Table 4). They bear little resemblance to AnthoRPamides I–IV except that Nv-RPamide I and II share the C-terminal
consensus LRPamide with Antho-RPamide II and Nv-RPamide III
shares the sequence PRPamide with Antho-RPamide I (Grimmelikhuijzen et al., 2004). Antho-RPamide I was shown to enhance
contractile activity and to increase the rate of spontaneous contractions in the sea anemone A. equina (Carstensen et al., 1992), an effect
similar to that of Antho-RFamide on the sea pansy muscle systems
(Anctil and Grimmelikhuijzen, 1989).
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Two putative peptides of the RWamide family (Table 4) were
detected in a predicted precursor protein (Table 3). Both, LVGRWamide (Nv-Rwamide I) and DRWamide (Nv-Rwamide II), are present in
three copies each. Their N-terminal residues differ from those of the
Antho-RWamides (Grimmelikhuijzen et al., 2004), including the lack
of a pyroglutamate. The Antho-RWamides have been immunolocalized in synaptic terminals of endodermal muscles of C. parasitica
(Westfall et al., 1995) and induce contractions of endodermal muscles
(McFarlane et al., 1991) presumably via calcium channel opening (Cho
and McFarlane, 1996).
Another family of peptides that generated much interest is the
cnidarian metamorphosins, which are involved in the metamorphosis of
planula larvae into mature animals (Leitz, 1998). Metamorphosin A
(MMA) was first isolated from the sea anemone A. elegantissima (Leitz et
al., 1994) and several more metamorphosins were later identified in the
same species as well as in Hydra (Leviev and Grimmelikhuijzen, 1995;
Takahashi et al., 1997; Leitz, 1998). They almost all share the C-terminal
sequence GLWamide which is biologically active. These peptides were
immunolocalized in larval sensory neurons and in endodermal neurons
of more mature stages (Gajewski et al., 1996). In addition to the eight
metamorphosins in A. elegantissima, the six in A. equina and the four in
Anemonia sulcata (Leitz, 1998), five putative metamorphosins (Table 4)
were identified in a single precursor protein (Table 3): four copies of
pEAGAPGLWamide (Nv-LWamide I), three of pEAGPPGLWamide
(Nv-LWamide II), six of GPPGLWamide (Nv-LWamide III),
three of GAPGLWamide (Nv-LWamide IV) and four of NAPGLWamide
(Nv-LWamide V). The N. vectensis peptides share best similarity with
the metamorphosins of Hydractinia echinata and Hym331 of Hydra
(two hydrozoans), and Ae-LWamide I of the sea anemone A. equina.
The diversity of this peptide family is only matched by that of the RFarelated family.
In addition to the uniquely cnidarian peptide families, there is an
uncommonly large retinue of neuropeptide receptors (39) presenting
homologies primarily with each other and, in some cases, weak
similarities with a suite of RFa- and tachykinin-related receptors
(Table 3). Therefore, they are listed here as unclassifiable. The presence
of these receptors is not surprising since they may be associated with the
various uniquely cnidarian peptides described above. It will be an
important challenge to express them with a view to identify their ligands
among the specific sea anemone peptides.
3.8.3. Galanin-related peptides
Galanin is a highly conserved peptide found in the nervous system
and gut of vertebrates (Tatemoto et al., 1983; Skofitsch and
Jacobowitz, 1985; Chartrel et al., 1995). No galanin-like peptide has
yet been identified in invertebrates, although galanin-like immunoreactivity was reported in the nervous system of a few invertebrates
(Lundqvist et al., 1991; Diaz-Miranda et al., 1996), including Hydra
(Yamamoto and Suzuki, 2001). Tree construction of RFa-related
receptors revealed a subclade of sea anemone receptors that clustered
with galanin receptors (Fig. 13A). These putative galanin-like
receptors are listed in Table 3.
