In gel mobility shift assays, in vitro translated LdEcR alone bound weakly to the pal1 ecdysone response ele-ment, although LdUSP alone did not, and this binding was dramatically enhance
Trang 1confirmation of ecdysone receptor and ultraspiracle from the Colorado potato beetle Leptinotarsa decemlineata
Takehiko Ogura1, Chieka Minakuchi1,*, Yoshiaki Nakagawa1, Guy Smagghe2and
Hisashi Miyagawa1
1 Division of Applied Life Sciences, Graduate School of Agriculture, Kyoto University, Japan
2 Laboratory of Agrozoology, Department of Crop Protection, Faculty of Bioscience Engineering, Ghent University, Belgium
The growth of insects progresses via unique
physiologi-cal events such as molting and metamorphosis Those
processes are strictly regulated by two peripheral
hor-mones, molting hormone (20-hydroxyecdysone; 20E)
and juvenile hormone (JH) 20E controls transcription
of target genes by interacting with molting hormone
receptor proteins, which bind to ecdysone response
ele-ments (EcREs) located upstream of the target genes
The transcriptional activation by 20E triggers signal
cascades, and the development is accomplished via
complex regulatory mechanisms [1] The heterodimer
of two nuclear receptors, ecdysone receptor (EcR) and ultraspiracle (USP), functions as a molting hormone receptor, and 20E is known to be a ligand for EcR USP is the homologue of vertebrate RXR [2,3] Amino-acid sequences of EcR and USP were first determined in the dipteran fruit fly Drosophila melano-gaster [4–6], and subsequently determined in other insects [7–25], as well as a crustacean [26] and a tick [27,28] These receptor proteins consist of regions
Keywords
ecdysone receptor (EcR); Leptinotarsa
decemlineata; ponasterone A; ultraspiracle
(USP); 20-Hydroxyecdysone
Correspondence
Y Nakagawa, Division of Applied Life
Sciences, Graduate School of Agriculture,
Kyoto University, Kyoto 606–8502, Japan
Fax: +81 75 7536123
Tel: +81 75 7536117
E-mail: naka@kais.kyoto-u.ac.jp
Present address
*Department of Biology, University of
Washington, Seattle WA 98195–1800, USA
(Received 27 April 2005, revised 13 June
2005, accepted 16 June 2005)
doi:10.1111/j.1742-4658.2005.04823.x
cDNA cloning of ecdysone receptor (EcR) and ultraspiracle (USP) of the coleopteran Colorado potato beetle Leptinotarsa decemlineata (LdEcR and LdUSP) was conducted Amino-acid sequences of the proteins deduced from cDNA sequences showed striking homology to those of other insects, especially the coleopteran yellow mealworm Tenebrio molitor Northern hybridization analysis showed a 12.4-kb message for the LdEcR A-isoform,
a 10.5-kb message for the LdEcR B1-isoform and a 5.7-kb message for the LdUSP, in fat body, gut, integument, testis and ovaries In developmental profile studies, expression of both the LdEcR and LdUSP transcript in integument changed dramatically In gel mobility shift assays, in vitro translated LdEcR alone bound weakly to the pal1 ecdysone response ele-ment, although LdUSP alone did not, and this binding was dramatically enhanced by the addition of LdUSP LdEcR⁄ LdUSP complex also showed significant binding to an ecdysone agonist, ponasterone A (KD¼ 2.8 nm), while LdEcR alone showed only weak binding (KD¼ 73.4 nm), and LdUSP alone did not show any binding The receptor-binding affinity of various ecdysone agonists to LdEcR⁄ LdUSP was not correlated to their larvicidal activity to L decemlineata From these results, it was suggested that multiple factors including the receptor binding affinity are related to the determination of the larvicidal activity of nonsteroidal ecdysone agon-ists in L decemlineata
Abbreviations
ANS-118, chromafenozide; DBH, dibenzoylhydrazine; EcR, ecdysone receptor; EcRE, ecdysone response element; 20E, 20-hydroxyecdysone; pIC50, reciprocal logarithmic value of IC50; PonA, ponasterone A; RH-0345, halofenozide; RH-2485, methoxyfenozide; RH-5849, N-tert-butyl-N,N¢-dibenzoylhydrazine; RH-5992, tebufenozide; RXR, retinoid X receptor; THR, thyroid hormone receptor; USP, ultraspiracle.
