From the Suntory Institute for Bioorganic Research,
Wakayamadai 1-1-1, Shimamoto-cho, Mishima-gun, Osaka 618-8503, Japan and the ¶ Department of Biological Science, Faculty of
Science, Hiroshima University, Kagamiyama 1-3-1, Higashihiroshima-city,
Hiroshima 739-8526, Japan
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ABSTRACT |
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Annetocin is a structurally and functionally
oxytocin-related peptide isolated from the earthworm Eisenia
foetida. We present the characterization of the annetocin
cDNA. Sequence analyses of the deduced precursor polypeptide
revealed that the annetocin precursor is composed of three segments: a
signal peptide, an annetocin sequence flanked by a Gly C-terminal
amidation signal and a Lys-Arg dibasic processing site, and a
neurophysin domain, similar to other oxytocin family precursors. The
proannetocin showed 37.4-45.8% amino acid homology to other
prohormones. In the neurophysin domain, 14 cysteines and amino acid
residues essential for association of a neurophysin with a
vasopressin/oxytocin superfamily peptide were conserved, suggesting
that the Eisenia neurophysin can bind to annetocin.
Furthermore, in situ hybridization experiments demonstrated
that the annetocin gene is expressed exclusively in neurons of the
central nervous system predicted to be involved in regulation of
reproductive behavior. These findings confirm that annetocin is a
member of the vasopressin/oxytocin superfamily. This is the first
identification of the cDNA encoding the precursor of an
invertebrate oxytocin-related peptide and also the first report of the
identification of an annelid vasopressin/oxytocin-related precursor.
The cyclic nonapeptides vasopressin
(VP)1 and oxytocin (OT) and
their structurally related peptides are well known as neurohypophysial hormones involved in osmoregulation and reproduction in all vertebrates (1-4). They are classified into the VP and OT families based on the
amino acid residue present at position 8: the VP family peptides
contain a basic amino acid, and the OT family peptides contain a
neutral amino acid at this position (1-4). Both the VP and OT family
peptides are present in all vertebrate species except the cyclostomes,
which have only the VP-related peptide vasotocin (2-4). The difference
in the polarity of this amino acid residue is believed to enable the VP
and OT peptides to interact with the respective receptor.
The structural organization of the precursor polypeptides of
neurohypophysial hormones is highly conserved in all vertebrates (2-4). The preprohormones are also structurally divided into the same
two classes. The mammalian VP family precursors are composed of four
regions: a signal peptide, a nonapeptide, a neurophysin, and a copeptin
domain. The architecture of non-mammalian precursors is quite similar,
except that the copeptin is not generated due to the absence of
post-translational cleavage in the precursor. Thus, a non-mammalian
neurophysin contains a C-terminal extended domain (2-4). The OT family
precursors are organized similarly, but they completely lack
copeptin(-like) domains, except for the isotocin precursor of the white
sucker fish, which also contains an extension of the C-terminal region
(2-4). Phylogenetic studies of the primary sequence of hormones, the
structural organizations of the hormone precursor, and gene structure
(5-10) led to the hypothesis that the VP and OT families separately
evolved from a common ancestral gene via gene duplication (2, 4). In addition, only vasotocin is present in the lowest vertebrate
cyclostomes (9), suggesting that a duplication of the ancestral gene
might have occurred after the evolutionary process of the Agenta (2, 4,
11).