This finding prompted a search for precursor proteins in which are
encoded immature galanin-like peptides. As the C-terminal residues
of galanin are required for high-affinity receptor binding of smooth
muscle preparations (Rossowski et al., 1990), the C-terminal consensus G(L/I/M)Tamide was subjected to PHI-BLAST analysis. This
yielded three predicted precursor proteins, one of which encoded
12 copies of the putative peptide GDTGITamide (Nv-Galanin I) and the
other two 8 copies (Nv_142194) and one copy (Nv_96465) of
pEGMTamide (Nv-Galanin II) (Table 4). Thus these sequences are
much shorter than the 29–30 aa of mammalian galanins. This
divergence of the sea anemone peptides is consistent with the lack
in the sea anemone galanin-like receptors (Fig. 13C) of many of the
key residues identified in mammalian galanin receptors (Church et al.,
2002; Lundström et al., 2007).
3.8.4. Tachykinin-related peptides
Vertebrate tachykinins (TKs) and invertebrate tachykinin-related
peptides (TKRPs) are present in neurons or gut endocrine cells and are
involved in various activities such as modulation of neuronal
excitability, induction of intestinal and oviduct contractions, and
developmental regulation (Otsuka and Yoshioka, 1993; Nässel, 1999;
Satake et al., 2003). There is no known TK or TKRP in cnidarians,
although substance P-like immunoreactivity was reported in Hydra
(Grimmelikhuijzen et al., 1981). However, the C-terminal TK consensus sequence FxGLM typical of substance P was not found in
N. vectensis. Instead a putative precursor protein was found that
apparently encodes a single copy of a TKRP with the C-terminal FxGyR
signature (Tables 3 and 4). In addition, a precursor protein was found
in which are encoded 16 copies of a putative TKRP with the
incomplete C-terminal motif GxR (Table 4). The N-terminal portion
of the sea anemone TKRPs shows no sequence similarity with any of
the known invertebrate TKRPs (Satake et al., 2003).
In contrast to the paucity of putative ligands, numerous TK-like
receptors were identified (Table 3). The 12 receptors showed
homology, and tended to cluster with invertebrate TK (24–30%
residue identity) and SIFamide receptors (27–33%) but slightly less
with vertebrate TKs (22–28%). The sea anemone sequences cluster
robustly with both invertebrate and vertebrate tachykinin receptors,
but not with RFamide-related receptors (Fig. 14A). Multiple alignments showed that several of the consensus residues of TK receptors
(Satake et al., 2003) are preserved in the TK-like sea anemone
receptors, but some of the residues important for ligand binding are
not (Fig. 14B). They are also absent in TK receptors from some of the
other invertebrates.
Fig. 14. Relationship of tachykinin-like receptors of N. vectensis. (A) Rooted NJ tree
showing that the sea anemone sequences cluster with known vertebrate and
invertebrate tachykinin receptors. (B) Alignment of sea anemone sequences with
tachykinin receptors. The represented sequence segment overlaps the third intracellular loop and sixth transmembrane region. Consensus motif (⁎) and residues involved
in agonist binding to human neurokinin A receptor are shown (letters).
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3.8.5. Peptides related to gonadotropin-releasing hormone (GnRH) and
vasopressin/oxytocin families
GnRHs form a highly conserved family of decapeptides that play a
major role in vertebrate reproduction (Seeburg et al., 1987). Several
conserved GnRH isoforms have also been identified in protochordates,
where they are also decapeptides (see Gorbman and Sower, 2003, for
review), and in mollusks in which are encoded GnRHs with 12 amino
acids (Iwakoshi et al., 2002; Zhang et al., 2008). Two forms of
biologically active GnRH-immunoreactive material were extracted
and partially purified in the sea pansy (Anctil, 2000), but the structure
of these putative peptides was not elucidated. The latter study also
reported the localization of immunoreactive GnRH in endodermal
neurons of both the sea pansy and the starlet sea anemone. Some of
these neurons were associated with gonad tissues and the sea pansy
GnRH-like extracts as well as LHRH caused an inhibition of peristaltic
contractions (Anctil, 2000).
