(Received for publication, April 18, 1994; and in revised form, October 21, 1994)
From the
Purification of a material immunoreactive to an antiserum against angiotensin II and present in the central nervous system of the pharyngobdellid leech Erpobdella octoculata was performed by reversed-phase high pressure liquid chromatography combined with both enzyme-linked immunosorbent assay and dot immunobinding assays for angiotensin II. Establishment of the amino acid sequence by Edman degradation, electrospray, and fast atom bombardement mass spectrometry measurements and enzymatic treatment by carboxypeptidase A indicated that this ``central'' angiotensin II-like material, the first one fully characterized in the animal kingdom, is an angiotensin II amide. This finding constitutes also the first biochemical characterization of a peptide of the angiotensin family in an invertebrate. Synthetic angiotensin II amide exerts, when injected in leeches, a diuretic effect and is, 1 and 2 h postinjection, 100-fold more potent than vertebrate angiotensin II.
An identification of the
proteins immunoreactive to an antiserum against angiotensin II
performed at the level of both central nervous system extracts and in vitro central nervous system-translated RNA products
indicated that in the two cases, two proteins were detected. Their
molecular masses, which were, respectively, 14 and
18 kDa for
the central nervous system extracts and
15 and
19 kDa for in vitro central nervous system-translated RNA products,
differ from that of angiotensinogen (
60 kDa), the precursor of
vertebrate angiotensin II.
In vertebrates, the renin-angiotensin system (RAS) ()has long been known to play a key role in body fluid
homeostasis (Gohlke et al., 1988). In this system, renin
cleaves the essentially inactive decapeptide angiotensin I (AI) from
angiotensinogen (A0). The further action by the angiotensin-converting
enzyme yields a simple 8-amino acid bioactive peptide, angiotensin II
(AII), the final product of this system. Nevertheless, the low amount
of renin in brain, spleen, lung, and thymus as compared with kidney,
adrenal, heart, testes, and submandibular gland suggests that, in
vertebrates, there are both RASs and non-RASs (Phillips et
al., 1993).
Angiotensin, which was first isolated and purified from plasma (Skeggs et al., 1956) has since been identified in many organs (Aguilera et al., 1981; Dzau, 1987; Hermann et al., 1982). The presence of ``central'' (brain) angiotensin has become widely accepted, notably with the finding of mRNA expression of angiotensinogen and renin in brain tissue (Lynch et al., 1986; Dzau et al., 1986).
In vertebrates, if peptides of the angiotensin family have been fully characterized, i.e. isolated and sequenced from extracts of kidney and skin (for a review, see Khosla(1985)), they have only been isolated from the brain. Indeed, despite the large volume of work with the brain peptide, no sequencing has been reported to date, and the question remains whether brain AII is identical to peripheral AII (Saavedra, 1992). If we consider the peptides of the angiotensin family isolated from the peripheral system, it is worth noting that the primary sequence of AI has been well preserved after the advent of the RAS except for variations at positions 1, 3, 5, and 9 (Khosla, 1985).
As for the existence of vertebrate endogenous brain angiotensins, evidence was given of the presence of AI, AII, and angiotensin III (AIII; fragment-(2-8) of AII), using a combination of HPLC and immunoassay (Hermann et al., 1982). AII, which comigrates with authentic AII, is the predominant peptide form of angiotensin found in the brain (Phillips et al., 1991). Its cellular localization has been reported (Saavedra, 1992). Nevertheless, it is worth mentioning that a high molecular weight AII (``big AII'') (Pohl et al., 1988; Phillips et al., 1991) has also been biochemically detected in the vertebrate brain.
The
physiological roles for peripheral and central vertebrate AII are
numerous (Saavedra, 1992), the best known being the fundamental role
that AII plays in the control of fluid balance. On the other hand,
little is known, to date, about this peptide in invertebrates except in Hirudinae. In this group of annelids, the internal medium of
freshwater leeches is hypertonic compared with the environment, which
leads to a constant osmotic water inflow compensated by the excretory
activity of the nephridia. Moreover, it has been demonstrated in two
sanguivorous leeches, Hirudo medicinalis (Zerbst-Boroffka,
1973) and Theromyzon tessulatum (Van der Lande, 1983), that a
profound diuresis, expressed by a loss of body mass, occurs during the
hours following a blood meal in order to eliminate water and ions in
excess in the ingested blood and thus to concentrate blood cells. One
of the substances involved in this physiological process would be AII,
which indeed exerts a diuretic effect when injected in the leech T.
tessulatum (Salzet et al., 1992a). Biochemical
identification of the central AII-like peptide in T. tessulatum revealed in HPLC a comigration of this peptide with vertebrate
AII, which suggests that it is structurally close to AII (Salzet et
al., 1993b). Nevertheless, the low levels of AII-like material in T. tessulatum, found at a maximum in mature animals (10
fmol/central nervous system) did not permit its chemical
characterization. Since these levels are
15-fold higher in mature
specimens of another leech, Erpobdella octoculata, this animal
was used for the biochemical characterization of the central AII-like
peptide in leeches.