Trang 2referred to as A⁄ B, C (DNA binding), D, E (ligand
binding) and F, which is consistent to other members
of the nuclear receptor superfamily Molecular
regula-tory mechanisms of transcriptional activation by 20E
were studied intensively in D melanogaster [29–33],
and were also reported for a dipteran, the yellow fever
mosquitoe Aedes aegypti [34], and lepidopterans, the
tobacco hornworm Manduca sexta [35–37] and the
silkworm Bombyx mori [38,39] On the other hand,
the natural ligand of USP is unknown, although recent
in vitro experiments indicated that JH binds to USP
and regulates transcriptional events [40–42]
Although 20E is a steroidal compound, some
syn-thetic ecdysone agonists which have no steroid
struc-ture are known Interestingly, it has been noted that,
while the binding affinity of ecdysteroids such as 20E
and its agonist, ponasterone A (PonA), is comparable
among insect species, that of nonsteroidal ecdysone
agonists, dibenzoylhydrazines (DBHs), are different
among insect orders [43] Recently, the X-ray crystal
structure of the ligand binding domain of EcR was
solved for the lepidopteran tobacco budworm
Helio-this virescens [44] Superimposition of PonA and a
DBH type compound, BYI06830, as bound to EcR
ligand binding domain, suggested that an aromatic
ring moiety of BYI06830 occupies a binding pocket
which is not fully shared with PonA Thus, there is a
possibility that the difference of binding affinity of
DBHs to receptors among insect species is due to the
difference of structures of the ligand binding pocket
In the other study, we demonstrated that the molting
hormone activities of ecdysone agonists measured in
cultured integument system of the lepidopteran rice
stem borer Chilo suppressalis are correlated to and
ruled by their respective receptor binding affinity to
in vitrotranslated EcR and USP proteins of C
suppres-salis[45] These recent results indicate that the
import-ance to investigate ligand–receptor interactions and
compare structures of molting hormone receptors
among insects is increasing for a better understanding
of the function of molting hormone in insect growth
and development
Previously, we performed structure–activity
relation-ship (SAR) studies of ecdysone agonists using C
sup-pressalis, the lepidopteran Spodoptera exigua and a
coleopteran field pest, the Colorado potato beetle
Leptinotarsa decemlineata[46–57] In those studies, the
larvicidal activity of DBHs against C suppressalis was
correlated with those against S exigua but not
correla-ted with those against L decemlineata, suggesting that
the receptor-binding of DBHs in L decemlineata is
dif-ferent to those in C suppressalis and S exigua The
aim of this study is to examine the SAR of ecdysone
agonists for the molecular interaction with the molting hormone receptor Here, we report (a) the determin-ation of primary amino acid structures of EcR and USP from L decemlineata (b) the analysis of mRNA expression profile of L decemlineata, EcR and USP, and (c) the measurement of the binding affinity of steroidal and nonsteroidal ecdysone agonists to the
in vitro translated receptor proteins Comparison of the receptor-binding affinity between various insects is expected to lead molecular bases for the divergence of the toxicity of ecdysone agonists
Results
cDNA cloning of LdEcR and LdUSP
A 379-bp fragment was amplified by RT-PCR using degenerate primers, and its sequence was determined A deduced amino acid sequence of the PCR product was homologous to a corresponding part of EcRs of other insects Then we subsequently conducted 5¢-RACE and 3¢-RACE, and sequences of 1337-bp and 998-bp frag-ments were determined, respectively Combining these sequences of PCR fragments, we deduced the whole cDNA sequence of LdEcR to be 2714-bp long The lon-gest open reading frame (ORF), which is followed by an in-frame termination codon, encodes a 565 amino acid peptide (Fig 1A) The deduced amino acid sequence has a structure typical for the nuclear receptor super-family We also amplified an 833-bp fragment by 5¢-RACE to determine a 2165-bp sequence A 488 amino acid sequence was deduced from this 2165-bp cDNA sequence, which was different to the 565 amino acid sequence only in a part of A⁄ B region (Fig 1B) A database search was conducted using the blast program (http://www.ncbi.nlm.nih.gov/BLAST/) and the longer sequence (565 amino acids) was found to be highly homologous to previously reported EcR A-isoform of other insects Thus we concluded that the longer cDNA encodes L decemlineata EcR A-isoform (LdEcR-A)
On the other hand, the shorter sequence (488 amino acids) was determined to be an EcR B1-isoform of
L decemlineata(LdEcR-B1)
In the same way, we determined a 1699-bp sequence
by combining sequences of 157-bp, 690-bp and 994-bp
