(Received for publication, October 2, 1995; and in revised form, December 27, 1995)
From the
Purification of a material immunoreactive to an antiserum
against the C-terminal part of the oxytocin (Pro-Leu-Gly-amide) and
present in the central nervous system of the Pharyngobdellid leech Erpobdella octoculata was performed by reversed-phase high
performance liquid chromatography combined with both enzyme-linked
immunosorbent and dot immunobinding assays for oxytocin. The amino acid
sequence of the purified peptide (Ile-Pro-Glu-Pro-Tyr-Val-Trp-Asp) was
established by Edman degradation and confirmed by electrospray mass
spectrometry measurement. When injected in leeches, purified or
synthetic peptides exert an anti-diuretic effect, the most effective
ranged between 10 pmol and 1 nmol. They provoked an uptake of water
1-2 h post-injection. Furthermore, electrophysiological
experiments conducted in the leech Hirudo medicinalis revealed
an inhibition of the potency of Na conductances of
leech skin by this peptide. Immunocytochemical studies with an
antiserum against synthetic oxytocin-like molecule provided the
cytological basis for existence of a neuropeptide, since large amounts
of immunoreactive neurons were detected in the central nervous systems
of E. octoculata. The purified molecule is both different to
peptides of the oxytocin/vasopressin family and is a novel neuropeptide
in the animal kingdom. It was named the leech osmoregulator factor
(LORF).
An identification of the proteins immunoreactive to an
antiserum against oxytocin performed at the level of both central
nervous systems extracts and in vitro central nervous
system-translated RNA products indicated that in the two cases, a
single protein was detected. These proteins with a molecular masses of,
respectively, 34 kDa (homodimer of 17 kDa) for the central nervous
systems extracts and
19 kDa for in vitro central nervous
system-translated RNA products were not recognized by the antiserum
against MSEL- and VLDV-neurophysin (proteins associated to oxytocin and
vasopressin), confirming that LORF did not belong to the
oxytocin/vasopressin family.
In annelids, the central nervous system (CNS) ()of Hirudinae is known to influence water balance (Rosca et
al., 1958; Czechowicz, 1968; Kulkarni and Nagabhushanam, 1978;
Malecha, 1983). In the rhynchobdellid leech Theromyzon tessulatum genital maturity is concomitant with a phasis of water retention
reflected by an increase in mass of the animals and correlated to a
c
lomic accumulation of yolk proteins (Baert et al.,
1991, 1992). This animal was used for a study of the control of water
balance.
Angiotensin II-amide (Salzet et al., 1995), GDPFLRF-amide (Salzet et al., 1994), and lysine-conopressin (Salzet et al., 1993a) have been isolated from CNS of the Pharyngobdellid leech Erpobdella octoculata. They generated when injected in T. tessulatum a decrease in mass of the injected animals, expressing a diuretic effect. On the other hand, an injection of an antiserum directed against oxytocin (OT) provoked a loss of mass in the injected leeches (Malecha et al., 1989a), pointing to an anti-diuretic role for substance(s) immunoreactive to anti-OT.
In E. octoculata, it is known that an OT-like
material is found in large amounts in the sex segmental ganglia (sex
SG), and that an extract of sex SG exerts an anti-diuretic effect when
injected in leeches (Salzet et al., 1993c). Cell counts of
OT-like cells in immature and mature animals indicated that the number
of immunoreactive cells was higher in immature specimens. Furthermore,
radioimmunoassays showed an amount of OT-like material 3-fold higher in
immature than in mature leeches. A biochemical study of an extract of
sex SG of mature E. octoculata demonstrated the presence of
two zones immunoreactive to a-OT. On the other hand, in immature E.
octoculata, an additional zone bearing 80% immunoreactivity
to a-OT was detected. For this additional zone in immature animal,
hypothesis of the presence of a possible fragment of the OT-like
precursor was given (Salzet et al., 1993c). In supernumerary
neurons of sex SG, OT-like material is colocalized with four RF-amide
peptides, i.e. FMRF-amide, FM(O)RF-amide, FLRF-amide, and
GDPFLRF-amide. FMRF-amide and GDPFLRF-amide as OT-like substance(s) are
involved in osmoregulation (Salzet et al., 1994).
