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First published online January 30, 2009
Journal of Experimental Biology 212, 542-549 (2009)
Published by The Company of Biologists 2009
doi: 10.1242/jeb.022889
The specific binding sites of eyestalk- and pericardial organ-crustacean hyperglycaemic hormones (CHHs) in multiple tissues of the blue crab, Callinectes sapidus
University of Maryland Biotechnology Institute, 701 E. Pratt Street, Columbus Center, Suite 236, Baltimore, MD 21202, USA
Author for correspondence (e-mail:
chung{at}comb.umbi.umd.edu)
Accepted 18 November 2008
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Summary |
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 TOP Summary INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONReferences |
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Key words: receptor binding assay, crustacean hyperglycaemic hormone, eyestalk, pericardial organ, hyperglycaemia
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INTRODUCTION |
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TOPSummaryINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONReferences |
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;
Webster, 1998;
Lacombe et al., 1999;
Böcking et al., 2002;
Chan et al., 2003;
Chen et al., 2005;
Fanjul-Moles, 2006;
Drexler et al., 2007). CHH in
many crustacean species is associated with multiple functions and
tissue-specific structural isoforms
(Yasuda et al., 1994;
Aguilar et al., 1995;
Chung and Webster, 1996;
Chang et al., 1999;
Ollivaux and Soyez, 2000;
Chung and Webster, 2004).
Eyestalk-CHH (ES-CHH), present in a moult stage-independent manner, is
associated with hyperglycaemia, osmoregulation and the inhibition of
ecdysteroid and methyl farnesoate synthesis
(Webster and Keller, 1986;
Liu et al., 1997;
Khayat et al., 1998;
Chung et al., 1999;
Webster et al., 2000;
Spanings-Pierrot et al., 2000;
Tsutsui et al., 2005). The
presence of CHH in the brain (eyestalk)–gut axis has also been reported
in Carcinus maenas (Kegel et al.,
1989; Weidmann et al., 1989;
Chung et al., 1999). In
contrast, insect ion transport peptide (ITP), a CHH-related neuropeptide
mainly produced in the corpora cardiaca, has a specific action on
Cl– transport in the insect ileum
(Phillips et al., 1998).
Multiple sequence analysis of the family of CHH neuropeptides generally
shows that the first 40 amino acid residues of these neuropeptides are well
conserved, compared with the C-terminal portion
(Böcking et al., 2002;
Chan et al., 2003;
Chen et al., 2005;
Fanjul-Moles, 2006). This
structural variation of CHHs is reflected in the glucose responses in
heterologous assays (Chung et al.,
1998; Chung and Zmora,
2008; Mosco et al.,
2008), indicating the importance of the C-terminal portion in
bioactivity. More specifically, amidation at the C-terminus plays a crucial
role in CHH-induced hyperglycaemia
(Katayama et al., 2002;
Mosco et al., 2008). In
insects, however, the biological importance in the N-terminal domain
(6–7 amino acids) was shown by expression of chimeric ITP in
Drosophila Kc1 cells that contained the N-terminal domain of
Penaeus japonicus CHH, and Schistocerca gregaria ITP
sequence with the N-terminus removed was inactive in ion transport in the
locust ileum (Zhao et al.,
2005).
Tissue-specific CHHs may be the products of alternative splicing of
multiple CHH genes (Dircksen et al.,
2001; Gu and Chan,
1998; Chen et al.,
2004), while cDNAs of PO-CHH isoforms have been reported
from various non-neuronal tissues (Chen et
al., 2004; Choi et al.,
2006; Lee et al.,
2007; Tiu et al.,
2007). However, the neuropeptide form of PO-CHH that is most
structurally related to insect ITP
(Phillips et al., 1998;
Drexler et al., 2007) has so
far been found only in the intrinsic multipolar cells in the POs of C.
maenas and Callinectes sapidus
(Dircksen et al., 2001;
Chung and Zmora, 2008).