of RT-PCR, 5¢-RACE and 3¢-RACE fragments A 384 amino acid sequence (Fig 1C) encoded by the longest ORF of the cDNA sequence has a structure typical for the nuclear receptor superfamily A database search with the blast program showed that this deduced sequence is highly homologous to other USPs Thus
we determined this sequence as L decemlineata USP (LdUSP)
Trang 3LdEcR-A and LdUSP amino acid sequences were
compared with EcR and USP sequences of other
insects, respectively (Table 1) The C region of
LdEcR shares a very high amino acid identity with
that of other EcR sequences (91–94%) The E region
of LdEcR is also highly homologous to those of
other EcRs (> 60%), especially to EcR-A from
coleopteran Tenebrio molitor (TmEcR-A, 91%) and
orthopteran Locusta migratoria (LmEcR, 89%) The
D region is homologous to those of T molitor and
L migratoria (78% and 70%, respectively), but less
homologous to those of others (< 38%) The A⁄ B
regions are rather diverse among all sequences
(< 52%) Similarly, the amino acid identity of the
C region of LdUSP is also very high among all
sequences (89–95%) Both D and E⁄ F regions are
also highly homologous to those of T molitor and
L migratoria (96% and 75%, 88% and 69%,
respec-tively), although they are less homologous to other
USPs A⁄ B regions of USPs are highly diverse (6–
45%), as observed for EcRs
mRNA expression profiles The spatial expression pattern of EcR mRNA was analyzed using total RNA prepared from the fat body, gut, integument and whole body of L decemlineata larvae at day 4 of the last (4th) instar A 12.4-kb mes-sage was detected by the LdEcR common probe in the integument and whole body, and slightly in the gut The mRNA of EcR was not detectable in the fat body (Fig 2A) We also demonstrated the temporal expres-sion pattern of LdEcR in the integument of 4th instar larvae As shown in Fig 2A, the EcR message steeply increased at day 4, then remained at the high expres-sion level until day 8 Total RNAs from the whole body of male and female adult, testis and ovaries as well as from L decemlineata cells were subjected to northern hybridization analysis Although the EcR message was detected in all tissues, the message was weak in adult males Expression of mRNA of LdEcR seems to be much higher in adult female than in adult male The EcR transcript abounds in L decemlineata
Fig 1 The deduced amino acid sequence of L decemlineata molting hormone receptor (A) The deduced amino acid sequence of LdEcR-A The DNA binding domain (DBD, C-region) is underlined The ligand binding domain (LBD, E-region) is underlined with dashes The junction of LdEcR-A and LdEcR-B1 is shown by an arrow head Gly164 and the downstream sequences are common between LdEcR-A and LdEcR-B1 The five amino acids encoded a 15-bp sequence that is absent in some cDNAs is boxed (B) The deduced amino acid sequence of the iso-form-specific region of LdEcR-B1 This sequence connects to Gly164 in (A) (C) The deduced amino acid sequence of LdUSP The DNA bind-ing domain (DBD, C-region) is underlined The ligand bindbind-ing domain (LBD, E ⁄ F-region) is underlined with dashes The sequence data of LdEcR-A, LdEcR-B1 and LdUSP have been submitted to the DDBJ ⁄ EMBL ⁄ GenBank nucleotide sequence database under the accession number AB211191, AB211192 and AB211193, respectively.
Trang 4cells A 10.5-kb transcript was also detected in all
tis-sues and developmental stages, although the signals
were very weak (Fig 2A) Northern hybridization
ana-lysis using LdEcR-A and LdEcR-B1 probes indicated
that the 12.4-kbp signal is mRNA of LdEcR-A, and
the 10.5-kbp transcript is LdEcR-B1 mRNA (Fig 2B)
We used LdEcR-A in the following experiments of our
study because the expression of LdEcR-A is much
higher than LdEcR-B1
Northern hybridization analysis was also conducted
with the probe for USP using the same series of total
RNA The expression pattern of LdUSP, as 5.7-kb
message, was similar to that of LdEcR-A over the dif-ferent tissues and developmental stages The very high expression was observed in the whole body of female adults (Fig 2A)
SDS/PAGE and gel mobility shift assay of
in vitro translated proteins LdEcR-A and LdUSP proteins were prepared by
in vitro transcription⁄ translation with [35S]methionine and subjected to SDS⁄ PAGE analysis (Fig 3) Molecular mass for LdEcR-A and LdUSP was
A
B
Fig 2 mRNA expression profiles of LdEcR
and LdUSP (A) LdEcR mRNA and LdUSP
mRNA expression in the fat body (FB), gut
(GUT), integument (INT) and whole body
(WB) at day 4 in the last larval instar, in the
INT at day 0, 2, 4, 6 and 8 in the last larval
instar, and in the adult male WB (#), female
WB ($), testis TES and ovary (OVA), and
L decemlineata cells BCIRL-Lepd-SL1
(CELL) For detecting LdEcR transcripts,
LdEcR common probe was used Ethidium
bromide staining of rRNA is shown as a
control for equal loading (B) The expression
of LdEcR-A and LdEcR-B1 mRNA in
integument of last instar larvae Temporal
expression profiles were studied using
isoform-specific probes.
