©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Structural Characterization of a Diuretic Peptide from the Central Nervous System of the Leech Erpobdella octoculata
ANGIOTENSIN II AMIDE (*)

(Received for publication, April 18, 1994; and in revised form, October 21, 1994)

Michel Salzet (1)(§) Philippe Bulet (2) Christian Wattez (1) Martine Verger-Bocquet (1) Jean Malecha (1)

From the  (1)Laboratoire de Phylogénie moléculaire des Annélides ER 87 CNRS, Groupe de Neuroendocrinologie des Hirudinées, Université des Sciences et Technologies de Lille, F-59655 Villeneuve d'Ascq Cédex, France and the (2)Institut de Biologie moléculaire et cellulaire, UPR 9022 CNRS, 15 rue Descartes, F-67084 Strasbourg Cédex, France

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

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.


INTRODUCTION

In vertebrates, the renin-angiotensin system (RAS) (^1)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.


MATERIALS AND METHODS

Animals and Dissection Procedure

Mature specimens of the pharyngobdellid leech E. octoculata, collected at Harchies (Belgium) and kept in the dark at 15 °C in pond water, were used for the isolation of the AII-like peptide.

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.

Antiserum

The polyclonal antiserum directed against AII (a-AII) was a kind gift of Dr G. Tramu (Laboratoire de Neurocytochimie fonctionnelle, Université de Bordeaux I, Talence, France). It was generated in a rabbit using synthetic human AII coupled to human serum albumin via glutaraldehyde. In radioimmunoassay experiments, cross-reaction of the antiserum was 100% for AII and fragment-(5-8) of AII, 3.13% for AIII, and 0.46% for AI. No cross-reaction was observed with fragment-(1-4) of AII and A0.

Immunoassays

Dot immunobinding assay (DIA) and enzyme-linked immunosorbent assays (ELISAs) based on the protocols of Salzet et al. (1992b, 1993a) were used to follow the AII-like activity during the purification procedures. Quantification of the AII-like peptide in CNS extracts was done by direct ELISA. For the two types of immunoassays, antiserum a-AII was employed at a dilution of 1:1000. As control, preadsorption of a-AII was carried out using homologous peptide. Prior to ELISA and DIA, a-AII, at its working dilution, was incubated overnight at 4 °C with synthetic AII (Sigma) (100 µg/ml undiluted a-AII).

Purification of the AII-like Peptide

A three-step procedure was used for this purification.

Step I, Sep-Pak Prepurification

4000 CNS were needed. Batches of 400 CNS were homogenized at 4 °C in 400 µl of 1 M acetic acid and sonicated (30 s) twice. Homogenates were centrifuged at 12,000 rpm for 30 min at 4 °C. After reextraction of the pellet, the two supernatants were combined and loaded onto Sep-Pak C(18) cartridges (500 µl of extract/cartridge; Waters) for solid phase extraction. After washing the cartridges with 5 ml of 1 M acetic acid, elution was performed with 5 ml of 50% acetonitrile in water acidified with 0.1% trifluoroacetic acid (Pierce). The eluted fractions were reduced 20-fold in a vacuum centrifuge (Savant) to remove organic solvent and trifluoroacetic acid. The total amount of AII-like material was quantified using AII ELISA.

Step II, Reversed-phase HPLC

The 50% elution fraction was taken up to 250 µl with acidified water (0.1% trifluoroacetic acid) and applied on a C(18)-peptide protein column (250 times 4.6 mm; Vydac), equilibrated with acidified water. Elution was performed with a discontinuous linear gradient of acetonitrile in acidified water from 0 to 15% in 10 min and from 15 to 45% in 30 min at a flow rate of 1 ml/min. The column effluent was monitored by absorbance at 226 nm, and the presence of AII-like material was detected by DIA.

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.

Step III, Final Purification

The AII-like material was applied twice on an ODS C(18) reversed-phase column (Ultrasphere, 250 times 2 mm; Beckman). The column was developed with a linear gradient of acetonitrile in acidified water from 0 to 60% in 60 min at a flow rate of 300 µl/min. The column effluent was monitored by absorbance at 226 nm, and the immunoreactive material was detected as above.

All HPLC purifications were performed with a Beckman Gold HPLC system equipped with a Beckman 168 photodiode array detector.

Amino Acid Sequence Analysis

Automated Edman degradation of the purified peptide and detection of phenylthiohydantoin-derivatives (PTH-Xaa) were performed on a pulse-liquid automatic sequenator (Applied Biosystems model 473A).

Mass Spectrometry

Electrospray Mass Spectrometry (ESMS)

The purified peptide was dissolved in water/methanol (50/50, v/v) containing 1% acetic acid and analyzed on a VG BioTech BIO-Q mass spectrometer (Manchester). Details of the method have been described elsewhere (Salzet et al., 1993a).