In this study, we now report the isolation and the characterization of an OT-like peptide from CNS of the Pharyngobdellid leech E. octoculata using HPLC purification procedures, automatic Edman degradation and electrospray mass spectrometry analysis, electrophysiological experiments, and immunocytochemical studies performed with an antiserum against synthetic oxytocin-like peptide. Finally, an identification of CNS proteins immunoreactive to an antiserum against OT at the level of both CNS extracts and in vitro CNS-translated products was also undertaken. This work demonstrated the existence of a novel neuropeptide involved in the process of osmoregulation. This molecule named leech osmoregulator factor (LORF) is unique in the animal kingdom.
Mature Rhynchobdellid leeches T. tessulatum were used in this study as a bioassay according to Salzet et al. (1995).
The fractions containing the immunological material were further applied to the same column with a shallower gradient of acetonitrile in acidified water from 0 to 15% in 10 min and from 15 to 45% in 40 min at a flow rate of 1 ml/min. After a 20-fold concentration by freeze drying, fraction aliquots of 0.5 µl were tested using DIA.
All HPLC purifications were performed with a Beckman Gold HPLC system equipped with a photodiode array detector Beckman 168.
All injected animals were kept at room temperature. To estimate the effect of an injection, leeches blotted on tissue paper were weighed to the nearest 0.1 mg at various time intervals following the injection (1, 2, 4, and 6 h). The change in body mass of the animals between the beginning of the experiment and the time of weighing was registered. Responses were expressed as percentages of mass variation (means ± S.D.). The efficiency of the product was determined by its capacity to elicit a variation of mass significantly different from that registered in controls.
Statistical analysis of data was done according to Salzet et al. (1993a). The confidence limit of the relative mean variation of mass was obtained according to Cochran(1977).
The specificity of the antiserum (a-LORF) was tested on consecutive sections mounted on different slides by preadsorbing this antiserum overnight at 4 °C with the homologous antigen at a concentration of 500 µg/ml pure antiserum.
Figure 1:
HPGPC elution profile of a
C Sep-Pak prepurified extract of 1000 central nervous
systems of E. octoculata. After solid phase extraction on
Sep-Pak C
cartridges, the fraction eluted by 50%
acetonitrile in acidified water (0.1% trifluoroacetic acid), containing
the oxytocin-like material, was loaded onto a HPGPC (Ultraspherogel,
7.5
300 mm, SEC200, Beckman) column. Elution was performed with
30% acetonitrile at a elution rate of 1 ml/min. The oxytocin-like
material was detected on aliquots of each fraction by the oxytocin-DIA. Bars indicate the immunoreactive material, and arrowheads, the eluted positions of molecular mass markers (a, hirudin; b, adrenocorticotropic hormone; c, angiotensin II; d,
tryptophan).
Figure 2:
Effect on the body mass of stage 3B T.
tessulatum of the injection (10 µl/leech) of 1 pmol of
purified oxytocin-like material from the immunoreactive zones (Z1, Z2
or Z3) detected after HPGPC separation. Controls received deionized
water. The loss of mass was determined at different times (1, 2, 4, and
6 h) after injection. Results are expressed as means ± S.D. Data
are from 20 injected animals, at each time point (experiment was
performed 4 times). Groups with an asterisk differ
significantly from controls ( =
0.05).
Figure 3:
Reversed-phase HPLC separation of
oxytocin-like material from the HPGPC column. The active and
immunoreactive fraction eluted from HPGPC column was loaded onto a
C-peptide-protein column (250 mm
4.6 mm; Vydac).
Elution was performed with a discontinuous linear gradient of
0-45% acetonitrile in acidified water (0.1% trifluoroacetic acid)
for 30 min, followed by a gradient of 45-80% acetonitrile in
acidified water (0.1% trifluoroacetic acid) for 10 min at a flow rate
of 1 ml/min. The oxytocin-like material was detected on aliquots of
each fraction by DIA. The bar indicates the immunoreactive
material.
Figure 4:
Final purification of the oxytocin-like
peptide. After three successive reversed-phase HPLC steps, the
oxytocin-like peptide was purified to homogeneity on a
Creversed-phase column (250 mm
2 mm; Beckman).
Elution was performed with a linear gradient of 0-60%
acetonitrile in acidified water (0.1% trifluoroacetic acid) for 60 min
at a flow rate of 0.3 ml/min. The asterisk indicates the peak
containing the purified oxytocin-like peptide, which was subjected to
an automated Edman degradation.