Table 1 Comparison of sequences Sequence comparison between (A) L decemlineata EcR A-isoform (LdEcR-A) and other EcR-A’s (B) LdUSP and other USPs Amino-acid identity against LdEcR-A and LdUSP is expressed as percentage in each region We could not com-pare F regions because their sequences are too short TmEcR-A: Tenebrio molitor EcR-A (GenBank accession number Y11533 [22]), LmEcR: Locusta migratoria EcR (AF049136), DmEcR-A: Drosophila melanogaster EcR-A (M74078, S63761), CsEcR-A: Chilo suppressalis EcR-A (AB067811), AamEcR-A2: Amblyomma americanum (AF020188), UpEcR: Uca pugilator EcR-A2 (AF034086), TmUSP: T molitor USP (AJ251542), LmUSP: L migratoria USP (AF136372), DmUSP: D melanogaster USP (X53417), CsUSP: C suppressalis USP (AB081840), UpUSP: U pugilator USP (AF032983).
Trang 5estimated to be 64 kDa and 49 kDa, respectively, from
the mobility in the gel The 64 kDa LdEcR-A protein
was consistent with the predicted size from the
deduced amino acid sequence (63.4 kDa) In the lane
of translation products of LdEcR-A, extra bands with
lower molecular weight were observed They were
probably degradation products of the full length
64 kDa protein Similar results were obtained for the
in vitro translation of USP of C suppressalis [13]
Otherwise, they might be products of internal initiation
or premature termination of translation The 49 kDa
LdUSP protein was slightly larger than the size
predic-ted from deduced amino acid sequence (43.1 kDa)
This might be the result of post-translational
modifica-tions
A gel mobility shift assay was conducted using
in vitro translated LdEcR-A and LdUSP proteins
(Fig 4) The mixture of LdEcR-A and LdUSP clearly
bound to the pal1 EcRE probe [58] Interestingly, a
weak signal was also detected for LdEcR-A alone
When a 100-fold excess of unlabeled competitor was
added, the band shift observed for the mixture of
LdEcR-A and LdUSP disappeared Drosophila hsp27
EcRE [59] probe gave the same results as pal1 (data
not shown) These results indicate that LdEcR-A and
LdUSP form the complex (LdEcR-A⁄ LdUSP) and
bind to the EcRE Addition of 20E to the reaction
mixture enhanced the probe-binding (data not shown)
as observed in D melanogaster [3], B mori [60],
Choris-toneura fumiferana [15] and Chironomus tentans [17],
although EcR alone did not show binding to EcRE in those studies
Ligand binding assay The specific binding of in vitro translated proteins to PonA was calculated by the difference between total binding and nonspecific binding as we previously reported [45] The dissociation equilibrium constant,
KD, of PonA was calculated from the saturation curve
of the specific binding and the Scatchard plot (Fig 5) The KDvalues of LdEcR-A and LdEcR-A⁄ LdUSP cal-culated from saturation curves were 72.6 and 2.8 nm, respectively
Receptor-binding affinity of ecdysone agonists to LdEcR-A⁄ LdUSP is shown in Table 2 The binding affinity of DBHs tested in this study was relatively low (< 6.00 in terms of pIC50) compared to that against
C suppressalis The SARs for binding affinities of
Fig 3 SDS ⁄ PAGE of in vitro translated LdEcR-A and LdUSP
pro-teins pET-23a (+) vector (lane 1), in vitro translated LdEcR-A (lane
2), LdUSP (lane 3) and LdEcR-A ⁄ LdUSP translated simultaneously
in the same tube (lane 4) with [ 35 S]methionine were separated on
10% SDS ⁄ PAGE gel.
Fig 4 Binding of LdEcR-A ⁄ LdUSP complex to the ecdysone response element (EcRE) In vitro translated LdEcR-A and ⁄ or LdUSP protein were incubated with 32 P-labeled pal1 EcRE and then applied for nondenaturing polyacrylamide gel electrophoresis Water (lane 1), a reaction mixture using pET-23a(+) vector substitute to LdEcR-A or LdUSP construct (negative control, lane 2), LdEcR-A (lane 3), LdUSP (lane 4) and LdEcR-A and LdUSP translated simulta-neously in the same tube (lane 5 and 6) were mixed with probes and loaded In the lane 6, 100-fold excess of the same EcRE oligo-nucleotide was added for competition experiment.