ES-CHH secretion from the sinus gland in response to stress has been shown
in many crustacean species (Chang et al.,
1998; Chung and Webster,
2005; Chung and Zmora,
2008; Kou and Yang,
1999; Webster,
1996). In C. sapidus, hypoxia induced increases in the
expression of PO-CHH and its neuropeptide concentration in
haemolymph, suggesting a putative role in the stress response
(Chung and Zmora, 2008).
Despite the observed increase in C. sapidus PO-CHH concentration in
the haemolymph during hypoxia, injection of native PO-CHH (10–20 pmol)
into C. maenas, C. sapidus or Machrobrachium rosenbergii
(350 pmol of recombinant PO-CHH, rPO-CHH) did not produce hyperglycaemia,
leaving its physiological function and target tissues as yet unidentified
(Chung and Zmora, 2008;
Dircksen et al., 2001;
Ohira et al., 2006).
As alluded to earlier, our previous findings of an increase in
PO-CHH expression and, moreover, the release of PO-CHH from animals
experiencing hypoxia indicates that it meets the crucial criteria of a
hormone, thus implying that there must be a target tissue(s). We used a cGMP
radioimmunoassay (cGMP RIA) in vitro and a traditional radioligand
binding assay with a combination of 125I-rPO-CHH and the membranes
of various tissues in order to identify putative target tissue(s). Moreover,
we investigated a possible in vivo role of PO-CHH in hyperglycaemia
by using 150–200 pmol (
30–50 nmol l–1) of
rPO-CHH, albeit a much higher concentration than the physiological levels
observed by Chung and Zmora (Chung and
Zmora, 2008). To validate these results, we tested native ES-CHH
as a reference neuropeptide.
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MATERIALS AND METHODS |
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TOPSummaryINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONReferences |
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). Animals at intermoult were used for these experiments
(Drach and Tchenigovtzeff,
1967).
Purification of native CHH from extracts of the sinus gland of C. sapidus and C. maenas
The sinus glands were dissected out from ice-cold adult males and females
of C. sapidus and kept at –80°C. Prior to purification, the
sinus gland (a batch of 50–100) was extracted in 2 mol
l–1 acetic acid by sonication (Branson, Danbury, CT, USA).
After centrifugation at 12,800 g for 10 min the supernatant
was separated by a two-step purification method as described elsewhere
(Webster, 1991). ES-CHH and
oxidized ES-CHH were identified by dot-blot analysis using C. sapidus
ES-CHH antiserum followed by mass determination
(Chung and Zmora, 2008). The
procedures for C. maenas were as stated in Webster
(Webster, 1991).
Recombinant rPO-CHH
Construction of expression plasmids
Two primers were designed based on the nucleotide sequence of the cDNA
encoding PO-CHH (DQ667141) (Chung and
Zmora, 2008). The construction of expression plasmids was followed
as described by Ohira and colleagues
(Ohira et al., 1999). In
brief, the forward primer
(5'-ATCCATGGCCCAGATTTACGACTCCTCCTGT-3',
Invitrogen, Carlsbad, CA, USA) contained 14 nucleotide residues encoding QIYDS
(the first five amino acid residues of mature PO-CHH). Two additional
nucleotides (AT and CC) were added: at the beginning and the
end of the NcoI site (italic) to prevent the removal of the terminal
nucleotide due to exonuclease activity of Taq DNA polymerase and to adjust the
reading frame, respectively. The reverse primer
(5'-ATGAATTCTTATCCTCTGATAGCATCCCTG-3', Invitrogen) also
contained 14 nucleotide residues encoding DAIRG (the last five amino acid
residues of mature PO-CHH) and two additional residues: an EcoRI site
(italic) and a stop codon (underlined). PCR was conducted with these primers
using a plasmid containing the PO-CHH cDNA as a template. The amplified cDNA
was subcloned into a pGEM T-Easy vector (Promega, Madison, WI, USA). After
release from the vector by NcoI/EcoRI digestion, the PO-CHH
insert was ligated into NcoI/EcoRI sites of a pET-Duet
expression vector (Novagen, Gibbstown, NJ, USA).