Figure 5:
Effect of the injection (10 µl/leech)
of synthetic (1 pmol) or of purified (1 pmol) LORF on the body mass of
the leech T. tessulatum at stage 3B. Controls received
deionized water. The loss of mass was determined at different times (2,
4, 6, and 8 h) after injection. Results are expressed as means ±
S.D. Data are from 40 injected animals at each time point (experiment
was performed four times). Groups with an asterisk differ
significantly from controls ( =
0.05).
Figure 6:
Effect of the injection of synthetic LORF
(10 µl/leech) at different concentrations (1-100 fmol,
1-100 pmol, and 1 nmol) on the body mass of the leech T.
tessulatum at stage 3B. Controls received deionized water. The
loss of mass was determined at different times (1, 2, and 4 h) after
injection. Results are expressed as means ± S.D. Data are from
40 injected animals at each time point (experiment was performed four
times). Groups with an asterisk differ significantly from
controls ( = 0.05).
Figure 7:
Transepithelial
Nacurrents measurement from the skin of leech Hirudo medicinalis. The tissue was clamped to zero with a
low-noise voltage clamp. Transepithelial resistance was calculated from
superimposed 10 mM-pulsed of 500 ms duration according to
Ohm's law. Amiloride or oxytocin-like peptide was applied to the
serosal solution.
Shortly after
serosal application of oxytocin-like peptide (5 µM), I decreased by 18.15 ± 0.94% (n = 3). 28.19 ± 2.74% of the amiloride-sensitive
portion of the short circuit was inhibited by oxytocin-like peptide,
and 34.8 ± 9.5% of the whole Na
-mediated
currents were blocked.
Figure 8: Frontal section through segmental ganglia of the nerve cord of E. octoculata, treated with anti-LORF preadsorbed (a and c) or not (b) with the homologous antigen (indirect immunoperoxidase). Preadsorption of the antiserum with LORF abolished the immunostaining. Bars = 50 µm.
Figure 9:
HPGPC elution profile of a protein extract
of 400 central nervous systems from E. octoculata. Elution
rate: 0.3 ml/min. Arrows indicate eluted positions of
standards in identical conditions of column and elution (a,
trypsin inhibitor (20 kDa); b, -lactalbumin (14.4 kDa); c, hirudin (7 kDa); d, oxytocin (1 kDa)). Zones
immunoreactive to a-OT are denoted by a bar (Za and Zb). Inset, photographs represent Western blot
analyses with a-OT of proteins contained in Za after SDS-PAGE in
nonreducing (A) and reducing (B) conditions. Small arrows indicate position of proteins immunoreactive to
a-OT; arrowheads on left of the immunoblot indicate
molecular mass standards.
Figure 10:
HPGPC elution profile of translated total
RNA extracted from 400 central nervous systems from E. octoculata. Elution rate: 0.3 ml/min. Arrows indicate eluted
positions of standards in identical conditions of column and elution (a, trypsin inhibitor (20 kDa); b, -lactalbumin
(14.4 kDa); c, hirudin (7 kDa); d, oxytocin (1 kDa)).
Zone immunoreactive to a-OT is denoted by a bar (Zc). Inset, photograph represents a Western blot analysis with a-OT
of proteins contained in Zc after SDS-PAGE in reducing conditions. Small arrows indicate position of proteins immunoreactive to
a-OT. Arrowheads on left of the immunoblot indicate
molecular mass standards.
After reversed-phase HPLC purification, the sequence of an OT-like peptide (IPEPYVWD) was established by a combination of automated Edman degradation and electrospray mass spectrometry measurement. Our data revealed that the 8-residue peptide present in the CNS of the leech E. octoculata is a novel neuropeptide, structurally different from peptides of the OT/VP family. This result constitutes the first report of such a peptide in the animal kingdom.
The isolation of this peptide with an antiserum against OT could be explained by the antiserum used. It was directed against the C-terminal part of OT (PLG-amide) and more precisely against dipeptides PL (30%) or PI (20%). PI is present in the purified OT-like peptide (IPEPYVWD) at the level of the N-terminal sequence. Similar results were also obtained in the mollusk Lymnaea stagnalis with an anti-lysine-vasopressin antiserum. The lysine-vasopressin antiserum directed against the sequence PKG-amide recognized the SKPFLRF-amide, peptide of the RF-amide family (De With et al., 1993). Although these results led us to think that the peptide isolated from E. octoculata CNS is not really an OT-like peptide, three lines of evidence established our results.
