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Isolation and expression of the ecdysteroid-inducible
angiotensin-converting enzyme-related gene in wing discs of
Bombyx mori
Guo-Xing Quan
a, Kazuei Mita
b, Kazuhiro Okano
c, Toru Shimada
d, Nanako Ugajin
a,
Zhao Xia
a, Noriko Goto
a, Eiji Kanke
a, Hideki Kawasaki
a,*aFaculty of Agriculture, Utsunomiya University, 350 Mine, Utsunomiya, Tochigi 321-8505, Japan
bGenome Research Group, National Institute of Radiological Sciences, Anagawa 4-9-1, Inageku, Chiba 263-8555, Japan
cLaboratory of Molecular Entomology and Baculovirology, Institute of Physical and Chemical Research,(RIKEN), Hirosawa 2-1, Wako
351-0198, Japan
dDepartment of Agricultural, Environmental Biology, University of Tokyo, Yayoi 1-1-1, Bunkyo-ku, Tokyo 113-8657, Japan
Received 20 May 1999; received in revised form 4 April 2000; accepted 6 June 2000
Abstract
We isolated a clone encoding a putative angiotensin-converting enzyme-related gene from the wing disc cDNA library of the silkworm,Bombyx mori(refer to asBmAcer). The predicted open reading frame encoded 648 amino acids with about 50% identities with theDrosophila melanogasterangiotensin-converting enzymeAnceandAcer. Northern analysis identified a 2.2-kilobase mRNA which was abundant in wing discs two days after the beginning of wandering. An accumulation of the transcript was observed approximately 2 h after 20–hydroxyecdysone (20E) exposure in vitro and was blocked slightly by a protein synthetic inhibitor. These data suggest that the transcription of theBmAcer gene is directly 20E-inducible.2001 Elsevier Science Ltd. All rights reserved.
Keywords:Angiotensin-converting enzyme; Metamorphosis;Bombyx mori; 20–hydroxyecdysone; Wing disc
1. Introduction
The Ashburner’s model (1974, 1991), which explained the action of ecdysone in Drosophilasalivary glands, was applied to imaginal discs (Natzle, 1993). Natzle offered the presence of two ecdysone-dependent pathways and the existence of early effector genes related to the initial cellular events leading to dramatic morphogenetic changes. As examples, he referred to 20E-inducible membrane bound polysome (IMP) genes inDrosophilaimaginal discs. The initiation of transcrip-tion of the IMP–E1 (IMP–early 1; Natzle et al., 1988), IMP–E2 (Paine-Saunders et al., 1990), and IMP–E3 (Moore et al., 1990) genes were induced by 20E in the presence of cycloheximide (CH) inDrosophilaimaginal discs in vitro. These genes, which are thought to encode a secreted protein or a protein working on cell rearrange-ment, belong to the group of early effector genes.
* Corresponding author. Fax:+11-81-28-649-5401.
E-mail address:[email protected] (H. Kawasaki).
0965-1748/01/$ - see front matter2001 Elsevier Science Ltd. All rights reserved. PII: S 0 9 6 5 - 1 7 4 8 ( 0 0 ) 0 0 1 1 2 - 0
The rate of cell proliferation increases in wing discs after the disappearance of hemolymph juvenile hormone in the last larval instar ofBombyx mori(Kurushima and Ohtaki, 1975), and the low concentration of ecdysteroids promotes cell proliferation after the wandering stage (Kawasaki, 1998). Cuticle deposition, evagination, the change in cell shape, and the appearance of newly syn-thesized peptides were observed in Bombyx wing discs two days after wandering (Kawasaki and Iwashita, 1987; Kawasaki, 1998), when the peak of ecdysteriod level was observed (Quan et al., 1998).