Expression of recombinant PO-CHH
The procedure for the expression of rPO-CHH in E. coli
Rosettagami(DE3)pLysS competent cells (Novagen) was as described
(Ohira et al., 1999). The
supernatant and the re-suspended insoluble material were separated by 18%
SDS-PAGE, and the rPO-CHH was found in both fractions (supplementary material
Fig. S1A). The soluble fraction was then purified by RP-HPLC (Waters, Milford,
MA, USA) on a C18 column (Gemini, 4.6 mmx150 mm, Phenomenex, Torrence,
CA, USA) with the following gradient conditions: 30–80% B over 30 min
(A: 0.11% TFA in water, B: 0.1% TFA in 60% acetonitrile and 40% water) at a
flow rate of 0.5 ml min–1. The peak was further examined by
dot-blot analysis using PO-CHH antiserum
(Chung and Zmora, 2008)
(supplementary material Fig. S1B).
The quantification of rPO-CHH was carried out using a Bio-Rad DC protein assay kit (Bio-Rad, Hercules, CA, USA) and a molecular mass of 8447.35 Da was determined by MALDI-TOF, which differed by 4.5 Da from the calculated mass of rPO-CHH (8443.2 Da, ABI Voyager DE Pro, Institute of Glycotechnology, Tokai University, Japan).
Iodination of CHH neuropeptides
Using chloramine T, 300 pmol of rPO-CHH or ES-CHH was iodinated with 300
µCi of 125I-Na (Amersham, Pittsburgh, PA, USA).
125I-rPO or 125I-ES-CHH was eluted on a pre-conditioned
PD-10 column (Bio-Rad) as described previously
(Chung and Webster, 1996) and
kept in a 1:1 ratio of glycerol at 4°C. The specific activity of both CHHs
was approximately 500–700 Ci mmol l–1.
Membrane preparations
Plasma membranes of various tissues were prepared by following the protocol
described previously (Chung and Webster,
1996). In detail, after dissection of the midgut, hindgut,
scaphognathites, gills, hepatopancreas and heart from anaesthetized ice-cold
juvenile crabs, the tissues were briefly rinsed in ice-cold crustacean saline
and homogenized in extraction buffer (300 mmol l–1 sucrose,
140 mmol l–1 NaCl, 10 mmol l–1 benzamidine,
and 10 mmol l–1 Hepes, pH 7.4) using a Polytron homogenizer
(IKA, Staufen, Germany). The homogenates were centrifuged at
500g for 10 min at 4°C and the supernatants were
re-centrifuged at 24,000g for 45 min at 4°C (Jouan,
Waltham, MA, USA). The resulting microsomal pellets were resuspended in the
binding assay buffer without BSA (140 mmol l–1 NaCl and 10
mmol l–1 Hepes, pH 7.4) and protein concentrations were
estimated as stated above. Membranes were aliquoted into 0.5 ml tubes and
stored at –80°C until further use.
Binding assays
Binding assays were carried out in 100 µl final volume following the
procedure described (Chung and Webster,
1996). Non-specific binding ranged from 20% to 30% of the total
binding. All triplicate assays were repeated at least twice.
cGMP measurements
Iodination of the cGMP analogue and the method of cGMP RIA are given
(Chung and Webster, 2006). In
brief, midgut, hindgut, scaphognathites, gills, hepatopancreas and heart
tissues were dissected from intermoult crabs and incubated for 45 min at room
temperature with 200 µl of Medium 199 at 900 mosmol l–1
adjusted with NaCl containing various concentrations of rPO-CHH (or medium
alone for control) in the presence of 0.25 mmol l–1
isobutylmethylxanthine. A set of tissues was also treated with 50 nmol
l–1 ES-CHH. After incubation, the tissues were collected in
100 µl of ice-cold 0.1 mol l–1 acetate buffer (pH 4.5) and
disrupted by brief sonication (Bronson, Danbury, CT, USA) then centrifuged for
10 min at 12,800 g at 4°C. The protein concentrations were
estimated as described above. RIA was used to determine cGMP levels of
25–50 µl of each acetylated sample as described previously
(Chung and Webster, 2006). The
data are given as pmolmg–1 protein.