The VP/OT superfamily peptides have also been characterized from
invertebrates, including insects (12), molluscs (13-16), and Annelida
(17, 18), as well as vertebrates (Table
I) (4). The peptides from invertebrates
are all amidated at the C terminus and share five residues, namely
Cys1, Asn5, Cys6, Pro7,
and Gly9 (Table I) (4). Annetocin has been isolated from
the lumbricid earthworm Eisenia foetida. The primary
sequence Cys-Phe-Val-Arg-Asn-Cys-Pro-Thr-Gly-NH2 is
homologous to sequences of OT-related peptides (18), and injection of
annetocin into the earthworm and leech results in induction of
egg-laying behavior (19). The similarity of not only the primary
structure but also the reproductive function implies that annetocin is
a member of the OT family. However, whether annetocin belongs to the
superfamily remains to be concluded since the organized structure of an
annetocin precursor polypeptide has not been characterized. In
invertebrates, the structure of the Lymnaea Lys-conopressin
precursor has been identified, demonstrating that the typical
architecture of the precursor of the VP/OT superfamily peptide is also
highly conserved in molluscs (20). This is the only identification of
the invertebrate VP/OT superfamily precursor, and thus, the
phylogenetic relationship of the VP/OT superfamily among invertebrates
has not been understood. To determine the feature of the precursor
polypeptide of the annelid VP/OT-related peptide and to investigate the
molecular evolution or divergence of the VP/OT superfamily in
invertebrates, we isolated and characterized the annetocin precursor
polypeptide cDNA. In this report, we present indisputable evidence
for the existence of the VP/OT superfamily in Annelida. The annetocin
precursor, very much like other precursors of the OT family, consists
of the typical three segments, i.e. a signal peptide,
annetocin, and a neurophysin-like domain with 14 cysteine residues
positioned identically to those of other neurophysins. The proannetocin
displays amino acid sequence identities between 37.4 and 45.8%. A
comparative study of amino acid sequences of prohormones also revealed
the presence of amino acid residues crucial for interaction of a
neurophysin with a hormone peptide in the Eisenia
neurophysin domain. Furthermore, in situ hybridization directly detected expression of the annetocin gene in neurons of the
subesophageal ganglion, which is known to be the central nervous tissue
of the earthworm, confirming the specific synthesis of annetocin in the
central nervous system as a neuropeptide. To the best of our knowledge,
this is the first report on the precursor structure of the invertebrate
OT-related peptide and also the first identification of cDNA
encoding the VP/OT-related peptide from Annelida, the most primitive
species from which a VP/OT-related peptide has ever been isolated.
Animals--
E. foetida lumbricid earthworms were
purchased from a fishing/bait store and kept in wet compost at
25 °C.
Oligonucleotides--
All nucleotides were ordered from Sawady Technology.
Total RNA Preparation--
Frozen partial earthworm heads (1 g)
were pulverized by grinding under liquid nitrogen. The ground tissues
were dissolved in 10 ml of TRizol reagent (Life Technologies, Inc.),
and total RNA was extracted according to the manufacturer's protocol.
3'-RACE--
All PCR amplifications were carried out in a
reaction mixture containing Taq polymerase (EX
Taq polymerase (Takara Shuzo) or rTaq DNA
polymerase (TOYOBO)) and 200 µM dNTP in a thermal cycler
(Perkin-Elmer GeneAmp PCR System 2400). First strand cDNA was
synthesized with the oligo(dT)-anchor primer supplied in the 5'/3'-RACE
kit (Boehringer Mannheim) and amplified using the anchor primer and the
first degenerate primer, TG(T/C)TT(T/C)GTI(A/C)GIAA(T/C)TG(T/C)CC (where I represents inosine), corresponding to the N-terminal part of
annetocin, Cys-Phe-Val-Arg-Asn-Cys-Pro. First-round PCR products were
reamplified using the anchor primer and the second degenerate primer,
GTI (A/C)GIAA(T/C)TG(T/C)CCIACIGGIGG, corresponding to the C-terminal
part of annetocin, Val-Arg-Asn-Cys-Pro-Thr-Gly-Gly, where the last Gly
was regarded as a C-terminal amidation signal. Both first- and
second-round PCRs consisted of 30 cycles for 30 s at 94 °C,
30 s at 51 °C, and 2.5 min at 72 °C (5 min for the last
cycle). The second-round PCR products were subcloned into a TA cloning
vector (Invitrogen) according to the manufacturer's instructions. The
DNA inserts of the positive clones were amplified by PCR using
universal M13 primers.
5'-RACE--
Template cDNA was synthesized using a primer
complementary to nucleotides 416-435 (ATTCCTTCGACAGCGCAGAC), followed
by dA-tailing of the cDNA using dATP and terminal transferase
(Boehringer Mannheim). The tailed cDNA was amplified using the
oligo(dT)-anchor primer and the gene-specific primer 1 (TCGACAGAT-GGTTCTCGAGG, complementary to nucleotides 349-368),
followed by reamplification of the first-round PCR products using the
anchor primer and the gene-specific primer 2 (GTGTTGACGAAGCAGAAGAAG,
complementary to nucleotides 309-328). Both first- and second-round
PCRs were performed for 30 cycles consisting of 30 s at 94 °C,
30 s at 55 °C, and 1.5 min at 72 °C. The second-round PCR
products were subcloned, and the inserts were amplified as described in
3'-RACE.