In this study, we report the isolation and characterization of an AII-like peptide from the central nervous system (CNS) of the pharyngobdellid leech E. octoculata using HPLC purification procedures, Edman degradation, mass spectrometry analyses, and enzymatic treatment. It is a carboxyl-terminally amidated octapeptide named AII amide. This is the first report on the characterization of both an AII-like peptide in an invertebrate and of a central peptide of the angiotensin family in the animal kingdom. This AII amide has a very potent diuretic effect and is thus involved in the control of leech hydric balance. Its high potency, compared with AII, is demonstrated. An identification of CNS proteins immunoreactive to an antiserum against AII at the level of both CNS extracts and in vitro CNS-translated products is presented. In addition, the significance of the existence of an AII amide in the angiotensin family is discussed in an evolutionary context.
After anaesthesia in 0.01% chloretone, animals were pinned out, dorsal side up, in leech Ringer's solution (Muller et al., 1981), and central nervous systems (CNS) were excised, immediately frozen in liquid nitrogen, and stored at -70 °C until use.
The fractions that contained 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 Beckman 168 photodiode array detector.
Leeches received an aqueous solution of either synthetic peptide corresponding to the isolated AII amide from E. octoculata (Neosystem) (lots 1-4) or synthetic AII (Sigma) (lots 5-8) at four different doses (0.01, 0.1, 1, and 10 nmol). Controls (lot 9) received deionized water. 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).
For
immunoprecipitation, 500 µl of supernatant were incubated overnight
under agitation at 4 °C in an immunoprecipitin complex (protein
A-Sepharose (Pharmacia Biotech Inc.) associated to a-AII) prepared as
follows. Five mg of protein A-Sepharose were suspended in
phosphate-buffered saline (PBS; 50 mM phosphate buffer, pH
7.4, 150 mM NaCl). The gel was allowed to swell for 1 h at
room temperature and was then washed briefly in PBS by centrifugation
4-fold. Ten µl of undiluted a-AII and 40 µl of PBS were then
successively added to the gel. After a 90-min incubation under gentle
agitation at room temperature, five washings in PBS were carried out by
centrifugation. Supernatant of CNS extracts (500 µl) was then added
to the immunoprecipitin complex. Incubation was conducted overnight
under agitation at 4 °C. The immunoprecipitin complex was washed
5-fold in PBS and then boiled in 1 SDS loading dye (bromophenol
blue 5%) supplemented with 5%
-mercaptoethanol. Proteins were
subjected to a Western blot analysis. For controls, the same
experimental procedure was employed except that a-AII was preadsorbed
by synthetic AII (100 µg of AII (Sigma)/ml of undiluted antiserum)
before being coupled to protein A-Sepharose.
Figure 1:
Reversed-phase HPLC separation of an
acidic 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 angiotensin II-like
material was loaded onto a C
-peptide protein column (250
4.6 mm; Vydac). Elution was performed with a discontinuous
linear gradient of 0-15% acetonitrile in acidified water (0.1%
trifluoroacetic acid) for 10 min, followed by a gradient of
15-45% acetonitrile in acidified water (0.1% trifluoroacetic
acid) for 30 min at a flow rate of 1 ml/min. The angiotensin II-like
material was detected on aliquots of each fraction by the angiotensin
II-DIA. The bar indicates the immunoreactive
material.
The
immunoreactive zone containing the AII-like material from 4000 CNS was
analyzed on the same column with a shallower gradient (data not shown).
Using DIA, three fractions (F1, F2, and F3) eluted between 26 and 28
min and immunoreactive to a-AII were resolved. However, ELISA indicated
that the major amount of AII-like material (98 ± 26 fmol/CNS)
was contained in the F2 fraction. F2 was further purified to
homogeneity by two successive and identical reversed-phase
chromatographies using the conditions described under ``Materials
and Methods.'' An immunoreactive peak to a-AII (Fig. 2) was
obtained at a retention time of 31 min. In the same conditions,
synthetic AII eluted from the column at a retention time of 31.8 min. A
quantification by ELISA indicated that we purified to homogeneity 87
± 12 fmol of AII-like material/CNS (final recovery of 50%).
Figure 2:
Final purification of the angiotensin
II-like peptide. After three successive reversed-phase HPLC steps, the
angiotensin II-like peptide was purified to homogeneity on a C reversed-phase column (250
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 angiotensin II-like peptide, which was subjected to an
automated Edman degradation.