A precursor protein was detected in the starlet sea anemone
(Table 3) in which two putative GnRH-like peptides appear to be
encoded (Table 4). Both isoforms are octapeptides. Nv-GnRH I (4 copies)
differs from Nv-GnRH II (one copy) by 4 aa in the N-terminal portion
of the sequences. Both retain the C-terminal consensus RPGamide
of mammalian GnRH and they share more similarity with LHRH than
with other GnRH isoforms, which is consistent with the higher affinity
of LHRH antibody compared with other (non-mammalian) antibodies
against sea pansy immunoreactive materials (Anctil, 2000). It would
be interesting to know whether the two sea anemone isoforms represent the two putative peptides extracted from the sea pansy.
The vasopressin/oxytocin family of peptides shares with GnRH
peptides the C-terminal Gamide. This is an important peptide family
associated with the vertebrate neuro-hypophyseal axis and with roles
in osmoregulation and reproduction (Acher, 1996). Several homologs
were identified in invertebrates where they are expressed in the
nervous system and may be involved in sexual behavior (Van Kesteren
et al., 1995; Kanda et al., 2005). Vasopressin/oxytocin immunoreactive
neurons have been visualized in Hydra (Grimmelikhuijzen et al., 1982;
Koizumi and Bode, 1991) and more recently two vasopressin-related
peptides were identified in Hydra that share the C-terminal tripeptide
Fig. 15. Relationship of GnRH-like/vasopressin-like receptors of N. vectensis. (A) Rooted NJ
tree showing two sea anemone sequences that cluster with an ensemble of GnRH and
vasopressin-related receptors. (B) Alignment of sea anemone sequences with GnRH and
isotocin/vasotocin receptors. The represented sequence segment is in the sixth
transmembrane region. Signature residue of rhodopsin GPCRs (⁎) and residues important
for receptor structure and ligand pocket formation (+) are shown. A tyrosine residue
involved that participates in ligand binding is also shown (Y).
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PRGamide with Arg-vasopressin but lack the N-terminal with the two
cysteines connected by a disulfide bond that characterize the family
(Morishita et al., 2003). The Hydra peptides appear to be involved in
neuronal differentiation (Takahashi et al., 2000).
In the starlet sea anemone, three putative prepropeptides were
identified that encode each one predicted vasopressin-related peptide
in one or several copies (Tables 3 and 4). As in the Hydra peptide sequences, the N-terminal cysteines are lacking, but the C-terminal
PRGamide of Arg-vasopressin/vasotocin is preserved. Nv-vasopressin II
extends the similarity with the Hydra peptides to LPRGamide, but
similarities end there. It is apparent that the cnidarian peptides form
a separate peptide family only remotely related with other invertebrate
vasopressin/oxytocin members which all include the N-terminal cysteine
residues found in the vertebrate orthologues (Kanda et al., 2005).
BLAST alignments yielded two relatively weak hits with GnRH and
vasopressin/oxytocin receptor families in N. vectensis (Table 3).
Sequence residue identity scores were also low (16–20% against
GnRH and vasotocin/isotocin receptors). Tree analysis shows that the
two receptors cluster with an assemblage of GnRH and vasopressinrelated receptors (Fig. 15A). Multiple alignments reveal that the sea
anemone sequences share some of the residues important for receptor
structure and ligand binding with GnRH receptors (Mamputha et al.,
2007) (Fig. 15B). Some of these residues are also shared by vasotocin/
isotocin receptors. The sea anemone sequences, therefore, may
represent ancestral forms that later diverged to form specific GnRH
and vasopressin/oxytocin receptors.
3.8.6. Melanocortin-related peptides
Melanocortins in vertebrates are derived from the complex precursor
proopiomelanocortin (POMC) (Hadley and Haskell-Luevano, 1999) and
are involved in a broad variety of functions in addition to their wellknown roles in pigmentary control (α-MSH) and in adrenal cortex
stimulation (ACTH) (Wikberg et al., 2000; Schioth, 2001). They are
present in neurons as well as in a variety of cell types where their release
leads to paracrine or autocrine responses. POMC precursors were
reported in a leech and a mollusk (Salzet et al., 1997; Stefano et al., 1991),
but their absence in the genome of Caenorhabditis elegans and of insects
suggests that if they are indeed present in invertebrates, it is the result of
lateral gene transfer from a chordate source (Dores and Lecaude, 2005).