Trang 6ecdysteroids were linearly correlated between L
decem-lineataand C suppressalis, whereas those of DBHs were
not (Fig 6A) No positive correlation was observed
between receptor-binding and larvicidal activity against
L decemlineatawith respect to DBHs (Fig 6B) [52,55]
Discussion
The comparison of EcRs and USPs
Three cDNAs encoding LdEcR-A, LdEcR-B1 and
LdUSP were obtained, and they had high amino acid
identity with EcR-A, EcR-B1 and USP of other
insects, respectively It is known that many insect
spe-cies have two or three EcR isoforms (EcR-A, EcR-B1
and EcR-B2), and their functions are different depend-ing on tissues, developmental stages and species [5,12,14,22,61,62] In D melanogaster, it was reported that EcR-B1 is predominantly expressed in larval tissues, and expression of EcR-A is predominant in imaginal discs [5] In B mori, C fumiferana, C sup-pressalis and M sexta, EcR-B1 was observed as the major isoform in larval stage, although expression pat-terns of EcR isoforms appeared to be diverse among these lepidopteran insects [12,14,61,62] Thus, functions
of EcR isoforms in larval stage might be different between lepidopteran and dipteran insects In this study, we showed that L decemlineata also possesses two isoforms, and the expression of LdEcR-A was much stronger than LdEcR-B1 (Fig 2A) Expression
of EcR-A was also predominant in larval tissue of coleopteran T molitor [22] These facts also indicate that the dominant isoform of EcR in larval develop-ment is different depending on tissues and insect orders Furthermore, LdEcR-A transcripts in the integument increased steeply at day 4 of 4th instar lar-vae (Fig 2A) We previously reported that the molting hormone titer in the hemolymph during 4th instar development of L decemlineata was constant until day
6 except for a small peak at day 4, and rapidly increased to the major peak between day 8 and day 9 [63] Thus, EcR-A transcripts in integument are pro-bably induced by the small peak of ecdysteroid on day
4, prior to the major hemolymph ecdysteroid peak The strong expression of EcR transcripts prior to the peak of ecdysteroid titer in hemolymph was also repor-ted in various insects such as D melanogaster and
M sexta [5,10,12,14,22,61] Therefore, expression of EcR mRNA could be up-regulated by the rising of ecdysteroid titer in hemolymph to secure sufficient responsibility to the peak of ecdysteroid titer Further studies for the mechanism of their transcriptional regu-lation would support the elucidation of different roles
of EcR isoforms in Coleoptera
Although we have obtained only one isoform from
L decemlineata, two USP isoforms, MsUSP-1 and MsUSP-2, were cloned from lepidopteran M sexta, and their different role on MHR3 promoter activation was shown [21,64] Dipteran Aedes aegypti and C ten-tans also possess two USP isoforms (A and USP-B) [17,65] Therefore, other USP isoforms might exist and contribute to the development of L decemlineata LdUSP showed higher conservation with two USP isoforms of the tick Amblyomma americanum (AamUSP-1 and AamUSP-2 [28]) than USPs of
A aegypti, C tentans and D melanogaster, but no significant difference was observed between homologies
to two USP isoforms LdUSP as well as T molitor
Fig 5 The binding affinity of ponasterone A (PonA) to in vitro
translated proteins In vitro translated LdEcR-A or LdEcR-A ⁄ LdUSP
were incubated with various concentration of [3H]-labeled PonA, in
the presence or absence of excess PonA Saturation
radioligand-binding curves and Scatchard plots are shown.
Trang 7and L migratoria USP showed higher homology to
RXR of human and mouse than USP isoforms of
dip-teran and lepidopdip-teran insects Thus, the functions of
USP of L decemlineata, T molitor and L migratoria
might have the similar function to RXR, being
differ-ent from those of Diptera and Lepidoptera The
LdUSP transcript showed similar developmental and
spatial expression profiles as LdEcR-A USP
tran-scripts also changed with the hemolymph ecdysteroid
titer in T molitor, C fumiferana and M sexta,
although their expression profiles were different from
the case of L decemlineata [15,21,23] On the contrary,
USP mRNA expression in the epidermis of C
suppres-salis and B mori was ubiquitous throughout the last
larval instar [13,66] Such a difference suggests that
hormonal regulatory mechanisms of USP transcription
are different among insect species
Previously, it was pointed out that there is a
con-served motif (motif-1) between T molitor EcR-A (amino
acids 29–39) and D melanogaster EcR-A (143–153)
The presence of conserved motif-2 between M sexta
EcR-A (60–79) and D melanogaster EcR-A (177–196)
was also pointed out in A⁄ B region of EcRs [22] The
sequence of amino acids 108–118 of LdEcR-A is
homo-logous to the motif-1, and this was also the case for EcR-A of coleopteran T molitor Interestingly, the sequence of amino acids 143–162 of LdEcR-A also has
a striking homology with the motif-2 It is different from
T molitor EcR-A, but consistent to EcR-A of lepidop-teran M sexta and C suppressalis EcR-A sequences of lepidopteran insects showed relatively lower homology
to LdEcR-A in comparison with EcR-A homologies between insects of other orders and coleopteran L de-cemlineata Thus, each of these two conserved motifs in
Table 2 Binding affinity of ecdysone agonists Binding affinity
(pIC50: M) of steroidal and nonsteroidal ecdysone agonists to the
receptor of L decemlineata (LdEcR-A ⁄ LdUSP) is shown
Com-pound 9: RH-5849, 10: halofenozide (RH-0345), 11: tebufenozide
(RH-5992), 12: methoxyfenozide (RH-2485), 13: chromafenozide
(ANS-118).