Figure 3: Electrospray mass spectrum of the purified angiotensin II-like peptide from the central nervous system of E. octoculata. Peaks at m/z = 348.9 and m/z = 523.8 are multiply charged ions with three or two charges corresponding to a mass of 1044.3 ± 1.4 Da. The peak at m/z = 571.3 corresponds to an internal mass standard (gramicidin).
A series of experimental results demonstrated that the carboxyl
terminus of the peptide is blocked by an amidation and thus that the E. octoculata AII-like peptide is an AII amide. First, during
Edman degradation, the sequencing yield obtained with purified AII-like
peptide was 82% versus 95.4% with synthetic AII. Second,
treatment of the purified AII-like peptide with carboxypeptidase A did
not affect the retention in HPLC. In contrast, after treatment of
synthetic AII with carboxypeptidase A, synthetic AII eluted earlier
(29.5 min versus 31.8 min). Third, the coinjection of purified
AII-like peptide and synthetic AII amide in an ODS C reversed-phase HPLC column revealed, after elution, a single peak
at a retention time of 31 min. In contrast, in the same conditions of
column and gradient, the coinjection of purified AII-like peptide and
synthetic AII revealed, after elution, two peaks at a retention time of
31 and 31.8 min, respectively. Fourth, FABMS measurements (Fig. 4) gave for the purified AII-like peptide a molecular mass
of 1045.6 Da and for synthetic AII a molecular mass of 1046.6 Da.
Figure 4: Fast Atom Bombardment mass spectra of the purified angiotensin II-like peptide from the central nervous system of E. octoculata (a) and of synthetic AII (b).
Figure 5:
Effect of the injection (10 µl/leech)
of synthetic angiotensin II (AII) or of synthetic angiotensin II amide
(AIIa), at different concentrations, 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 80 injected animals at each time point (experiment was performed 4
times). Groups with an asterisk differ significantly from
controls ( = 0.05).
Comparative analysis also indicated that the dose of AII required to obtain a similar response, i.e. the same loss of mass significantly different from that of controls, had to be, compared with AII amide, 10-fold higher at 4 h postinjection or 100-fold higher at 1 h and 2 h postinjection.
Thus, these experimental results revealed that the amidated form of AII is far more potent than the nonamidated form of AII.
Figure 6:
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, angiotensin II (1 kDa)). Zone
immunoreactive to anti-angiotensin II (a-AII) is denoted by a bar (Z1). Inset photograph represents a Western blot analysis
with a-AII of proteins contained in Z1. Small arrows indicate
position of proteins immunoreactive to a-AII, arrowheads to
the left of the immunoblot indicate molecular mass
standards.
Figure 7: Immunoblot analysis after immunoprecipitation, with anti-angiotensin II (a-AII) preadsorbed (a) or not preadsorbed (b) with synthetic angiotensin II of central nervous system extracts from E. octoculata. Total proteins were separated by SDS-polyacrylamide gel electrophoresis, transferred to a polyvinylidene difluoride membrane, and reacted with a-AII. Small arrows indicate position of proteins immunoreactive to a-AII; arrowheads to the left indicate molecular mass standards.
Figure 8:
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, angiotensin II (1 kDa)). Zone immunoreactive to
anti-angiotensin II (a-AII) is denoted by a bar (Z2). Inset photograph represents a Western blot analysis with a-AII
of proteins contained in Z2. Small arrows indicate position of
proteins immunoreactive to a-AII. Arrowheads to the left of the immunoblot indicate molecular mass
standards.
The central AII-like material present in the CNS of E. octoculata was characterized as an AII amide. This result constitutes the first report of the presence of a peptide of the angiotensin family in an invertebrate. It also represents the first central peptide of the angiotensin family fully characterized in the animal kingdom. Indeed in vertebrates, in contrast to peripheral angiotensin, which has been characterized in different classes, sequencing for a central angiotensin has never been provided.
From a sequence comparison of the first eight amino acids of peripheral AI isolated from skin or kidney in different classes of vertebrates (Khosla, 1985), a sequence that corresponds to AII, it emerges that this sequence has been well preserved since the advent of RAS except for minor variations at positions 1, 3, and 5. In position 1, the N-terminal residue is either Asp or Asn. Moreover, two cases are of interest: the peptide of the skin of the australian frog Crinia georgiana, where the N terminus has been elongated by the tripeptide Ala-Pro-Gly-, and the peptide of the kidney of the snake Elaphe climocophora, where the N-terminal residue is acylated. In positions 3 and 5, the valine residue is always found except in the skin of C. georgiana, where it has been replaced with Ile at position 3, and in the kidney of some mammals (human, horse, pig, mice, rat, rabbit, dog, guinea pig), where it has been replaced with Ile at position 5.