DNA Sequencing--
All nucleotide sequences were determined
using Big-Dye sequencing kits (Perkin-Elmer) and an automated DNA
sequencer (Perkin-Elmer Model 373A) and analyzed on GENETYX-MAC
software (Software Development). Universal M13 primers or gene-specific
primers were used to sequence both strands.
Northern Blot Hybridization--
A full-length digoxigenin
(DIG)-labeled annetocin precursor cDNA was synthesized using a DNA
labeling kit (Boehringer Mannheim) and was used as a probe for Northern
blot analysis. Total RNA was separated on a denaturing
formaldehyde-containing 1% agarose gel and fixed onto Hybond
N+ membrane (Amersham Pharmacia Biotech) by UV irradiation.
Hybridization and detection were carried out according to the
manufacturer's standard procedure (Boehringer Mannheim). RNA size was
estimated using DIG-labeled RNA molecular markers (Boehringer Mannheim).
In Situ Hybridization--
An earthworm head was dissected and
incubated in 4% paraformaldehyde/PBS (10 mM sodium
phosphate buffer (pH 7.5) and 0.9% NaCl) at 4 °C overnight. After
washing five times with 10 mM sodium phosphate (pH 7.5) and
0.1% Tween 20 at 4 °C for 30 min, the fixed head was dehydrated in
ethanol and benzene and embedded in 96% polyester wax (BDH).
Transverse sections 6-µm thick were cut, arranged on
3-aminopropyltriethoxysilane-coated slides (Mathunami), and dried for
5 h at 50 °C and further overnight at room temperature. The
sections were deparaffinized in xylene (2 × 5 min, room
temperature) and rehydrated in ethanol and PBS, followed by successive
treatment with a 10 µg/ml proteinase K solution (Nakalai Tesque; 10 min, room temperature), PBS (3 × 5 min, room temperature), 4%
paraformaldehyde/PBS (10 min, room temperature), PBS (3 × 5 min,
room temperature), 0.2 N HCl (10 min, room temperature),
PBS (3 × 5 min, room temperature), 0.25% acetic anhydride in 0.1 M triethanolamine HCl buffer (pH 8.0) (10 min, room
temperature), and PBS (3 × 5 min, room temperature). The sections
were then incubated for 2 h at 50 °C in prehybridization medium
containing 50% formamide, 10% dextran sulfate (Sigma), 1% blocking
reagent (Boehringer Mannheim), 5× SSC (1× SSC = 0.15 M NaCl and 0.015 M sodium citrate (pH 4.5)),
and 50 µg/ml denatured herring sperm DNA. To prepare a sense or
antisense probe, 53-mer oligonucleotides complementary or identical to
a preproannetocin cDNA located between nucleotides 152 and 204 were
tailed at the 3'-terminus by DIG-11-dUTP using a DIG oligonucleotide
tailing kit (Boehringer Mannheim). Hybridization was performed for
16 h at 50 °C in hybridization medium containing a 1 pmol/ml
concentration of each labeled probe. Hybridized sections were washed
with 50% formamide and 2× SSC (2 × 20 min, room temperature),
2× SSC (2 × 20 min, 50 °C), and 0.2× SSC (2× 20 min,
50 °C). After incubation in blocking buffer diluted with washing
buffer prepared from the DIG Wash and Block Buffer Set (Boehringer
Mannheim) for 1 h at room temperature, slides were incubated in a
1:1000 dilution of alkaline phosphatase-conjugated anti-digoxigenin Fab
(Boehringer Mannheim) for 1 h at room temperature. Subsequently,
slides were washed in washing buffer (2 × 20 min, room
temperature); covered with a chromogen mixture consisting of 100 mM Tris (pH 9.5), 100 mM NaCl, 10 mM MgCl2, 337.5 µg/ml nitro blue tetrazolium,
and 175 µg/ml 5-bromo-4-chloro-3-indolyl phosphate; and kept at room temperature in the dark until sufficient color had developed (~36 h).
Staining was stopped by washing with 10 mM Tris (pH 7.5)
and 1 mM EDTA.