Fast Atom Bombardment Mass Spectrometry (FABMS)

Positive FABMS was carried out using a ZAB-HF double focusing mass spectrometer (mass range 3200 Da at 8 kV ion kinetic energy) and recorded on a VG 11/250 data system (VG Analytical Ltd., Manchester). The spectrophotometer was equipped with a saddle field atom gun (Ino Tech Ltd., Teddington). Ionization of the sample was performed with 1 mA of 8 kV energy xenon atom beam. The underivatized peptides were dissolved in deionized water containing 5% acetic acid or in neutral deionized water. The matrices (Sigma) were 1-thioglycerol, glycerol, metanitrobenzylalcohol, and magic bullet (dithiothreitol:dithioerythritol, 5:1, w/w). One µl of matrix was deposited on a stainless steel target, and the peptide in solution was added. Time between dissolution and the first scan acquisition was about 30 s. Mass calibration was carried out using a saturated solution of NaI in glycerol. Wide range single scans of the small peptides (mass less than 2500 Da) were produced by magnetic scanning at 8 kV accelerating voltage (scan time 8 s from 400 to 1200 Da) at resolution 1004.

Carboxypeptidase Treatment

Purified peptide (100 pmol) was dissolved in 200 µl of 50 mM Tris/HCl, pH 8, and 1 µg of carboxypeptidase A (Boehringer Mannheim) was added. Enzymatic digestion was carried out for 8 h at 37 °C and then stopped by adding 20 µl of acidified water (0.1% trifluoroacetic acid). The mixture was then dried in a vacuum centrifuge and redissolved in 50 µl of 20% acetic acid. The sample was then subjected to reversed-phase HPLC. Positive control was performed with 100 pmol of synthetic AII (Sigma) treated in the same experimental conditions as above.

Biological Assay

The bioassay (Malecha, 1983) was conducted on T. tessulatum, rhynchobdellid leeches bred in the laboratory and fed on ducks. Leeches at stage 3B, a stage that corresponds to an important water retention phasis, were distributed in nine lots of 20 animals having an identical mean body mass before being injected subepidermally (10 µl of solution/leech).

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).

AII-like Protein Identification

Central Nervous System Protein Extracts

CNS in batches of 400 were homogenized at 4 °C in 400 µl of Tris-buffered saline (TBS; 50 mM Tris-HCl, pH 7.4, 150 mM NaCl) supplemented with 2% EDTA and 1 mM phenylmethylsulfonyl fluoride and sonicated (30 s) twice. Each homogenate was centrifuged at 12,000 rpm for 30 min at 4 °C. The pellet was reextracted a second time, and the two supernatants were combined and subjected either to an immunoprecipitation or to high performance gel permeation chromatography (HPGPC).

Protein Purification

For HPGPC, the supernatant was applied to a high performance gel permeation column (SEC2000, Ultraspherogel, 7.5 times 300 mm; Beckman) eluted with 30% acetonitrile at a flow rate of 300 µl/min. The effluent was monitored at 215 nm. Eluted fractions were concentrated 5-fold by freeze drying and tested by AII ELISA. Positive fractions were then subjected to electrophoresis and Western blot analysis.

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 times SDS loading dye (bromophenol blue 5%) supplemented with 5% beta-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.

In vitro Translated Products of Central Nervous System RNA Extracts

CNS in batches of 400 were subjected to a total RNA extraction by the guanidium isothiocyanate method (Sambrook et al., 1989). Total RNA was then subjected to a translation in a mixture containing 30 µl of rabbit reticulocyte lysate (Amersham Corp.) and 20 µl of a solution of total RNA (30 µg) for 1 h at 30 °C. Translation was stopped on ice. The translated products were subjected to HPGPC as described above. Fractions immunoreactive to a-AII were then subjected to a Western blot analysis.

Western Blot Analysis

SDS-polyacrylamide gels were prepared according to Laemmli(1970) except that the separating gel consisted of a 10-25% polyacrylamide gradient slab gel. Molecular mass standards, purchased from Sigma, were as follows: serum albumin, 67 kDa; ovalbumin, 45 kDa; carbonic anhydrase, 30 kDa; trypsin inhibitor, 20 kDa; and alpha-lactalbumin, 14.4 kDa. The sample buffer contained beta-mercaptoethanol. After electrophoresis, proteins were transferred to a polyvinylidene difluoride membrane (Immobilon P; Millipore Corp.) and reacted with a-AII as described earlier (Salzet et al., 1993b). Control of specificity was realized by preadsorbing a-AII overnight at 4 °C with synthetic homologous peptide (100 µg of AII (Sigma)/ml of undiluted antiserum).


RESULTS

AII-like Peptide Isolation

CNS of E. octoculata were subjected to peptide extraction in 1 M acetic acid at pH 2. ELISA revealed the presence in the crude extract of CNS of a quantity of AII-like material estimated at 165 ± 25 fmol/CNS. The crude extract was purified using Sep-Pak C(18) cartridges. The fraction eluted by 50% acetonitrile in acidified water (0.1% trifluoroacetic acid) was reduced 20-fold by freeze drying and applied to a C(18) reversed-phase HPLC column (Fig. 1). The total amount of AII-like material detected at this step of purification was 134 ± 38 fmol/CNS (recovery of 80%). Eluted fractions tested by DIA revealed a zone immunoreactive to a-AII at a retention time between 23-25 min, corresponding to that of 28-30% acetonitrile (Fig. 1). Results obtained after using a-AII preadsorbed by synthetic AII established the specificity of the immunodetection.