This view is supported in the present study as no POMC gene was
identified in the genome of the starlet sea anemone. Instead, the
transcripts of two putative preprohormones were found (Table 3) that
encode peptides sharing C-terminal residues with α-MSH. The
transcript Nv_38399 includes six copies of the predicted pentapeptide
GAVPVamide and four copies of the variant SAVPVamide (Table 4), both
of which share the last two C-terminal residues with α-MSH. The other
transcript includes nine copies of a predicted nonapeptide sharing with
α-MSH only the C-terminal residue Vamide (Table 4).
BLAST searches yielded nine transcripts in the sea anemone
genome that matched with melanocortin receptors (Table 3), especially with the MCR5 subtype with which they cluster in a MECA tree
construct (Fig. 12A). Four coral GPCRs were recently found to form a
sister clade to MECA members and to show more consensus residues
for ligand binding with melanocortin than with other MECA receptors
(Anctil et al., 2007). In the present study multiple alignments revealed
that the sea anemone receptors share even more consensus residues
with MCR5s (not shown). The finding that only α-MSH-like peptides
are predicted in the sea anemone genome is consistent with reports
that α-MSH binds with greater affinity on MCR5s than on other MCRs
(Wikberg et al., 2000).
3.8.7. Insulin-related peptides
Insulin is one of the best known hormones and it plays an important
role in vertebrate energy metabolism. While the structure, function and
location of synthesis of insulins are highly conserved in vertebrates,
insulin-related peptides (ILPs) of higher invertebrates form a
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Fig. 16. Relationships of insulin-like growth factors and insulin-like peptide receptors of N. vectensis. (A) Unrooted NJ tree showing a clustering of sea anemone sequences with
insulin-like growth factors but not with insulin-like peptides. (B) Unrooted NJ tree showing that a sea anemone sequence shares with a hydra sequence an outgroup position in
relation to various insulin-related receptors. (C) Alignment of sea anemone sequences with B and A chains of insulin-related peptides. Conserved motifs and residues are highlighted
below the multiple alignment. The cleavage sites are marked (DR...SR). (D) Alignment of sea anemone sequences with various insulin-related receptors. The represented sequence
segment is on the C-terminal side of the leucine repeat region. Residues that form with tryptophan 176 of human IGF1 receptor a pocket for ligand interaction are shown (+).
structurally diverse group encoded by large multi-gene families that are
expressed in the central nervous system (Smit et al., 1998). There is
evidence that ILPs, in addition to a hormonal role, are synaptic
modulators (Chiu et al., 2008). Some of the invertebrate ILPs include
peptides related to the insulin-like growth factors (IGFs) which have
widespread growth-promoting effects in vertebrates (Rotwein, 1991),
especially in the nervous system (Ye and D'Ercole, 2006). In the fruitfly
and the nematode C. elegans IGF signaling is also involved in the
regulation of aging (Tatar et al., 2003; Honegger et al., 2008).
An insulin-like receptor was cloned in Hydra that showed an
expression pattern consistent with a role in promoting growth and
morphogenetic patterning (Steele et al., 1996). This suggested that an
insulin-related molecule was involved but none has been identified so
far in any cnidarian species. Here two sea anemone transcripts appear
to be insulin-related peptide orthologues that appear to be closer to
IGFs from lower vertebrates than to vertebrate or invertebrate ILPs in
phylogenetic analysis (Fig. 16A). Multiple alignment showed that, in
spite of their low identity scores (15–23%), their organization is very
similar to that shared by ILPs and IGFs (Smit et al., 1998), including
the N-terminal signal sequence, the B and A chains separated by the
DR/SR cleavage sites and the conserved motif sequences CGxxL in B
chain and CCxxxC in A chain (Fig. 16C).
