(Received for publication, May 15, 1995; and in revised form, July 3, 1995)
From the
Four nonapeptides that inhibit juvenile hormone synthesis have
been isolated by four high performance liquid chromatographic steps
from extracts of the brain of the field cricket, Gryllus
bimaculatus. The primary structures of these peptides were
assigned by Edman degradation and mass spectrometry as
Gly-Trp-Gln-Asp-Leu-Asn-Gly-Gly-Trp-NH (Grb-AST B1),
Gly-Trp-Arg-Asp-Leu-Asn-Gly-Gly-Trp-NH
(Grb-AST B2),
Ala-Trp-Arg-Asp-Leu-Ser-Gly-Gly-Trp-NH
(Grb-AST B3), and
Ala-Trp-Glu-Arg-Phe-His-Gly-Ser-Trp-NH
(Grb-AST B4). Each
of the peptides shows high sequence similarity to the
locustamyoinhibiting peptide (Lom-MIP), but is structurally different
from all the allatostatins so far identified. The synthetic
allatostatins Grb-AST B1-4 are potent inhibitors (50% inhibition
at 10
to 7
10
M)
of juvenile hormone III biosynthesis by corpora allata from 3-day-old
virgin females of G. bimaculatus using an in vitro bioassay. At 10
M, Grb-AST B1 also
strongly inhibits juvenile hormone III biosynthesis by corpora allata
from 2-day-old adult males and 1-day-old (males and females) and
4-day-old (females) last instar larvae of G. bimaculatus. The
inhibitory effect of Grb-AST B1 was also evident on corpora allata from
a related species, Acheta domesticus. Inhibition of juvenile
hormone synthesis by Grb-AST B1-4 is reversible.
Inhibition of juvenile hormone (JH) ()biosynthesis by
corpora allata (CA) in vitro has provided the basis for the
isolation of a family of allatostatins
(Tyr-Xaa-Phe-Gly-Leu-NH
) from brain extracts of various
cockroach species (Diploptera punctata, Periplaneta
americana, and Blattella
germanica)(1, 2, 3, 4, 5, 6) .
In D. punctata and P. americana, molecular cloning
has led to the isolation of cDNA that encodes a precursor polypeptide
containing 13 and 14 potential allatostatic sequences, respectively,
including those formerly identified through conventional purification
techniques(7, 8) . In D. punctata, the
expression of the allatostatin gene in midgut endocrine cells was also
demonstrated(9) . In the blowfly, Calliphora
vomitoria, six peptides with sequence similarity to cockroach
allatostatins have been identified; these inhibit JH III biosynthesis
in cockroaches, but do not inhibit synthesis of JH III bisepoxide in
the fly itself(10, 11) . An allatostatin that is
structurally unrelated to the cockroach allatostatins has been purified
and characterized from heads of pharate adults of the moth Manduca
sexta(12) .
Very recently, we identified two peptide inhibitors of JH III biosynthesis from extracts of the brain of the field cricket, Gryllus bimaculatus, which were designated Grb-AST A1 and Grb-AST A2, respectively(13) . Each of the peptides shows C-terminal amino acid sequence similarity to cockroach allatostatins and blowfly callatostatins. In this report, we describe the isolation and primary structure of four nonapeptides from brains of G. bimaculatus that are potent inhibitors of JH III synthesis in vitro by CA from virgin adult females, males, and last instar larvae of the species. These peptides are structurally different from all allatostatins so far identified, but are homologous to locustamyoinhibiting peptide (Lom-MIP), a peptide that inhibits the spontaneous contractions of the hindgut and oviduct of Locusta migratoria(14) . We propose that this family of peptides represents a novel type of neurohormones that inhibit JH biosynthesis in crickets.
Figure 1: Chromatographic steps of cricket allatostatin isolation. Horizontalbars in the chromatogram of the second HPLC run indicate fractions with allatostatic activity. Whitecolumns in the chromatograms of the fourth HPLC runs indicate allatostatic activity of the corresponding fractions (tested at 100 brain equivalents). See ``Materials and Methods'' for experimental details.