Characterization of cDNA Encoding an Annetocin Precursor
Polypeptide--
In an attempt to obtain annetocin precursor
polypeptide cDNA fragments, we performed a 3'-RACE experiment using
degenerate primers corresponding to the N-terminal part of annetocin
(Cys-Phe-Val-Arg-Asn-Cys-Pro) and the anchor primer (see
"Experimental Procedures"). To increase abundance and specificity,
the first-round PCR products were further amplified with degenerate
primers corresponding to the C-terminal part of annetocin
(Val-Arg-Asn-Cys-Pro-Thr-Gly-Gly) and the same anchor primer. Here, the
C-terminal amide group was thought to be derived from a C-terminal Gly
residue that is well known as the typical amidation signal.
Electrophoresis of the second-round PCR mixture revealed a single
product of ~0.5 kilobases (data not shown). Sequencing of the
subcloned second-round PCR products showed that all clones had
essentially identical nucleotide sequences, except for minor
differences in the 3'-terminal sequence, probably attributable to
utilization of the alternate polyadenylation signal AATAAA and various
lengths of the poly(A) tract. The predicted amino acid sequence
comprised a cysteine-rich domain preceded by the endoproteolytic
dibasic sequence Lys-Arg after a partial annetocin sequence derived
from the second-round PCR primers, indicating that an annetocin
precursor might be organized similarly to precursors of the VP/OT
superfamily. To determine the 5'-end sequence, we performed 5'-RACE
using specific primers for the clone (see "Experimental
Procedures"). A single product of ~0.3 kilobases (data not shown)
was obtained and sequenced after subcloning and amplification as
described for the 3'-RACE products and contained two putative ATG
initiation codons in addition to a TGA stop codon upstream of the first
ATG codon. PCR products amplified using different polymerases had
identical nucleotide sequences, confirming that these cDNA clones
were not generated by artifacts. By combining nucleotide sequences
determined by 3'- and 5'-RACE, the entire cDNA sequence encoding a
preproannetocin was identified. Fig. 1
shows the complete sequence of the longest cDNA. The annetocin precursor cDNA is composed of 668 nucleotides containing a 417-base pair single open reading frame flanked by a short 5'-untranslated sequence of 58 base pairs and a 3'-untranslated sequence of 193 base
pairs followed by various lengths of poly(A) tail. The open reading
frame region begins with two putative start codons present at positions
59 and 86 and terminates with a stop codon at position 473. Both
putative initiation codons conform to the Kozak rule (AAAATGG and
AACATGA) (22). Two polyadenylation signals (AATAAA) were found in the
3'-untranslated region at positions 637 and 647. Nucleotide sequence
analysis of all clones indicated that the second polyadenylation signal
was used relatively more frequently than the first one; however, the
biological significance of this remains to be elucidated. Northern blot
analysis of total RNA using a DIG-labeled preproannetocin cDNA as a
probe detected a single band of ~0.8 kilobases (Fig.
2A) even after longer exposure (data not shown), suggesting that the annetocin gene produces a single
transcript. The apparent migration of the 0.8-kilobase sequence was
well in accordance with the estimated length of the cDNA,
confirming that the longest cDNA sequence identified by combination
of 3'- and 5'-RACE includes a full-length nucleotide sequence encoding
an annetocin preprohormone.
Amino Acid Sequence of the Deduced Annetocin
Preprohormone--
The open reading frame region encodes a 139-residue
polypeptide with a predicted molecular mass of ~14.6 kDa. Amino acid
sequence analysis revealed that the structural organization of the
annetocin precursor polypeptide was quite homologous to that of the
VP/OT-related preprohormones: the precursor was composed of a signal
peptide, a nonapeptide, and a neurophysin domain, as shown in Fig.
2B. The annetocin transcript was predicted to be translated
with the Met present at position 1 or 10 since the nucleotide sequences surrounding the two start codons are entirely consistent with the Kozak
rule as described above. Hydropathy plot analysis of preproannetocin
identified the N-terminal sequence as the most hydrophobic (Fig.
2C), suggesting that this region serves as a signal peptide.