2. Materials and methods
2.1. Experimental animals
Bombyx morilarvae of the C108 strain (from National Institute of Sericultural and Entomological Science of Japan) were reared at 25°C under a photo period of 12 h of light and 12 h of dark. Since the 4th larval ecdysis occurs during the scotophase, the newly molted 5th instar larvae were segregated immediately after the onset of the photophase (Sakurai, 1984), and this day was designated as day 0. Under these conditions, most larvae began wandering in the late photophase of day 6–6.5.
2.2. cDNA library construction and sequencing
The cDNA libraries were prepared from 0 (W0) and two days (W2) after the beginning of wandering in the 5th instar larvae to compare the cDNAs of the two stages. Two cDNA libraries were constructed in a Uni– Zap XR vector as described previously (Quan et al., 1998) using 5 µg of poly(A)+RNA from the wing discs of W0 and W2. From both cDNA libraries, 999 cDNA clones respectively, were randomly sequenced from the 59 end about 700 bp length respectively.
AfterE. coliXL1–Blue cells were grown individually in 2.5 ml of LB broth containing ampicillin, the plasmid DNAs were isolated by an automated DNA extractor (Model PI–100, Kurabo Industries Ltd.). A sequencing reaction using the dye-primer method (DYEnamic Energy Transfer T3 dye-primer kit, Amersham) was car-ried out by another robot (Catalyst 800, ABI). The flu-orescence-labeled DNAs were applied to an automated DNA sequencer (Model 377XL, ABI).
The BLAST (Basic Local Alignment Search Tool) program was used to search the GenBank (SWISS– PROT) sequence repository for identity (Altschul et al., 1990). Polypeptide sequences were aligned using the ClustalW (Thompson et al., 1994). Pairwise alignments of the DNA sequences were carried out using the LALIGN program in the FASTA package (Pearson and Lipman, 1988; Pearson, 1990).
2.3. Rapid amplification of the cDNA 59 terminal end (59RACE)
Total RNA prepared from the W2 stage using ISOGEN (Nippongene) was reverse-transcribed with the SP1 primer (TAAGCTGGCCCTGTTCTT) whose complementary sequence of positions 310 to 327 is underlined in Fig. 1.; the primer was then coupled with the anchor oligonucleotide at the 59 end using the 59RACE kit (Gibco BRL, Life Technologies). The PCR was performed with a complementary sequence to the anchor and the SP2 primer (GGTCGCTTTGTCC AGATGCA) whose complementary sequence of
pos-itions 280 to 300 is underlined in Fig. 1. The PCR pro-ducts were purified and cloned into a pBluscript SK(2) vector (Stratagene).
2.4. Sequencing analysis of the rest of the BmAcer gene
PCR products after 5’RACE were cloned into Bluscript SK- and were sequenced from both direction. WS72 clone was digested with EcoRI and XhoI, and each fragment (1105 bp, 1107 bp) was subcloned into Bluscript SK- and sequenced from both direction. Sequence was operated using Thermo Sequenase fluor-escent labeled primer cycle sequencing Kit with 7-deaza-dGTP (Amersham pharmacia, Tokyo) by an automatic DNA sequencer (DSQ-1000L, Shimadzu, Tokyo).
2.5. Northern blot analysis
Total RNAs were extracted from the wing discs, fat bodies, mid guts and silk glands of the 5th instar larvae and wing tissues of pupae. Fifteen (wing) or twenty (other tissues) micrograms of RNA were separated by electrophoresis on an agarose gel containing formal-dehyde and transferred onto a nylon membrane (Hybond N+, Amersham). The membrane was hybridized with the 268 bp PCR product, which was random-labeled by incorporation of digoxigenin–11–dUTP (Boehringer Mannheim) with primers indicated as dotted lines in Fig. 1. After PCR, the product was cloned into a pBluescript SK-vector and sequenced. After hybridization at 55°C, washes were performed successively for 10 min in 2×SSC with 0.1% SDS twice, then for 20 min in 0.1×SSC with 0.1% SDS at 68°C.