In vivo glucose bioassay
Juvenile blue crabs at intermoult were injected with 100 µl of rPO-CHH
(150–200 pmol) or native ES-CHH (10 pmol), while control animals
received equal volumes of crustacean saline. At time t=0 (prior to
injection), 100 µl of haemolymph was withdrawn in a marine anticoagulant at
ratio of 1:1 as described before (Chung
and Zmora, 2008). At time intervals of 30, 60, 90 and 120 min, the
haemolymph was collected and glucose levels were determined using a glucose
oxidase assay (Webster, 1996).
Due to high individual variation in the level of initial glucose, the data
were converted into percentage increment and the results were pooled from
three separate experiments.
Statistical analysis
Statistical significance was tested using the GraphPad InStat program
(GraphPad Software, San Diego, CA, USA).
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RESULTS |
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TOPSummaryINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONReferences |
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75 fmol) of 125I-ES-CHH or 125I-rPO-CHH with 100
µg of membranes (final volume, 100 µl) from the following tissues:
scaphognathites, abdominal muscles, heart, midgut, hindgut, gills and
hepatopancreas. Non-specific binding was estimated by the addition of 10 pmol
of cold ES-CHH and rPO-CHH at 10–7 mol
l–1.
|
Fig. 1B shows the number of
specific binding sites of 125I-rPO-CHH. All tissue membranes tested
except hepatopancreas contained specific binding sites for
125I-rPO-CHH, with varying concentrations from the highest in
scaphognathites, 11.5(±0.7)x10–11 mol
l–1 mg–1 protein (N=6), to the
lowest in gills, 2.12(±0.50)x10–11 mol
l–1 mg–1 protein (N=6). These
specific 125I-rPO-CHH binding sites in the membranes of abdominal
muscles and gills were also partially displaced by the addition of cold ES-CHH
at 10–7 mol l–1, giving 65% and
30% of the specific binding, compared with that of cold rPO-CHH,
respectively.
Binding characteristics of 125I-ES-CHH in the membranes of hepatopancreas and gills
The specific binding sites of 125I-ES-CHH in the membranes of
hepatopancreas and gills were further characterized in terms of saturation and
displacement (Figs 2A,B,
respectively). These membrane sites were tested for saturation with increasing
concentrations of 125I-ES-CHH from 5.5x10–11
to 8.1x10–10 mol l–1, whereas values
for non-specific binding were obtained by the addition of cold ES-CHH at
10–7 mol l–1. The KD
values were 1.68(±0.34)x10–10 mol
l–1 for hepatopancreas and 2.40(±0.42)x
10–10 mol l–1 for gills. The calculated
number of maximal binding sites (Bmax) was
3.8(±0.5)x10–11 mol l–1
mg–1 protein for hepatopancreas and
1.2(±0.2)x10–10 mol l–1
mg–1 protein for gills, giving a similar Hill co-efficient of
1.0 for the two.
|
Binding characteristics of 125I-rPO-CHH in the membranes of abdominal muscles
Based on the results shown in Fig. 1A
and B, the membrane of abdominal muscles was chosen for further
analysis of the binding characteristics of 125I-rPO-CHH and
125I-ES-CHH, as it was easy to collect a large amount compared with
scaphagonathites. Fig. 3A,B
shows the saturation of the specific binding sites of 125I-rPO-CHH
(Fig. 3A) and
125I-ES-CHH (Fig.
3B) with Bmax values of
1.3(±0.2)x10–10 and
7.8(±0.1)x10–11 mol l–1 mg
protein, respectively. The calculated KD was similar at
1.3(±0.2)x10–9 moll–1 for
125I-rPO-CHH and 1.1(±0.2)x10–9 mol
l–1 for 125I-ES-CHH, while the Hill co-efficient
of both CHHs was 1.0.