Insulin and IGF receptors are closely related members of the
tyrosine-kinase receptor superfamily. Three putative insulin-related
receptors were identified in the sea anemone genome (Table 3) which
showed surprisingly low residue identity scores against the Hydra
orthologue (13–22%). A tree was constructed suggesting that the
cnidarian receptors (Hydra and sea anemone) form an outgroup in
relation to a broad range of vertebrate and invertebrate insulin receptors
clustered with vertebrate IGF receptors (Fig. 16B). The general structure
of the ILP/IGF receptors, including the leucine-rich repeats, the α and β
chains, the single transmembrane domain and the tyrosine kinase
domains, is preserved in the sea anemone receptors. Residues involved
in formation of the receptor pocket for ligand interaction in IGF, but not
in insulin, receptors (Garrett et al., 1998) are largely shared by one of the
sea anemone orthologues (Fig. 16D).
3.8.8. Glycoprotein hormone-related receptors
Vertebrate gonadotropins and thyrotropin are glycoprotein hormones that affect the differentiation and growth of gonads and the
thyroid gland by binding to leucine-rich repeat-containing GPCRs
(LGRs) (Pierce and Parsons, 1981; Braun et al., 1991; Van Loy et al.,
2008). Although LGRs were cloned and characterized in a snail (Tensen
et al.,1994), the fruitfly (Hauser et al.,1997) and the nematode C. elegans
(Kudo et al., 2000), no natural ligand has been found in these
invertebrates. Similarly, a LGR was cloned in the sea anemone A.
elegantissima which shows extensive similarity to the mammalian LGRs
(Nothacker and Grimmelikhuijzen, 1993), but no ligand has been
identified. The localization of invertebrate LGR expression is unknown
except in the snail where LGR expression was found in CNS neurons
(Tensen et al., 1994).
In the starlet sea anemone seven LGRs were identified in the genome
(Table 3) but no ligand resembling vertebrate gonadotropins or
thyrotropin was found. In tree analysis one sea anemone transcript
clustered with thyrotropin and gonadotropin receptors and another
with invertebrate glycoptotein receptors (Fig. 17A). Other transcripts
failed to cluster with any of the receptor subclasses. Multiple alignments
reveal that the structural features of LGRs are shared by the sea anemone
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Fig. 17. Relationship of glycoprotein-like receptors of N. vectensis. (A) Rooted NJ tree
showing that two sea anemone sequences are nested either with vertebrate or invertebrate
glycoprotein receptors whereas others fall outside of all receptor subclasses. (B) Alignment
of sea anemone sequences with various glycoprotein receptors. The represented sequence
segment overlaps the sixth transmembrane region and third extracellular loop. A classspecific motif for the different glycoprotein hormone receptors (+) and residues important
for ligand pocket formation (letters) are shown.
receptors, such as the large N-terminal extracellular ectodomain
involved in ligand binding and composed of tandem suites of leucinerich repeat motifs, and the 7-transmembrane domain typical of GPCRs at
the C-terminal region (not shown). Residues important for ligand
pocket formation (Moyle et al., 2004; Vassart et al., 2004) are conserved
in some of the sea anemone sequences (Fig. 17B). Some of the sequences
share, along with the glycoprotein hormone receptor of A. elegantissima,
a conserved motif with fruitfly and sea urchin homologs (Fig. 17B). The
transcript Nv_80429 shared 45% residue identity with the sea anemone
A. elegantissima glycoprotein hormone, and Nv_204412 shared 37–40%
with sea star and shrimp orthologues.
3.9. Evolutionary and functional implications
Although this genomic survey predicts the occurrence in the starlet
sea anemone of many of the sets of transmitter systems available to
higher metazoans, it also reveals some unexpected findings. The major
surprises are the presence of numerous gene transcripts related to
cholinergic function but the absence of metabotropic acetylcholine
receptors, the paradox between the dearth of specific biogenic aminesynthesizing enzymes and the large number of aminergic receptors,
an indolaminergic system based on melatonin but lacking specific
serotonergic contribution, and the greater diversity of represented
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neuropeptide families than anticipated from known cnidarian peptides previously identified by extraction and purification.
