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(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, In vertebrates, the renin-angiotensin system (RAS) ( 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 ( 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
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
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
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
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
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 (
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,
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
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 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.
Volume 270,
Number 4,
Issue of January 27, 1995 pp. 1575-1582
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
ANGIOTENSIN II AMIDE (*)
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
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.
)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).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.
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.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
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-peptide protein
column (250 
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.Step III, Final Purification
The AII-like material
was applied twice on an ODS C reversed-phase column
(Ultrasphere, 250 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.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).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, 7.5 300 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. 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.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 
-lactalbumin, 14.4 kDa. The sample buffer
contained -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).
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 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 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.
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.
50%).
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.
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.
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.
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.
= 0.05).
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).
-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.
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.
-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.
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.
))
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.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
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