The cleavage site of the signal peptide may be the
Ala32-Cys33 bond (or
Ala23-Cys24 if the Met at position 10 is used
as the initiating signal), as seen in other precursor polypeptides of
the VP/OT superfamily. Furthermore, this prediction is supported by the
Amino Acid Sequence Comparison of a Proannetocin with Other
Prohormones of the VP/OT Superfamily--
The amino acid sequence of
the annetocin prohormone is aligned with the sequences of other
species' prohormones of the VP/OT superfamily in Fig. 3. The
neurophysin domain of the annetocin prohormone includes the longest
sequence between the cleavage site Lys-Arg and the third conserved
cysteine residue (position 41). Furthermore, seven amino acids are
present between the ninth and tenth cysteines of the invertebrate
prohormones, whereas the vertebrate counterparts contain five or six
amino acids in the corresponding region. Generally, lower animal
prohormones seem to include longer sequences in these regions.
Comparative sequence analyses showed that several amino acids such as
Arg23, Gly34, Glu67,
Pro73, and Gly93 in the annetocin prohormone
are completely conserved in the neurophysin domains of any species, in
addition to the 14 cysteine residues. Other than the nonapeptide
sequences flanked by the Gly C-terminal amidation signal and the
endoproteolytic Lys-Arg site, only the Glu67-Asn68-His69-Leu70-Ser71-Thr72-Pro73
region is relatively homologous to the corresponding regions in
vertebrates and Lymnaea. The total amino acid sequence of
the annetocin prohormone is 37.4-45.8% homologous to the sequences of
other prohormones (Table II).
Interestingly, the similarity of the annetocin prohormone to the
Lymnaea Lys-conopressin prohormone is not significantly
distinct from the similarities to the prohormones of vertebrates. This
result is somewhat surprising because we expected the amino acid
homology of the invertebrate prohormone to another relatively close
invertebrate or lower vertebrate (for example, cyclostomes) prohormone
to be much higher than to advanced vertebrate prohormones. However, the
highest and lowest similarities were found with the bovine vasopressin
prohormone (45.8%) and the white sucker vasotocin I prohormone
(37.4%), respectively. These findings suggest that the amino acid
sequence of the invertebrate VP/OT superfamily prohormone may show a
remarkable interphyletic difference that is not correlated with
phyletic distance.
Expression of the Preproannetocin Gene in the Central Nervous
System--
Localization of annetocin precursor mRNA in the
central nervous system of E. foetida was directly observed
by in situ hybridization to 6 µm serial sections of the
earthworm anterior end using an antisense DIG-labeled 53-mer
oligonucleotide probe. Positive staining in the cytoplasm of neurons
was observed only when using the antisense probe, but was not seen
either when using the sense probe or no probe or in RNase-treated
sections (data not shown). Furthermore, specificity of the antisense
oligomer for hybridization was confirmed by Northern blot analysis
(data not shown). Taken together, the positive signals observed in the
cytoplasm of neurons represented specific hybridization with the
annetocin mRNA. As shown in Fig. 4,
several positively stained neurons were detected exclusively in the
subesophageal ganglia, whereas no neurons present in the cerebral
ganglia or ventral ganglia were stained (data not shown). Moreover, the
annetocin gene was shown to be expressed almost symmetrically in two
separate regions of the subesophageal ganglia, although the
physiological significance of this phenomenon has yet to be examined. A
total of at least 10 positively stained neurons were observed in these
regions. These results demonstrate specific expression of the annetocin
gene in the subesophageal ganglia. A recent immunohistochemical study
has also demonstrated that annetocin-like immunoreactive neurons are
localized in the same regions (31), which is in agreement with our
data. In addition, it has been well established that the subesophageal
ganglion plays an important role in regulation of egg-laying behavior
and that the anterior part of the earthworm (rather than the posterior part) is involved in reproductive movement (32). Thus, localization of
the annetocin transcript in the subesophageal ganglia and restricted distribution of annetocin-like immunoreactivity in the anterior part
are compatible with the involvement of annetocin in reproductive behavior.
Although VP/OT-related peptides have been isolated from several
invertebrates, the organizational structure of the precursor polypeptide has never been characterized, except for that of the Lymnaea preproconopressin (20). Consequently, the molecular evolution or diversity of the invertebrate VP/OT superfamily peptides and the phylogenetic relationships between vertebrates and
invertebrates remain to be clarified. This is the first report of the
characterization of cDNA encoding an invertebrate OT-related
peptide, annetocin isolated from the earthworm E. foetida.