No.
N N H O
O Xn
Yn
Binding affinity
L decemlineata
12 3,5-(CH3)2 2-CH3-3-OCH3 5.94
13 3,5-(CH 3 ) 2 2-CH 3 -3,4-(CH 2 ) 3 O- 5.77
A
B
Fig 6 Relationships among biological activities (A) Receptor-bind-ing affinity (pIC50: M) of ecdysone agonists against the receptor of
L decemlineata is compared to that of C suppressalis Triangles: ecdysteroids Circles: DBHs (B) Receptor-binding affinity (pIC 50 : M) and larvicidal activity (pLD50: m M ⁄ insect) of DBHs against
L decemlineata [52,55] were compared.
Trang 8A⁄ B region might play different roles and be important
for determining the function of the EcR-A, which is
dif-ferent among insect species
The functional analysis of LdEcR-A and LdUSP
The gel mobility shift assay showed that the complex
of in vitro translated LdEcR-A and LdUSP proteins
bound to EcREs, indicating that cDNAs cloned in this
study encode functional EcR and USP LdEcR-A
alone also bound to EcREs, although the binding was
much weaker than that of LdEcR-A⁄ LdUSP The
binding experiment of EcR and USP proteins to
EcREs was also conducted in dipteran D melanogaster
[2], A aegypti [65] and C tentans [17], and lepidopteran
B mori [60], C suppressalis [13] and C fumiferana
[15] In those experiments, EcR alone did not bind to
EcRE, which is different from the result of this study
The degree of mobility shift of a band which is caused
by monomeric binding of LdEcR-A alone should be
much smaller than that by LdEcR-A⁄ LdUSP
How-ever, the degree of band retardation by addition of
A alone was a little larger than that of
LdEcR-A⁄ LdUSP Therefore LdEcR-A alone appeared to
bind to EcREs as a homodimer Homodimeric binding
to DNA sequences is reported for vertebrate THR,
which shows similar characteristics to EcR [67,68]
Furthermore, it is concerned that the determinant of
the binding type of nuclear receptors to its response
element, namely monomer, homodimer and
heterodi-mer, is the nucleic acid sequence of the hormone
response element [69] Thus, perhaps pal1 and hsp27
probes, which are not intrinsic EcREs of L
decemlin-eata, enable LdEcR-A to form a homodimer
From the ligand binding assays using [3H]PonA, the
KD value of ponA for LdEcR-A⁄ LdUSP is 2.8 nm
The KDvalue was close to that for CsEcR-B1⁄ CsUSP
(C suppressalis) and DmEcR⁄ DmUSP (D
melanogas-ter), which have been reported to be about 1.0 nm
[3,45] Thus, it was shown that in vitro translated
LdEcR-A⁄ LdUSP heterodimers are capable of
inter-acting with ligands with high affinity, and possess a
required ability for a receptor
The ligand binding affinity of LdEcR-A/LdUSP
As shown in Fig 6A, receptor-binding affinities of
ecdysteroids were well correlated between
LdEcR-A⁄ LdUSP and CsEcR ⁄ CsUSP, whereas this is not
the case for DBHs As shown in Table 1, sequence
homology of ligand binding domains between
LdEcR-A and lepidopteran CsEcR-A is considerably
lower than those between LdEcR-A and coleopteran
TmEcR-A Therefore, the difference between struc-tures of ligand binding domain is most likely respon-sible for the difference in receptor-binding affinities
of DBHs among different insect orders On the other hand, the ligand binding domain should have a sub-stantial conservation in the structure which is neces-sary for ecdysteroid binding regardless of insect orders, because 20E is believed to be the most active form of molting hormone in all insects The crystal structure analysis of EcR of lepidopteran H virescens indicated the amino acid residues which are import-ant for the binding with PonA and a DBH analog BYI06830 [44] We examined the conservation of these amino acid residues among several insects by comparing the sequences of the ligand binding domain of EcRs (Fig 7) As expected, amino acids which have been shown to be important for ecdyster-oids-binding are highly conserved among all insects Thus, probably the structure of EcR ligand binding domain is also conserved among insects for arran-ging these amino acid residues in proper location to accommodate an ecdysteroid molecule However, amino acids which have been shown to be important for binding with BYI06830 are also well conserved among EcRs The difference in receptor-binding affinity of DBHs among insect orders might rather
be attributed to the amino acid residues which are considered to be involved in binding to both types
of ligands Amino acids which are corresponding to Met429 and Thr451 of LdEcR-A represent the pos-sibility, as they are different between EcR-As of lepi-dopteran and other insects Further studies such as point mutation and X-ray crystal structure analysis
of various EcRs will elucidate the factors responsible for difference of the binding of DBHs to EcRs among insects
We previously reported that larvicidal activity and receptor-binding of DBHs are correlated very well in C suppressalis, suggesting that receptor-binding affinity of DBHs is concerned to rule the strength of their