Figure 2:
Dose response for inhibition of JH III
biosynthesis by Grb-AST B1 (closed circles), B2 (open
circles), B3 (closed squares), B4 (open
squares), and Lom-MIP (closed triangles) for CA from
3-day-old adult virgin female G. bimaculatus. Percentage
inhibition was calculated as (1 - (2nd incubation/1st
incubation)) 100. Data are mean values of 5-15
determinations. For clarity, standard error is not shown. JH III
biosynthesis by untreated glands was
35-40
pmol
h
CA
in this
experiment.
A
computer search for similar peptides revealed sequence similarities
with locustamyoinhibiting peptide (Lom-MIP) (Table 1)(14) . Sequence similarity between Grb-AST B1
and Lom-MIP is 78%. Synthetic Lom-MIP, however, showed a maximal
inhibitory activity on JH III biosynthesis of crickets that was
significantly lower than that of Grb-AST B1-4 (compare values for
50% inhibition; Fig. 2and Table 1). On the other hand,
half-maximal inhibition (which is different from 50% inhibition since
the glands are not inhibited up to 100%) is reached at
5
10
M. This is only slightly higher than the
respective doses of Grb-AST B1-4 required for half-maximal
inhibition. This means that Lom-MIP and Grb-AST B1-4 may bind
equally effectively to the cricket allatostatin receptor, but that
Lom-MIP causes a weaker response. The adipokinetic peptide of G.
bimaculatus (Grb-AKH)(20) , which shares the two
C-terminal amino acids with Grb-AST B1-3 (Table 1), showed
30% inhibition at 10
M, the highest
concentration soluble in the incubation medium (data not shown).
Figure 3:
Reversibility of inhibition of JH III
biosynthesis by Grb-AST B1 (closed circles), B2 (open
circles), B3 (closed squares), and B4 (open
squares) for CA from 3-day-old adult virgin females. The second
incubation was carried out in medium containing 10M allatostatins. Controls (closed triangles)
were incubated in normal medium during each incubation period. See
``Materials and Methods'' for experimental details. Rates of
JH III biosynthesis during the first incubation (between 35 and 45
pmol
h
CA
in this
experiment) were set to 100%. Data are means ± S.E. of 10
determinations.
Figure 4:
Dose response for inhibition of JH III
biosynthesis by Grb-AST B1 for CA from 2-day-old adult males ()
and 3-day-old adult virgin females (
). Data are means ±
S.E. of 5-15 determinations. JH III biosynthesis by untreated
male CA was
30
pmol
h
CA
. Data for
females are taken from Fig. 2.
The search for neuropeptides with allatostatic activity in G. bimaculatus has led to the isolation of four nonapeptides
with sequence similarity at the C terminus (Gly-Xaa-Trp-NH;
Xaa = Gly in B1-3 and Ser in B4) and a common amino acid
at position 2 (Trp). In addition, three of the four peptides
(B1-3) have in common amino acids at positions 4 and 5 (Asp-Leu).
These peptides have been designated G. bimaculatus allatostatic neuropeptides B1-4 (Grb-AST B1-4) in
accordance with their biological activity and with the widely accepted
nomenclature for invertebrate neuropeptides(21) . The B
designation has been used because another class of allatostatic
neuropeptides has been recently presented in G. bimaculatus,
Grb-AST A1 and A2 (13) , which are members of the
Tyr-Xaa-Phe-Gly-Leu-NH
allatostatin family first found in D. punctata(1) .
The primary structures of the four
allatostatic neuropeptides show homology to the locustamyoinhibiting
peptide (Lom-MIP), which was isolated from brain-corpora
cardiaca-CA-subesophageal ganglion extracts of the locust L.
migratoria(14) . Nonapeptides Grb-AST B1-3 resemble
Lom-MIP in having Trp at position 2 (also in Grb-AST B4), Asp-Leu at
positions 4 and 5, and Gly-Trp-NH at the C terminus.
Lom-MIP suppresses the spontaneous contractions of the hindgut and
oviduct of L. migratoria and of the hindgut of a cockroach, Leucophaea maderae(14) . The C terminus
Gly-Trp-NH
in Lom-MIP and in Grb-AST B1-3 is in
common with the myoinhibiting peptide Ala-Pro-Gly-Trp-NH
isolated from the snail Lymnea stagnalis(22) .
In addition, Grb-AST B1-3 show some carboxyl-terminal sequence
similarity to Grb-AKH, which ends in
Thr-Gly-Trp-NH
(20) . None of Grb-AST B1-4
shows sequence similarity to either of the known allatoactive
neuropeptides.