The preproannetocin was found to consist of a signal peptide, annetocin
(flanked by a Gly C-terminal amidation signal and a Lys-Arg dibasic
endoproteolytic sequence), and a neurophysin domain. Of particular
significance is that 14 cysteine residues that play a crucial role in
constructing the correct tertiary structure of a neurophysin are
completely conserved in the Eisenia neurophysin domain. All
cysteines are positioned almost identically to those of the known
neurophysin domains. These are all typical characteristics of the VP/OT
superfamily precursors, leading to the conclusion that annetocin is a
member of the VP/OT superfamily. This is the first evidence for the
presence of the VP/OT superfamily in Annelida, supporting the
presumption that the ancestral gene encoding the VP/OT superfamily
preprohormones with the principal structure was present in the stem
group Archaemetazoa, from which invertebrates diverged ~600 million
years ago (4, 20).
We also observed, by in situ hybridization, the localized
expression of the annetocin gene in ~10 specific neurons located relatively symmetrically in the subesophageal ganglia, demonstrating the specific expression of the annetocin gene in the earthworm central
nervous system. Combined with findings obtained by an immunohistochemical study of annetocin (31), these results support the
suggestion that annetocin serves as a neuropeptide regulating reproductive behavior and indicate that the principal regulatory mechanism in reproductive function and the structure of the OT-related peptide are conserved between invertebrates and vertebrates. Also of
interest is how the annetocin gene expression is regulated. In mammals,
multiple transcription factors and hormone receptors are involved in
the regulation of expression of the VP (33-36) and OT (37-43) genes.
Although the regulation mechanism of the invertebrate VP/OT superfamily
peptide gene has yet to be determined, the involvement of annetocin in
the control of reproductive events that are thought to be periodical
suggests that expression of the annetocin gene is regulated both
positively and negatively by several inducible transcription factors.
Otherwise, regulatory expression of an annetocin receptor gene may play
a major role in the induction of egg-laying behavior. Investigation of
the annetocin gene promoter region could provide important information about the regulation mechanism of annetocin gene expression. To identify the gene structure and promoter components, genomic DNA analyses of the annetocin gene are in progress.
Annetocin is structurally and functionally related to the OT family
peptides. It has been revealed that the organizational structure of the
annetocin precursor also has the characteristics of the OT family
rather than the VP family since a copeptin(-like) domain (or a
C-terminal extended domain of a neurophysin) that is present in the VP
family precursor is absent in proannetocin. On the other hand, the
precursor of structurally VP-related Lys-conopressin of
Lymnaea has been shown to possess a C-terminal extended
domain (20). These findings might lead to the speculation that
invertebrate precursors are also divided into two types, the VP and OT
family precursors, on the basis of the structural organization, similar to the vertebrate precursors. Nevertheless, this classification is
unlikely to be true of the invertebrate VP/OT superfamily because invertebrates are believed to contain only one VP/OT-related peptide. Van Kesteren et al. (15) showed that Lymnaea
Lys-conopressin, structurally related to VP, exhibits an oxytocin-like
activity and that only a single VP/OT superfamily gene is present.
Similar data have been obtained in our study of the physiological
effects of VP/OT-related peptides on the
leech,2 demonstrating that
annetocin induces egg-laying behavior to almost the same extent as
Lys-conopressin that was isolated from the leech (17). These data
support the notion that the amino acid at position 8 is not essential
for physiological functions of the invertebrate VP/OT superfamily
peptides (15). Thus, classification of the invertebrate VP/OT-related
precursors into the VP or OT family on the basis of the physiological
function of the peptide and the residue at position 8 is impossible.
Taken together, it appears most likely that the apparent OT-related
structure of the annetocin precursor was generated probably due to a
point mutation in the ancestral gene, causing incidental interruption of the precursor sequence immediately after the fourteenth cysteine, which was not a selective pressure.
However, the possibility of unknown structural generality for
precursors of the invertebrate VP/OT-related peptide cannot be
excluded. Identification of structures of precursor polypeptides from
other invertebrates including Aplysia, octopus, and leech would contribute to the establishment of universal characteristics of
VP/OT-related precursors of invertebrates or at least in the same phylum.