larvicid-al activity [45] Among DBHs, it was reported that hlarvicid-alo- halo-fenozide (RH-0345) has a high insecticidal activity against coleopteran field pests such as Popillia japonica and L decemlineata, but tebufenozide (RH-5992), meth-oxyfenozide (RH-2485) and chromafenozide (ANS-118) were not so potent against these insects [43] Thus, based
on the case of DBHs in C suppressalis, the receptor-binding affinity of RH-0345 was expected to be high in
L decemlineata However, the receptor-binding affinity
of RH-0345 and the other three DBHs to
LdEcR-A⁄ LdUSP was low (Table 2) Furthermore, the binding affinity of RH-2485 and ANS-118 was higher than that
of RH-0345 This means that the receptor-binding
Trang 9affinity of DBHs is not a major factor to determine the
larvicidal activity in L decemlineata To make this
point clear, we tested the receptor-binding affinity of
other DBHs against LdEcR-A⁄ LdUSP (Table 2) The
measured binding affinity of 12 DBHs (1–12; Table 2)
did not show any correlation to the larvicidal activity
(Fig 6B) Furthermore, our previous SAR study
demonstrated that hydrophobicity of compounds is
important to larvicidal activity of DBHs against C
sup-pressalis; thus the hydrophobicity is important for
receptor-binding affinity However, the hydrophobicity
of DBHs was not correlated to their receptor-binding
activity to LdEcR-A⁄ LdUSP, although existence of
optimal hydrophobicity for larvicidal activity of DBHs
against L decemlineata was shown in our previous
study [52,55] Therefore, other factors such as
absorp-tion through the membrane and metabolism in the
insect body might play a very important role Otherwise,
although DBHs are considered to show potency as
agonists of 20E in C suppressalis by binding to
EcR⁄ USP, there is a possibility that different
mecha-nisms, such as neurotoxicity and the existence of other
receptors, give influence on the larvicidal activity of
DBHs in L decemlineata Further study such as X-ray
crystal structure analyses of EcR⁄ USP and metabolic
analyses of DBHs in various insects would confer new
knowledge of the mode of action of DBHs
A recent study of phylogenic analysis suggests that
EcR and USP have coevolved during diversification
of insects [70], which is supported by the result of this study (Table 1) It was also suggested that EcRs and USPs of lepidopteran and dipteran insects are under a strong acceleration of evolutionary rate in comparison with those of insects in other orders This raises a possibility that the results of studies for EcRs and USPs of lepidopteran and dipteran insects are not necessary applicable for those of insects in other orders Therefore, although a number of stud-ies has been conducted in lepidopteran and dipteran insects, more detailed studies on EcR and USP of insects in other orders and other ecdysozoan are necessary for precise understanding of their physiology
In conclusion, we successfully performed cDNA cloning of EcR and USP of L decemlineata, and
in vitro translation of corresponding receptor proteins The translated proteins could bind to EcREs and ecdysone agonists as heterodimers, indicating that they are functional molting hormone receptors of
L decemlineata As L decemlineata is a major pest in agriculture world-wide, the receptor proteins isolated
in this study can be very helpful to develop effective compounds in high throughput assays and SAR stud-ies Furthermore, although EcR and USP proteins have been isolated from various insects, the down-stream of the ecdysone signaling pathway which is triggered by their activation function of transcription still remains to be elucidated The isolated genes and
Fig 7 Comparison of the ligand binding domain sequence of EcRs Amino-acid sequences are compared for ligand binding domain of EcRs LdEcR: L decemlineata EcR, TmEcR: T molitor EcR, LmEcR: L migratoria EcR, D melanogaster EcR, CcEcR: Ceratitis capitata EcR (Gen-Bank accession number AJ224341), AaeEcR: A aegypti EcR (P49880), CtEcR: Chironomus tentans EcR (P49882), CsEcR: C suppressalis EcR, CfEcR: C fumiferana EcR (U29531), HvEcR: H virescens EcR (O18473), BmEcR: B mori EcR (P49881) Sequences are separated in three groups according to insect orders The upper three sequences are EcRs of insects belonging to orders other than Diptera and Lepidop-tera The middle four (shaded with light gray) are EcRs of dipteran insects, and the lower four are that of lepidopteran insects Amino-acid residues which are important for the binding with PonA are shaded with dark gray, and that of BYI06830 are boxed [47] The position of LdEcR-A Met429 and Thr451 are shown by arrow heads Activation function 2 (AF-2) is indicated with dots Amino acids which correspond
to a-helical structures are also indicated.