The use of another a-OT
characterized by Tramu et al. (1983), which recognized both
the N-terminal and the C-terminal parts of OT, gave identical results
(data not shown). Moreover, from preliminary experiments conducted on
the biological activity of the purified peptide from E.
octoculata, it appears that this purified peptide is involved in
the control of the hydric balance. Injection of 10 pmol, 100 pmol, or 1
nmol of synthetic OT-like peptide provoked an uptake of water in
injected T. tessulatum. Furthermore, electrophysiological
experiments conducted in H. medicinalis establish the
inhibition of potency sodium conductance of leech skin by the OT-like
peptide. Leech skin has proven to be a valuable model for the study of
ion transport in invertebrate tight epithelia (Weber et al.,
1993, 1995). It is known that Na transport in leech
skin can be modulated by the second messenger cAMP via an increase in
the number of Na
channels. However, in leech skin
oxytocin-like peptide failed to activate the cAMP second messenger
system, as could be seen by a decreasing effect on transepithelial
short circuit current. Oxytocin-like peptide inhibited the currents
through amiloride-sensitive or amiloride-insensitive Na
conductances. At present, it remains unclear by which mechanism
oxytocin-like peptide enroles its inhibiting potency on Na
conductances of leech skin. Two mechanisms seem to be possible:
1) a direct action of the peptide on the channel protein from the
outside of the cell, or 2) coupling of oxytocin-like peptide to a
membrane bound receptor and subsequent signal transduction to the
inside of the cell, followed by a regulation of Na
channels from within the cell. Finally, immunocytochemical
studies performed with an antiserum raised against synthetic
oxytocin-like peptide indicated a high specific immunostaining of
neurons in segmental ganglia of the nerve cord. For all these reasons,
the purified oxytocin-like peptide was named the leech osmoregulator
factor (LORF).
The LORF sequence (IPEPYVWD) is included within the
N-terminal part of a respiratory pigment, the myohemerythrin (protein
of 14 kDa) of the sipunculid Themiste zostericola (Klippenstein et al., 1976): GWDIPEPYVWDESFRV . . . It
also presents 77% sequence homology with a fragment of the N-terminal
part of a yolk protein (
14 kDa), the ovohemerythrin of the leech T. tessulatum: YDIPEPFRWDESF . . . (Baert et al.,
1992). Several hypotheses can be advanced for these data. 1) LORF is a
novel neuropeptide; 2) it is a contaminant from hemerythrin present in
blood and c
lomic fluid. However, several findings argue in favor
of the first hypothesis, i.e. LORF is a novel neuropeptide.
First, immunocytochemical studies performed with an antiserum against
synthetic LORF revealed neurons immunoreactive to a-LORF in the nerve
cord. Moreover, some of these neurons reached projections in direction
to neurohemal area, indicating a neurohormonal role (Hagadorn, 1958;
Orchard and Webb, 1980) of LORF. Moreover, most of supernumerary
neurons of the sex segmental ganglia that were immunoreactive to a-OT
(Salzet et al., 1993c) are also immunoreactive to a-LORF.
Second, a-LORF and a-OT did not recognize the ovohemerythrin in Western
blot or in ELISA and a-ovohemerythrin, the LORF. Third, LORF is active
on osmoregulation of leeches. Our experiments, conducted at the protein
level with CNS extracts or RNA-translated products, indicated that the
OT-like precursor is a homodimer protein of
17 kDa in CNS extract
and a protein of
19 kDa in translated RNA. These proteins were not
recognized with a-(MSEL and VLDV)-neurophysin, associated proteins in
the case of OT or VP (Acher et al., 1985), confirming that
LORF is not a peptide of this family. No recognition of these proteins
by a-RF-amide was found, although these molecules are colocalized with
LORF in supernumerary neurons of sex SG (Salzet et al.,
1993c). Fourth, molecular mass of ovohemerythrin (14. 4 kDa) is
different to OT-like proteins.
According to its localization in sex segmental ganglia of the nerve cord, it could be postulated that this peptide could also act on reproduction. Selective ablations of either sex segmental ganglia or cerebroïde ganglia of T. tessulatum at stage 3A, stage just before water retention phasis, indicated that only the lack of cerebroïde ganglia blocks the water phasis retention and oogenesis. LORF could exert different biological activities in function of its localization in T. tessulatum CNS. However, further experimental testing are needed to conclude.
The whole of our results establish that LORF is a novel neuropeptide, never previously isolated in the animal kingdom. Further experimental testing with a-LORF would be performed either in other phyla of invertebrates or in vertebrates, in order to investigate the presence of this neuropeptide in course of evolution.