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(18) 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(18)-peptide protein column (250 times 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(18) reversed-phase column (250 times 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.



AII-like Peptide Characterization

After the final purification step, a fraction aliquot of the immunoreactive material present in F2 was submitted to Edman degradation. The sequence, established on 680 pmol of purified AII-like peptide with a sequencing yield of 82%, was Asp-Arg-Val-Tyr-Ile-His-Pro-Phe (Table 1). The primary structure of the E. octoculata AII-like peptide is fully superposable on that of authentic AII. However, measurement of the molecular mass (Fig. 3) of the leech AII-like peptide by ESMS gave an m/z of 1044.3 ± 1.4 Da, differing by 1 Da from the monoisotopic molecular mass (1046.6 Da) calculated from the amino acid sequence determined by Edman degradation. This result predicted that the leech AII-like peptide possesses an amidated carboxyl terminus, the calculated monoisotopic molecular mass of AII amide being 1045.6 Da.




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(18) 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).



Biological Activity of the Leech AII-like Peptide

A comparative analysis (Fig. 5) of the repercussion on the leech mass variation of an injection of either synthetic AII amide or AII indicated that for the doses administered (0.01, 0.1, 1, and 10 nmol) and times postinjection considered (1, 2, and 4 h), synthetic AII amide was always effective, except at 4 h postinjection for the dose of 0.01 nmol. For the highest doses of AII amide assayed (1 and 10 nmol), a very close response (20% of loss of mass) was registered 2 and 4 h postinjection. In contrast, synthetic AII was ineffective at the doses of 0.01 and 0.1 nmol but effective at the doses of 1 and 10 nmol. It has to be noted that at a given postinjection time (1, 2, or 4 h postinjection) a dose of 1 or 10 nmol of AII exerts a similar effect.


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 (alpha = 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.

Identification of Proteins Immunoreactive to a-AII

In CNS Extracts

Extracts of CNS with TBS were fractionated on HPGPC. The collected fractions were assayed with AII ELISAs. A specific immunoreactive zone (Z1) corresponding to proteins with a molecular mass between 10 and 25 kDa was obtained (Fig. 6). Proteins contained in Z1 were then subjected to Western blot analysis with a-AII. Two proteins immunoreactive to a-AII, with molecular masses of 14 and 18 kDa, respectively, were detected in CNS extracts. An immunoprecipitation with a-AII preadsorbed or not preadsorbed with synthetic AII was conducted on protein extracts of CNS (Fig. 7, lanes a and b). In these conditions, after Western blot analysis with a-AII, the two proteins of 14 and 18 kDa immunoreactive to a-AII were detected when using a-AII not preadsorbed with synthetic AII (Fig. 7, lane b) but not when using a-AII preadsorbed with synthetic AII (Fig. 7, lane a).


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, alpha-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.



In CNS RNA-translated Products

After extraction of total RNA and transcription in rabbit reticulocyte lysate, translated proteins were treated in the same way as the protein extracts from the CNS with TBS. After HPGPC, an immunoreactive zone (Z2) corresponding to proteins with molecular masses ranging from 10 to 25 kDa is detected (Fig. 8). The proteins contained in Z2 were further subjected to a Western blot analysis. Two proteins with molecular masses of 15 and 19 kDa, respectively, were detected.


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 alpha-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.




DISCUSSION

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. (^2)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.


FOOTNOTES

*
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed: Laboratoire de Phylogénie moléculaire des Annélides ER 87 CNRS, Groupe de Neuroendocrinologie des Hirudinées, Université des Sciences et Technologies de Lille, SN3, F-59655 Villeneuve d'Ascq Cédex, France. Tel: 33-2043-4054; Fax: 33-2043-6849.

(^1)
The abbreviations used are: RAS, renin-angiotensin system; AI, angiotensin I; A0, angiotensinogen; AII, angiotensin II; HPLC, high pressure liquid chromatography; CNS, central nervous system; DIA, dot immunobinding assay; ELISA, enzyme-linked immunosorbent assay; PTH, phenylthiohydantoin; ESMS, electrospray mass spectrometry; FABMS, fast atom bombardment mass spectrometry; TBS, Tris-buffered saline; HPGPC, high performance gel permeation chromatography; PBS, phosphate-buffered saline.

(^2)
M. Salzet, P. Bulet, C. Wattez, M. Verger-Bocquet, and J. Malecha, unpublished data.


ACKNOWLEDGEMENTS

We are indebted to Dr. J. A. Hoffmann (Institut de Biologie Moléculaire et Cellulaire, UPR 9022 CNRS, Strasbourg, France) for the facilities he provided to us for the peptide sequencing. We also thank Dr. A. Van Dorsselaer, (Laboratoire de spectrométrie de masse bioorganique, UA 31 CNRS, Strasbourg, France) for the mass spectrometry determination. The technical assistance of A. Desmons, G. Montagne, M-C. Slomianny, and N. Thesse is kindly appreciated.


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