|
The competitiveness of the specific binding sites of 125I-rPO-CHH or 125I-ES-CHH was assayed by adding cold rPO-CHH and/or ES-CHH at concentrations from 5x10–11 to 5x10–7 mol l–1 (Fig. 3C). The calculated IC50 values were 2.8(±0.3)x10–9 and 5.0(±0.9)x10–7 mol l–1 for the homologous competition of rPO-CHH and ES-CHH, respectively. With heterologous neuropeptides, the IC50 values were estimated at >1 µmol l–1 for both CHHs.
cGMP measurements
The effect of ES-CHH (50 nmol l–1) and rPO-CHH (100 nmol
l–1) on cGMP production was examined in the following
tissues: Y-organs, foregut, midgut, hindgut, scaphognathites, abdominal
muscles, gills, hepatopancreas and heart. The results were converted into fold
increment of control. As shown in Fig.
4, ES-CHH (open bars) produced varying degrees of stimulation of
cGMP from 3.5- to >20-fold in all tissues tested except midgut, with the
lowest in hepatopancreas (3.5-fold) and the greatest in Y-organ and
hindgut (>20-fold). The incubation of rPO-CHH with foregut, midgut,
hindgut, scaphognathites, abdominal muscles and heart did produce a 1.5- to
4.0-fold increase in the level of cGMP
(Fig. 4, filled bars), whereas
gut tissues produced the greatest response of 3- to 4-fold. Abdominal muscles
had the lowest response of only 1.5-fold. The level of cGMP in gills, Y-organs
and hepatopancreas was not affected by incubation with rPO-CHH at 100 nmol
l–1.
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In vivo glucose bioassay
The injection of 20 pmol of native PO-CHH into the juvenile crabs
(60–90 mm carapace width, weighing between 30 and 60 g) gave calculated
in vivo concentrations of 3–7 nmol l–1,
providing the haemolymph volume is 10% of bodyweight. This concentration of
3–7 nmol l–1 did not produce hyperglycaemia in
haemolymph of C. sapidus (Chung
and Zmora, 2008). Based upon the results obtained in the current
study (binding assays and cGMP response), we conducted further in
vivo assays using a higher dose at a final concentration of 30–50
nmol l–1 by injecting 150–200 pmol of rPO-CHH, 10 pmol
of ES-CHH or crustacean saline as a control. The results obtained from the
time course response of CHH injections on the glucose level in haemolymph are
shown in Fig. 5: rPO-CHH
induced hyperglycaemia by significantly elevating glucose to 270±26%
(N=18) after 30 min compared with that at t=0
(P<0.01) and at 30 min of ES-CHH injection (P<0.05).
This elevated level of glucose by rPO-CHH was maintained to the end of the
incubation (120 min, 353±46%; N=18; P<0.01). The
hyperglycaemic response of ES-CHH was positively related to incubation time,
as the level of glucose was significantly and steadily raised from
182±15% (N=18, P<0.05) at 30 min to
850±180% (N=18, P<0.001) at 120 min. At 90 and 120
min, ES-CHH produced much higher hyperglycaemia than rPO-CHH. Animals that
received crustacean saline showed 120–170% increases throughout the
experimental period, with the highest level of 172±15% (N=15,
P<0.05) at 60 min, compared with that of control at
t=0.
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|
DISCUSSION |
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TOPSummaryINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONReferences |
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We produced rPO-CHH in E. coli for the binding assay, as it
readily generates a large amount of protein. We reasoned that rPO-CHH with an
extended Ala residue at the N-terminus binds its binding sites and is
bioactive, as a previous report has shown that recombinant moult-inhibiting
hormone (rMIH) produced using the same procedure was biologically active and
retained binding capacity to its receptors
(Okumura et al., 2005;
Asazuma et al., 2005). The
study of the structure and activity of CHH-related neuropeptides revealed that
the C-terminus is more critical for bioactivity than the N-terminus, as the
cyclization of Gln to pyroglutamate at the N-terminus or the extended Ala at
the N-terminus does not affect the secondary structure, binding capacity or
bioactivity (Chung and Webster,
1996; Katayama et al.,
2002; Katayama et al.,
2003; Katayama et al.,
2004; Okumura et al.,
2005; Asazuma et al.,
2005).