The ability of Grb-AST B1-4 to inhibit JH III
biosynthesis in crickets is almost an order of magnitude lower than
that of Grb-AST A1 and A2 (Table 1). Maximal inhibition (reached
at an allatostatin concentration of 10
M), however, ranges from 60 to 80% for both classes. In
the assay on CA from 3-day-old virgin females, the peptides showed 50%
inhibition of JH III synthesis at 1
10
to 7
10
M. This suggests that cricket CA
are highly sensitive to both types of cricket allatostatic
neuropeptides when they are at the height of activity during
middle-late vitellogenesis (day 3 after imaginal moult). In D.
punctata, an inverse relationship between the activity of the CA
and their sensitivity to allatostatins was observed (3, 23) . Also in crickets, CA with lower basal rates
of JH III biosynthesis (e.g. CA from 5-day-old females) might
be more sensitive to allatostatins, but the rate of JH synthesis by
these CA is often very inconsistent, which makes these CA unsuitable
for testing allatoinhibiting substances.
Testing synthetic peptides
or HPLC fractions for only 2 h was necessary because otherwise total
incubation time would have been too long and often resulted in reduced
rates of JH III biosynthesis during the last incubation period. ()Compared with the 3-4-h incubation used by many
other authors(1, 5, 17) , the 2-h incubation
may slightly reduce sensitivity of the assay, but was sufficient to
show fast response to and reversibility of allatostatic material.
From a structural point of view, it is interesting to note that a
change at position 7 (Ala instead of Gly as found in Lom-MIP) reduces
the inhibitory activity. On the other hand, a change at position 8 (Ser
instead of Gly as in Grb-AST B4) has no negative effect on the
allatostatic activity: Grb-AST B4 turned out to be the most active of
the four B allatostatins. Grb-AKH, which shares only the two C-terminal
amino acids with Grb-AST B1-3, had only weak allatostatic
activity. The occurrence of Trp at position 2, which is a common
feature of all B allatostatins and of Lom-MIP, might indicate the
importance of this residue with regard to the biological activity of
these peptides. The information about the bioactivity therefore might
be encoded not only in the C-terminal region, as shown for the
Tyr-Xaa-Phe-Gly-Leu-NH allatostatin
family(3, 24) .
One may ask whether Grb-AST B1-4 can be termed ``allatostatins'' since the first isolation of a member of this peptide family (Lom-MIP) used a bioassay based on myoinhibiting activity of the HPLC fractions (14) . In crickets, the allatostatic activity is the only effect demonstrated so far; therefore, the term allatostatin seems to be justified.
Grb-AST
B1-4 proved to be neither sex/stage- nor species-specific.
Although extracted only from brains of adult females, they inhibit JH
III biosynthesis by CA from adult males and last instar larval males
and females as well. The allatostatic activity of the B allatostatins
was also shown in a closely related cricket species, the house cricket, A. domesticus. Interspecific allatostatic activity was also
shown for the Tyr-Xaa-Phe-Gly-Leu-NH allatostatin family (25, 26) .
The existence of two types of
allatostatic neuropeptides in G. bimaculatus makes it more
difficult to understand their modes of function. As yet, nothing is
known about changes in brain and hemolymph titer of allatostatic
neuropeptides, e.g. during the female reproductive period of G. bimaculatus. Isolation and quantification of allatostatic
receptors will be needed, the levels of which may be largely
attributed to changes in CA sensitivity for allatoregulating
peptides(8) . The occurrence of multiple allatostatin species
might suggest the existence of individual receptors for each species of
molecule. It remains to be determined, however, whether each
allatostatin species is associated with a different receptor. It even
remains to be determined whether allatostatins are the principal
regulators of JH biosynthesis in vivo. The immunocytochemical
localization of Tyr-Xaa-Phe-Gly-Leu-NH allatostatin-like
peptides in the central nervous system as well as in midgut cells of
many invertebrates (8, 27) suggests that allatostatins
are multifunctional neuropeptides, even though inhibition of JH
biosynthesis by CA in vitro was the only bioassay utilized in
their isolation. Sequence homology, as found between Lom-MIP and the
family of allatostatic neuropeptides presented in this paper, may
indicate a myomodulatory role of these peptides, as was also discussed
for the Tyr-Xaa-Phe-Gly-Leu-NH
allatostatin
family(28, 29) .