Proannetocin shows 37.4-45.8% amino acid homology to the VP/OT
superfamily prohormones. Unexpectedly, the amino acid sequence of
proannetocin is not so highly homologous to that of another invertebrate prohormone, Lymnaea proconopressin. These data
indicate the interphyletic high molecular diversity of the invertebrate VP/OT superfamily peptide precursors. Thus, to understand the invertebrate molecular diversity of the VP/OT superfamily and the
phylogenetic relationship between vertebrates and invertebrates, investigation of intraphyletic molecular evolution is necessary. Comparative studies of the VP/OT superfamily prohormones also revealed
that Arg23, Gly34, Glu67,
Pro73, and Gly93 plus the 14 cysteines are
conserved in all neurophysin domains, despite amino acid diversity
among species, as shown in Fig. 3. These findings suggest that the 14 cysteines and the amino acids described above are strictly involved in
the function(s) of the Eisenia neurophysin common to
vertebrates or that substitutions of these amino acids can be a
critical selective pressure. It is well known that specific disulfide
bridges formed by highly conserved cysteine residues are responsible
for functional conformation of the vertebrate neurophysin requisite for
interaction with the hormone peptide (27-30). This suggests that the
neurophysin of the annetocin precursor can construct a conformation
principally similar to those of vertebrates. Recently, the crystal
structure of the complex of bovine neurophysin with oxytocin has been
determined, revealing amino acid residues in neurophysin and oxytocin
essential for electrostatic and multiple hydrogen bonding interactions
with each other (28-30). In particular, the In this study, we have shown the first characterization of cDNA
encoding the precursor of the annelid VP/OT superfamily peptide, annetocin. Our data have provided the first evidence for conservation of the structural organization of the VP/OT superfamily precursor in
annelids and also have enabled the investigation of the invertebrate VP/OT superfamily precursors in greater detail, indicating that an
invertebrate neurophysin can interact with the nonapeptide owing to the
presence of amino acid residues essential for oxytocin-neurophysin complex formation.
INTRODUCTION
Top
Abstract
Introduction
References
The invertebrate VP/OT-related peptides and species from which the
peptide was characterized (13-18)
EXPERIMENTAL PROCEDURES
RESULTS
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Fig. 1.
Nucleotide sequence and deduced amino acid
sequence of the annetocin cDNA. The annetocin sequence is
underlined. The dibasic cleavage site is boxed.
The 14 conserved cysteine residues are indicated in black.
The AATAAA potential polyadenylation signals are indicated in
boldface.
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Fig. 2.
Northern blot analysis of total RNA
(A), schematic representation of the annetocin
precursor polypeptide (B), and hydropathy plot
analysis of the predicted precursor (C). In
A, total RNA was extracted from the anterior part of the
earthworm, and ~25 µg of RNA was subject to Northern blot
hybridization using a DIG-labeled annetocin cDNA probe. RNA
molecular markers are shown on the left in kilobases (kb).
In B, the hydrophobic leader sequence is labeled
S. The annetocin sequence and neurophysin-like domain are
represented by the hatched and black
bars, respectively. The endoproteolytic sites are shown as
black lines. In C, the plot was
generated according to the method of Kyte and Doolittle (21) using
GENETYX-MAC software.
3,
1 rule (23). The annetocin sequence CFVRNCPTG, flanked by Gly as
an amidation signal and the typical dibasic processing sequence
Lys-Arg, was found to follow a signal sequence. The following moiety
showed the properties of a neurophysin domain. The striking feature of this neurophysin domain is that 14 cysteine residues are positioned almost identically to those in the neurophysin domains of other VP/OT-related prohormones (Fig. 3). This
result suggests that an essential tertiary structure of the
Eisenia neurophysin domain is also highly conserved because
disulfide pairings by 14 cysteine residues in the neurophysin domain
are believed to play a significant role in conformation essential for
interaction with a hormone peptide (27-30). A copeptin-like region
(another typical domain in the prohormones of the VP family, but not in
those of the OT family) is entirely absent in preproannetocin since a
TGA stop codon is found immediately after the fourteenth cysteine.
These findings indicate that the principal architecture of VP/OT
superfamily preprohormones is also highly conserved in Annelida and
that a precursor polypeptide of annetocin is structurally closer to
precursors of OT-related peptides since a C-terminal extension is not
seen in the preprohormones. In addition, no consensus sequence for any
other post-translational modification such as glycosylation or
phosphorylation was found in the precursor polypeptide.
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Fig. 3.