To our surprise, approximately half of rPO-CH was recovered in the
supernatant, suggesting that the protein was properly folded in its native
structure (Anfinsen, 1973). The
dot-blot analysis showed that the protein purified by an RP-HPLC that was
present in the supernatant was recognized by PO-CHH antiserum
(Chung and Zmora, 2008). The
4.2 Da difference by MALDI-TOF between the calculated and estimated mass is
acceptable, as it lies within a 0.05% margin of error.
Specific binding sites for rPO-CHH and ES-CHH were commonly found in the
membranes of various tissues of C. sapidus: scaphognathites,
abdominal muscles, heart, midgut, hindgut, gills and hepatopancreas, most of
which were considered to be target tissues of ES-CHH
(Webster, 1993;
Kummer and Keller, 1993;
Chung and Webster, 1996;
Chung and Webster, 2003;
Chung and Webster, 2006). We
proposed that scaphognathites may be the target tissues of PO-CHH
(Chung and Zmora, 2008) after
considering the anatomical structure and location of POs in animals
(Maynard, 1961).
Interestingly, it appeared that cold rPO-CHH partially competed to displace
125I-ES-CHH from 50–60% of the binding sites that are present
in membranes of scaphognathites and abdominal muscles, while it did not
compete for specific binding sites in the other tissues.
As for 125I-rPO-CHH, all the membranes tested exhibited specific
binding sites, with the greatest number occurring in the scaphognathites and
the least in the hepatopancreas. Based on the number of binding sites in the
tissues, scaphognathites, abdominal muscles and midgut are considered to be
the major target tissues for rPO-CHH. Interestingly, the specific binding
sites for 125I-rPO-CHH in the membranes of scaphognathites were
displaced with homologous neuropeptide but not with ES-CHH. In the abdominal
muscles, 65% of specific binding sites of 125I-rPO-CHH and of
125I-ES-CHH were competed out by adding cold ES-CHH and rPO-CHH,
respectively. Hepatopancreas, gills, hindgut, heart and midgut are the major
target tissues of ES-CHH of C. sapdius, as reported for C.
maenas (Webster, 1993;
Kummer and Keller, 1993;
Chung and Webster, 2006).
All tissues except abdominal muscles showed two types of CHH binding site
with a varying degree of compatibility to the heterologous counterpart. Those
present in heart, gut and hepatopancreas are not compatible with heterologous
CHH, but are specific to the homologous neuropeptide. Gills and
scaphognathites possess the major receptor types of ES-CHH and PO-CHH,
respectively, while their minor forms, the sites of PO-CHH binding in gills
and ES-CHH binding in scaphognathites, are likely to be compatible with
heterologous neuropeptides. Interestingly the abdominal muscle that was
considered to be one of the major target tissues of ES-CHH
(Keller and Sedlmeier, 1988)
contains binding sites compatible with both CHHs, as similar levels of binding
in the presence of heterologous neuropeptides were found. It is reported that
tissues or cells co-express two similar receptors for follicle stimulating
hormone (FSH): one is specific to FSH, while the other is promiscuous, binding
to either human chorionic gonadotropin or thyrotropin stimulating hormone
(Costagliola et al., 2005). In
this case, the sequence similarity between these ligands is only 40%
(Costagliola et al., 2005).
Thus, considering the sequence identity between two CHHs (>60%), the CHH
receptor in abdominal muscles may be promiscuous by binding to both
neuropeptides, but with different affinities.
The specific binding sites of ES-CHH in the membranes of hepatopancreas and
gills of C. sapidus showed two typical binding characteristics:
saturation and displacement, which is similar to those found in the
hepatopancreas of Orconectes limosus and C. maenas and the
gills and Y-organs of C. maenas in terms of the values of
Bmax and KD
(Kummer and Keller, 1993;
Webster, 1993;
Chung and Webster, 2006). It
appears that the receptors for ES-CHH present in the gills and hepatopancreas
of these species are rather similar in terms of affinity, despite the possible
difference in the receptor density of each tissue.