Trang 10proteins will also be available to study
protein–pro-tein interactions in such hormonal regulatory
path-ways, and be helpful to understand the complicated
physiology of insects
Experimental procedures
Chemicals
Ecdysone and 20E were purchased from Sigma Chemical
Co (St Louis, MO, USA) and PonA was from Invitrogen
Corp (Carlsbad, CA, USA) Tritiated ponasterone A
([3H]PonA, 150 CiÆmm)1) was purchased from American
Radiolabeled Chemicals Inc (St Louis, MO, USA)
Ecdy-steroids (cyasterone and makisterone A) and all DBHs were
from our stock samples [52,55]
Isolation of RNA from L decemlineata
Larvae and adults of L decemlineata were reared as
des-cribed previously [63] A L decemlineata cell line
(BCIRL-Lepd-SL1), which was established from female pupae, was
routinely maintained as described previously [71] Total
RNA was isolated from the whole bodies and tissues of last
instar (4th) larvae and adults using TRIzol (Gibco BRL,
Grand Island, NY, USA) as described previously [13]
BCIRL-Lepd-SL1 was also used for isolation of total
RNA Poly (A)-rich RNA was purified from the total RNA
using mRNA Purification Kit (Amersham Bioscience Corp.,
Piscataway, NJ, USA)
RT-PCR
Reverse-transcription was conducted using ReadyÆToÆ
GoTM T-Primed First-Strand Kit (Amersham Bioscience
Corp.) for total RNA isolated from the fat body and integument of last instar larvae Three forward and reverse degenerate primers were designed for EcR based
on amino acid sequences conserved in C-E regions of other EcRs (Table 3) In the same way, two forward and one reverse degenerate primers were designed for USP using the homology in C region of other USPs (Table 3) The first PCR for EcR was conducted using LdEcR-F1 and LdEcR-R1 primers (annealing temperature: 48C) Subsequently, the second and the third nested PCR were performed with LdEcR-F2 and LdEcR-R2 primers (52C) and with LdEcR-F3 and LdEcR-R3 primers (46C), respectively The first PCR with LdUSP-F1 and LdUSP-R1 primers and the second nested PCR with LdUSP-F2 and LdUSP-R1 primers were conducted for USP Annealing was performed at 48C and 46 C, respectively
Rapid amplification of cDNA ends Poly (A)-rich RNA extracted from L decemlineata cells was subjected to the 5¢- and 3¢- rapid amplification of cDNA ends (RACE) with SMARTTM RACE cDNA amplification kit (Clontech, Palo Alto, CA, USA) For both of EcR and USP, two reverse primers for 5¢-RACE and two forward primers for 3¢-RACE were designed (Table 3) 5¢-RACE for EcR was executed by PCR with primer LdEcR-RR1, and 3¢-RACE for EcR was per-formed with LdEcR-RF1, respectively, according to manu-facturer’s instructions 5¢-RACE and 3¢-RACE were followed by nested PCR using LdEcR-RR2 (annealing temperature: 66C), LdEcR-RF2 (66 C), respectively In the same way, 5¢-RACE for USP was executed with LdUSP-RR1, and 3¢-RACE for USP with LdUSP-RF1 Each RACE reactions were followed by nested PCR
Table 3 Primers used in this study Degenerate primers and 5¢- and 3¢-RACE primers are shown The term N means a mixture of A, T, G and C In the same way, D (A, G, T), H (A, C, T), K (G, T), M (A, C), R (A, G), S (C, G), W (A, T) and Y (C, T) means a mixture of deoxynucleo-side.
Degenerate primers
5¢-RACE
3¢-RACE