Comparison of amino acid sequences of the
VP/OT superfamily prohormones. The proannetocin is aligned with
the prohormones of the Lymnaea conopressin (20), bovine
vasopressin (24) and oxytocin (25), chicken vasotocin (8), lungfish
vasotocin and [Phe2]mesotocin (10), white sucker
vasotocin II (26) and isotocin I (7), and lamprey vasotocin (9). The
nonapeptide sequences and the 14 highly conserved cysteine residues are
indicated in boldface, and the 14 cysteines are also
boxed. Asterisks denote identical amino acid
residues among all precursors. Gaps marked by hyphens were
inserted to optimize homology.
Amino acid sequence homology of proannetocin to other
vasopressin/oxytocin superfamily prohormones
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Fig. 4.
Localization of the annetocin
transcript. In situ hybridization of an antisense
DIG-labeled 53-mer oligonucleotide probe to fixed 6-µm sections of
the earthworm anterior detected the presence of annetocin mRNA in
the cytoplasm of neurons located in the subesophageal ganglia.
Arrows indicate positively stained neurons. Scale
bar = 20 µm.
DISCUSSION
-carboxyl group of
Glu59 in the bovine prohormone (corresponding to
Glu67 in proannetocin) has been shown to participate in an
electrostatic interaction with the N-terminal amino group of oxytocin.
Moreover, involvement of the side chain of Arg20
(corresponding to Arg23) in the optimal location of the
-carboxyl group of Glu59 has been proposed (28, 30).
Conservation of these amino acid residues in all prohormones suggests
that the Eisenia neurophysin can bind to annetocin in a
manner similar to the oxytocin-bovine neurophysin complex. In addition,
a homology modeling study generated the putative tertiary structure of
the annetocin-Eisenia neurophysin complex, quite similar to
that of the oxytocin-bovine neurophysin complex.3 In this putative
complex, most hydrogen bonds corresponding to those of the
oxytocin-bovine neurophysin complex and the electrostatic interaction
of Glu67 with the
-amino group of annetocin were
observed. Previously, Tyr at position 60 or 61 in the vertebrate
prohormones was thought to be requisite for binding to the nonapeptide
(44), and the Lymnaea neurophysin was proposed to fail to
interact with Lys-conopressin because the sequence Asn-Tyr-Leu in most
vertebrate neurophysins has been replaced by an N-linked
glycosylation sequence Asn-Asp-Ser (sequence 62-64 in the
Lymnaea prohormone) (20). However, the crystal structure of
the complex has shown that Tyr61 is not involved in the
interaction between a neurophysin and a nonapeptide hormone (29, 30).
This indicates that the invertebrate neurophysins can also be
associated with the peptides. The important amino acid in oxytocin
essential for interaction is Tyr2, which has an aromatic
ring situated in a "tight pocket" (29, 30). Annetocin contains a
phenylalanine instead of tyrosine at this position (Table I). A side
chain of phenylalanine has aromatic or hydrophobic characteristics
quite analogous to those of tyrosine, although it is unable to form a
hydrogen bond, implying that annetocin may possess the potential to
bind to the neurophysin in a manner similar to oxytocin. A homology
modeling study also showed the possibility of such tight packing of the
Phe2 ring of annetocin.3 In combination, these
phenomena suggest that the nonapeptide binding property of a
neurophysin is also conserved in invertebrates. To investigate the
function of the Eisenia neurophysin and the binding mode for
the annetocin-neurophysin complex, characterization of the
Eisenia neurophysin molecule is now in progress.
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FOOTNOTES |
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* The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
The nucleotide sequence reported in this paper has been submitted to the DDBJ/GenBankTM/EBI Data Bank with accession number AB014478.
§ To whom correspondence should be addressed. Tel.: 81-75-962-3743; Fax: 81-75-962-2115; E-mail: Hiroyuki_Minakata{at}suntory.co.jp.
2 Y. Fujino, T. Nagahama, T. Oumi, K. Ukena, F. Morishita, Y. Furukawa, O. Matsushima, M., Ando, H. Takahama, H. Satake, H. Minakata, and K. Nomoto, manuscript in preparation.
3 H. Satake, K. Kami, and H. Minakata, unpublished results.
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ABBREVIATIONS |
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The abbreviations used are: VP, vasopressin; OT, oxytocin; RACE, rapid amplification of cDNA ends; PCR, polymerase chain reaction; DIG, digoxigenin; PBS, phosphate-buffered saline.
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REFERENCES |
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