The IC50 values of C. maenas ES-CHH and oxidized ES-CHH
were interesting, as they were one or two orders of magnitude lower than that
of C. sapidus ES-CHH. The greatest variation in the sequence of CHHs
of C. sapidus and C. maenas, which is a common feature among
the family of CHH neuropeptides, lies in the C-terminal tail
(Phillips et al., 1998;
Webster, 1998;
Lacombe et al., 1999;
Böcking et al., 2002;
Chan et al., 2003;
Chen et al., 2005;
Fanjul-Moles, 2006), thus
implying the importance of this region in species-specific receptor
recognition and, thus, for bioactivity. However, the C-terminal synthetic
peptide of C. sapidus ES-CHH did not compete with
125I-ES-CHH, suggesting that the C-terminal fragment alone is not
structurally sufficient for binding. As for the oxidized C. sapidus
ES-CHH with an additional mass of 16 Da
(Chung and Zmora, 2008), the
exact position of oxidation at the Met residue is unknown, since three Met
residues are present in the primary amino acid sequence, at positions 47, 53
and 59. The Met residue at position 47 resides within a globular structure
that is formed by three intradisulphide bridges
(Katayama et al., 2003).
However, Met positions 53 and 59 which are conserved in most crab species are
located in the C-terminal tail; thus one of these residues may be the target
of oxidation. Nonetheless, it seems that the oxidation of a single Met residue
interferes with binding to its receptors, which results in a low value of
IC50 as shown in Fig.
2C. This fact may further imply the importance of the C-terminus
in binding as well as the bioactivity of CHHs
(Chung et al., 1998;
Katayama et al., 2002;
Mosco et al., 2008). Such
differences in the values of IC50 support our previous observation
that injection of C. maenas ES-CHH or oxidized C. sapidus
ES-CHH induced only 20–30% hyperglycaemia of ES-CHH
(Chung and Zmora, 2008).
As shown in Fig. 1A,B, the
abdominal muscle membrane contained specific binding sites for both rPO-CHH
and ES-CHH with comparable KD values. However, they show
differences in Bmax and IC50 values of the
displacement: two times more and 50 times higher 125I-rPO-CHH
values than those of 125I-ES-CHH, respectively. As shown in the
insets of Fig. 3A,B, the Hill
coefficient (1.0) indicates that binding of each neuropeptide to these
sites occurs in a non-cooperative manner. Interestingly, the membrane of
scaphognathites (shown in supplementary material Fig. S2) revealed
IC50 values of 4.5 E–10 mol l–1
for rPO-CHH, but >1µmoll–1 for ES-CHH, suggesting that
this respiratory tissue may be the primary target tissue of PO-CHH, as
proposed previously (Chung and Zmora,
2008). The binding sites of each neuropeptide were poorly
displaced by their heterologous counterpart.
Compared with the values of Bmax and KD for 125I-ES-CHH binding in the membranes of hepatopancreas and gills as shown in Fig. 2, the specific binding sites of this neuropeptide in the abdominal muscles exhibited one order of magnitude less affinity but were 4–7 times greater in number. Low affinity but great binding capacity suggests that hepatopancreas and gills have a similar type of receptor to that in the major target tissues for ES-CHH of C. sapidus. However, the CHH receptor present in the abdominal muscles appears to be capable of binding to both CHHs, but with a preference for PO-CHH over ES-CHH.
The cGMP response to ES-CHH and rPO-CHH was similar as reported in C.
maenas (Dircksen et al.,
2001; Chung and Webster,
2006). In particular rPO-CHH had no effect on cGMP production in
the Y-organ, gills and hepatopancreas of C. sapidus. The lack of cGMP
response in hepatopancreas was anticipated, in that it showed the least number
of specific binding sites for rPO-CHH (Fig.
1B). The abdominal muscles contain the specific binding sites of
rPO-CHH, yet the stimulation of cGMP production was only 1.5-fold. The second
messenger of rPO-CHH is not known in these muscles, but cGMP is not likely to
be the second messenger of this CHH. ES-CHH of C. sapidus, on the
other hand, stimulated an 7-fold increase in cGMP in this tissue, and a
similar result was reported in Homarus americanus
(Goy, 1990), therefore
indicating that it may be the second messenger.