As vertebrate brain AII comigrates in HPLC with authentic AII (Phillips and Stenstrom, 1985), the molecule of the angiotensin family characterized in E. octoculata, which did not migrate at the same retention time as authentic AII, differs from vertebrate brain AII. Indeed by a combination of ESMS and FABMS, enzymatic treatment with carboxypeptidase A and coelution of the purified and the synthetic AII amide, we established that the AII-like peptide isolated from the CNS extracts of E. octoculata presents a carboxyl-terminal amidation. The presence of a molecule of AII amide in an animal belonging to the oldest group of metazoan Coelomates (annelids) leads us to think that the molecule of central AII has been well conserved in the course of evolution. Of particular interest is a comparison of the sequence of the leech molecule of the angiotensin family, characterized from the CNS, with that of the first eight amino acids of peripheral AI isolated from the kidney in different classes of vertebrates. It reveals an almost complete identity in the structure between the leech molecule and the first eight amino acids of peripheral AI of human, horse, pig, mice, rat, rabbit, dog, and guinea pig, the two sequences differing only by the presence of a carboxyl-terminal amidation in leeches. Moreover, compared with avians, reptiles, amphibians, and teleosts (Khosla, 1985), 6 out of 8 amino acids are constant; only amino acids in position 5 and sometimes in position 1 differed.
In vertebrates, a
high molecular weight AII (big AII), with a molecular weight of
5-7-kDa, has been detected in the brain (Pohl et al.,
1988). Such a big AII has not been detected in E. octoculata CNS extracts, but two proteins, one of 14 kDa and the other
of
18 kDa, were identified. Two hypotheses can be proposed to
explain this result: either these two proteins are tightly related, the
smaller one being a product of degradation of the larger one, or they
represent two distinct proteins. Three lines of evidence favor the
latter hypothesis. First, after immunoprecipitation of a CNS extract
obtained in the presence of protease inhibitors, these two proteins
immunoreactive to a-AII were also detected. Second, after extraction of
total CNS RNA and transcription in rabbit reticulocyte lysate, two
proteins immunoreactive to a-AII with a molecular mass of
15 and
19 kDa, respectively, were detected. They were slightly larger
than the ones detected in CNS extracts, which could be because of the
presence of a signal peptide necessary when the mature peptide has to
be secreted. Third, immunocytochemical data indicate in E.
octoculata a dual localization in both glial cells and neurons of
the CNS AII-like material. (
)Nevertheless, only a molecular
biology approach could permit us to definitively conclude in favor of
the existence of two proteins immunoreactive to a-AII in the CNS of E. octoculata.
From preliminary experiments conducted on the biological activity of the molecule of AII amide isolated from E. octoculata, it appears that this AII amide is involved in the control of hydric balance of leeches where the presence of diuretic (Salzet et al., 1993a; Salzet et al., 1994) and antidiuretic (Salzet et al., 1993c) neuropeptides has been reported. In leeches, the existence of diuretic hormone(s) was suspected for a long time. Indeed, in H. medicinalis, an 8-fold increase in the excreted urine volume is registered 15 min after a blood meal (Zerbst-Boroffka, 1973), which can be compared with the situation in blood-feeding insects, where a profound diuresis is necessary immediately after the blood meal to eliminate fluids (Schooley, 1993). On the other hand, there is an involvement of peptides of the angiotensin family in the control of diuresis of leeches. Indeed, in T. tessulatum, the AII-like peptide amount increases just after a blood meal, and AII has, when injected in T. tessulatum, a diuretic effect (Salzet et al., 1992a). As demonstrated in this paper, the AII amide of E. octoculata exerts a diuretic effect when injected in leeches. Calculation of efficacies shows that AII amide possesses, when injected in T. tessulatum, 10-100 times the efficacy of AII, emphasizing the importance of the carboxyl-terminal amidation for triggering the biological activity. Concerning the involvement of peptides of the angiotensin family in the control of hydric balance, it has to be noted that in vertebrates, AII may be either diuretic or antidiuretic, depending on the dose administered (reviewed by Gray and Erasmus(1989)). The existence in leeches of several molecules with a diuretic function (lysine conopressin (Salzet et al., 1993a), GDPFLRF amide (Salzet et al., 1994), and AII amide (this paper)) is not surprising. Indeed, in vertebrates it has been shown that neuropeptides interact on each other in order to control hydric balance, e.g. centrally administered AII produces vasopressin release (Saavedra, 1992). Nevertheless, so far we do not know whether in leeches these molecules with a diuretic function act on hydric balance indirectly or directly, either on nephridia, as is the case for FMRF amide in H. medicinalis (Wenning et al., 1993) and/or on the tegument and/or on the gut epithelium.