The predicted presence of nicotinic, but not muscarinic, receptors
suggests that acetylcholine or an acetylcholine-like substance functions only as a fast-acting transmitter by gating ion channels in sea
anemones. Acetylcholine, while having no electrophysiological effect
on jellyfish motor neurons (Spencer, 1989), induces muscle contractions in sea anemones (Mendes and Freitas, 1984), thus suggesting
that it acts as a transmitter at neuromuscular junctions. It would be
interesting to examine whether acetylcholine is released by sea
anemone motor neurons and acts rapidly on individual muscle cells
through nicotinic-like receptors. Whatever functional role acetylcholine may have, the range of transcripts associated with amino acid
transmitters strongly indicate that the latter are more heavily involved
in controlling activities in sea anemones than acetylcholine. In this
cnidarians may not differ from other invertebrates.
The failure to find transcripts for specific rate-limiting enzymes
involved in the synthesis of catecholamines and serotonin suggests that
if conventional biogenic amines are produced by sea anemones, their
output must be sporadic and their biosynthetic pathways circuitous.
Alternately, amine derivatives unique to cnidarians may be produced by
hitherto undiscovered enzymes and act on receptors selective for them.
The sea anemone genome includes a large number of aminergic-like
receptors that defy inclusion within the vertebrate classification
schemes. For example, none of the non-melatonin receptors could be
unambiguously identified as dopamine, noradrenaline, serotonin or
histamine receptors. This is consistent with earlier reports of cloned
aminergic-like receptors in the sea pansy which, when expressed, failed
to be activated by a wide range of aminergic compounds (Bouchard
et al., 2003, 2004). The sum of evidence strongly suggests that
aminergic-like transmitters unique to sea anemones act on a wide
range of receptors to effect diverse biological responses.
The large number of transcripts related to melatonin function is
puzzling in view of the scarcity of literature on melatonin in invertebrates
(Vivien-Roels and Pévet, 1993). Because many anthozoans harbor
dinoflagellate symbionts and dinoflagellates are known to produce
melatonin, the possibility arises that the presence of melatonin in the
host is the result of interspecific exchange (Hardeland and Poeggeler,
2003). However, the specific presence of immunoreactive melatonin in
sea pansy neurons (Mechawar and Anctil, 1997) and the absence of
symbionts in the scarlet sea anemone argue against this view. The three
isoforms of HIOMT in the genome and the three ESTs related to HIOMT
strongly suggest that HIOMTs are expressed and melatonin is produced
by sea anemones. On the other hand, why are there so many melatoninlike receptors? It is striking that these receptors are unambiguously
matched with vertebrate melatonin receptors in BLAST alignments at the
exclusion of other aminergic receptors, whereas no clear ligand candidate
can be deduced for the other aminergic receptors, including the precursor
of melatonin formation, serotonin. This suggests that melatonin as a
transmitter and its receptors preceded the emergence of serotoninergic
and other conventional aminergic systems in early metazoan evolution.
While the large number of melatonin-like receptors may reflect
melatonin's involvement in diverse biological activities of sea anemones,
they may also serve as melatonin-binding proteins to stabilize melatonin
for a role as anti-oxidant. No day–night rhythm of melatonin production
was detected in the sea pansy (Mechawar and Anctil, 1997) and it has
been proposed that the role of melatonin as an antioxidant preceded its
involvement in circadian time-keeping early in evolution (Hardeland and
Poeggeler, 2003).
While 110 transcripts in the genome are associated with peptidergic
transmitters, they are outnumbered by non-peptidergic transcripts
(167). This may reflect the functional importance of nonpeptidergic
transmitters in cnidarians, but as only a few of these transmitter
candidates so far were demonstrated to be released from neurons,
peptides still appear to be the dominant neurotransmitters. So far, of all
the known vertebrate and invertebrate neuropeptide families, only
286
M. Anctil / Comparative Biochemistry and Physiology, Part D 4 (2009) 268–289
RFamide-related peptides were identified in cnidarians. The other
identified cnidarian peptide families possess carboxyterminal signatures that are unique to the phylum (Grimmelikhuijzen et al., 2004).