Previously, 10–20 pmol injections of native PO-CHH (3–5 nmol
l–1) lacked hyperglycaemic activity
(Dircksen et al., 2001;
Chung and Zmora, 2008).
However, we reasoned that injection of a higher concentration of PO-CHH,
although not its physiological level, may be required to induce hyperglycaemia
in haemolymph due to the following findings: (1) the limited specific binding
sites found in the hepatopancreas (Fig.
1B); (2) the relatively small cGMP response of PO-CHH, compared
with that of ES-CHH; and (3) the structural differences in the C-terminal
tail. As stated above, the Met of ES-CHH at positions 53 and 59 that is
required for high affinity binding to receptors and bioactivity
(Chung and Zmora, 2008) is
replaced by Val and Pro, respectively, in PO-CHH. Moreover, the C-terminus of
PO-CHH is not amidated, as its importance in hyperglycaemia has previously
shown (Katayama et al., 2002;
Mosco et al., 2008).
Therefore, we pursued an in vivo time course study of haemolymph
glucose by the injection of rPO-CHH at 30–50 nmol l–1.
The hyperglycaemia caused by the injection of a high concentration of rPO-CHH
was immediate, as the elevated level of glucose at 30 min after injection was
significantly greater than that of ES-CHH. Overall the hyperglycaemic response
by rPO-CHH, like cGMP production, was truncated compared with that caused by
ES-CHH. We were not able to use native PO-CHH as a control for rPO-CHH due to
the lack of material, but ES-CHH elevated the glucose to the highest level at
90–120 min after injection, similar to earlier reports
(Chung and Webster, 1996;
Chang et al., 1998;
Chung et al., 1998;
Dircksen et al., 2001;
Chung and Zmora, 2008).
These truncated responses of cGMP and hyperglycaemia to PO-CHH are
comparable with the levels of hyperglyacemia caused by the injection of C.
meanas CHH and oxidized ES-CHH (Chung
and Zmora, 2008). The difference in IC50 values of
C. meanas CHH, oxidized ES-CH and rPO-CHH (as shown in
Fig. 2C,
Fig. 3C), and their truncated
hyperglycaemia response suggest that the structural difference in the
C-terminus may affect the efficacy and potency of CHH molecules. This is in
accordance with previous reports of the importance of the C-terminal portion
of the family of CHH neuropeptides in binding to specific receptors, as shown
in the structure–activity of the moult-inhibiting hormone and CHH
(Katayama et al., 2002;
Katayama et al., 2004;
Mosco et al., 2008).
In this paper, we have shown the co-presence of the specific binding sites
of ES- and PO-CHH in multiple tissues of C. sapidus. As stated above,
the tested tissues of C. sapidus probably contain at least three
types of CHH receptor: specific receptors for (1) ES-CHH and (2) PO-CHH, and
(3) one promiscuous receptor. Thus, the tissues which contain the promiscuous
receptor bind to each CHH and also displace homologous and heterologous CHH.
As for the second messenger, in contrast to ES-CHH which utilizes cGMP in most
tissues where its binding sites were found
(Chung and Webster, 2006),
PO-CHH appears to modulate a different second messenger(s), particularly in
muscles. It will be interesting to examine in future studies how the
structures of these receptors differ from each other within a species as well
as among different species, as it is reported that there is a co-evolution of
binding specificity in families of homologous ligands and their receptors
(Moyle et al., 1994;
Costagliola et al., 2005).
Based on our previous observation (Chung
and Zmora, 2008) and the current binding study, we infer that the
co-presence of ES- and PO-CHH receptors in these tissues may be a common
feature in crustaceans.
|
Acknowledgments |
|---|
|
Footnotes |
|---|
* Present address: Department of Applied Biochemistry, Institute of
Glycotechnology, Tokai University, 1117 Kitakaname, Hiratsuka, Kanagawa
259-1292, Japan 
|
References |
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TOPSummaryINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONReferences |
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