While this survey of the starlet sea anemone genome confirms the
existence of these peptides, it reveals also the existence in the sea
anemone of peptides related to a wide range of vertebrate peptide
families. Therefore, while it appears that some peptide families
(RIamide, RNamide, RPamide, RWamide and LWamide) turned out to
be evolutionary dead-ends, many others (related to galanins, tachykinins, GnRHs, vasopressins, melanocortins, insulins and glycoprotein
hormones) had already emerged in various guises in cnidarians and
radiated in higher metazoans.
Despite the importance of neuropeptides in these animals, no
peptidergic receptor has yet been characterized in cnidarians. This survey
reveals the presence in the sea anemone genome of numerous receptor
transcripts predicted to bind every class of putative peptide ligands
belonging to known peptide families. It is particularly striking that
RFamide-, tachykinin- and melanocortin-related receptors numerically
outmatch their potential ligands (Table 3), suggesting the potential
diversity of peptidergic signaling in sea anemones. Numerous other
neuropeptide receptors are unclassifiable and may add to the diversity of
signaling systems in cnidarians. There are many uniquely cnidarian
neuropeptides the receptors of which are unknown, and even more
neuropeptide receptors for which there is no clue on the nature of their
ligands. A major research program will be required to match them together.
When surveying phylogenetic trees constructed from the sea
anemone transmitter-related transcripts it becomes clear that in most
cases the latter form compact outgroups to other invertebrate and/or
vertebrate orthologues. This is expected as sea anemones are considered
to be representatives of the sister group to bilateral animals. Invertebrate
orthologues are also expected to be closer to the corresponding sea
anemone sequences than vertebrate counterparts, but in fact many sea
anemone transmitter-related protein classes appear to be closer to
vertebrate than to invertebrate counterparts. This is especially true of
glutamate NMDA and metabotropic receptors, GABA ionotropic receptors, excitatory amino acid transporters, aminergic receptors, adenosine
and melanocortin (MECA) receptors, RFamide-related and galanin
receptors, and insulin-related growth factors. In these cases only
protochordate and echinoderm sequences among invertebrates share
the closeness with vertebrate sequences, thus suggesting homology of
sea anemone sequences predominantly with deuterostome counterparts. This probably reflects gene loss by protostome descendants of the
common ancestors of cnidarians and bilaterians, such as nematodes and
arthropods (Technau et al., 2005), rather than any special evolutionary
closeness between cnidarians and deuterostomes.
Functionally important domains, motifs and residues of the predicted
proteins also reflect the phylogenetic distance between sea anemones
and higher metazoans. A leitmotiv of this survey is the limited extent to
which these key sequences and residues are preserved in sea anemones.
The best cases of preservation for some of the transcript isoforms are
found in ionotropic receptors (nicotinic, GABAergic, P2X), amino acid
transporters; to a lesser extent, DOPA decarboxylase (dopamine formation) and monoamine transporters, RFamide-related receptors, insulin
growth factor-like receptors and leucine-repeat GPCRs. The highly
conserved residues of ionotropic receptors and transporters may reflect
severe constraints on membrane protein configurations associated with
transmembrane molecular transport. In the case of the other sea
anemone sequences where the usual vertebrate-like signatures are
incomplete to large extents, it will remain to be investigated whether
these sequences represent unique classes of cnidarian proteins with
different substrate– or ligand–protein interactions from those known to
exist in higher invertebrates and in vertebrates.
The findings of this genomic survey of sea anemone transmitter
systems suggest that chemical transmission was already complex in
cnidarians, in agreement with a survey showing the diversity of
developmental signaling genes in anthozoans (Technau et al., 2005).
The diversity of chemical transmitter systems in sea anemones could
not have been anticipated on the basis of the small range of behaviors
and effector activities available to these animals. It remains to be seen
how much of this genetic diversity is actually expressed and functionally implemented in cnidarians.
Acknowledgements
This research was supported by a Discovery grant from the Natural
Sciences and Engineering Research Council of Canada to the author.
I am grateful to Professor C.J.P. Grimmelikhuijzen, University of
Copenhagen, for his constructive criticism and comments on an
earlier version of the manuscript.
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