2.6. In vitro culture of wing discs
Wing discs dissected out from the fifth instar day 6 larvae were rinsed with phosphate-buffered saline (PBS) and cultured according to the method of Kawasaki (1989). The Grace’s medium (Gibco BRL, Life Technologies) was supplemented with 10% fetal calf serum (GIBCO BRL, Life Technologies). Twenty–hyd-roxyecdysone (SIGMA CHEMICAL CO.) stock solution in ethanol was added and used at the indicated final con-centrations. The control discs were treated with equival-ent volumes of ethanol alone.
Fig. 1. The cDNA nucleotide and predicted protein sequence of theBmAcergene. TheBmAcercDNA contains a 1944 bp open reading frame. The stop codon is indicated by asterisks, and the polyadenylation signal is underlined. The PCR–oligonucleotide primer sites used to generate the 268 bp Northern blot hybridization probe are indicated by the dotted lines. The putative signal peptide is marked in a broken line, the potential N-linked glycosylation sites are underlined, and the conserved HEXXH motif and E405and D409, which relate to enzymatical activity, are indicated
by fat letters. The sequence ofBmAcerreported in this paper has been deposited in the Gene–Bank database (accession No. AB026110).
performed as described above except for a hybridization temperature of 42°C.
3. Results
3.1. Cloning and sequence of a Bombyx angiotensin-converting enzyme-related (BmAcer) gene
Gene-expression patterns significantly depend on the tissues as well as the developmental stages in multi-cellular organisms. The cDNAs from which the ESTs (expressed sequence tags) are derived are present in
libraries in proportion to the level of mRNA in the tissues from which the library has been prepared (Marra et al., 1998). The frequency of the cDNA clone of each gene reflects the level of mRNA.
determined. Fig. 1 presents the nt sequence and its deduced amino acid sequence for the 648 amino acids with a hydrophobic N-terminal domain, which is the sig-nal peptide for the secreted protein. A homology search of the deduced amino acid sequence indicated that the sequence shows a high homology with the angiotensin-converting enzyme (ACE) superfamily, a 51% identity with Drosophila Ance, and a 46% identity with Droso-phila Acer and human ACE. Since several of the func-tional sites of ACE such as the intact ACE Zn2+ -bind-site motif (H–E–X–X–H) and the two amino acids (Glu, Asp) relating to enzymatical activity (Williams et al., 1994) are conserved in the deduced amino acid sequence, as shown in Figs. 1 and 2, it is likely that we isolated the cDNA clone encoding a Bombyx homolog of ACE designated as Bombyx angiotensin-converting enzyme-related gene (BmAcer).
3.2. Developmental expression of BmAcer mRNAs
To characterize the transcripts found during wing disc morphogenetic processes, the 268 bp cDNA of BmAcer
amplified by PCR with the primers described in Fig. 1 was used as a probe in Northern analyses. Hybridization with the probe gave three bands of 2.2 kb, 4.0 kb and considerably higher molecular weight (faint). Judging from the sizes of the ACE transcripts of other species, the 2.2 kb appears to be the transcripts of the BmAcer
gene, while the other bands remain unknown. During the feeding period of the fifth larval instar, the BmAcer
transcript could not be detected. At the W1 stage, one day after the beginning of wandering, the BmAcer tran-script began to accumulate, and a maximum peak in the transcriptional levels reached at day eight (W2); then a sharp decrease in the transcript levels was then observed at the end of cocoon spinning and the transcript was not detectable at the larval–pupal ecdysis (P0) as shown in Fig. 3.
3.3. Accumulation of the BmAcer mRNAs in vitro
A Northern blot was prepared from day six wing discs cultured in vitro for 12 h with concentrations of 20E from 1028 M to 1024 M. In order to allow for the
dis-sociation of endogenous hormone/recepor complexes (half life equals 30 min at 25°C; Yund and Fristrom,
Fig. 2. Comparison of the amino-acid sequence around the conserved motif relating to enzymatical activity deduced from the nucleotide sequence of cDNA forBombyxwith reported ACEs. The numbering corresponds to the first amino acid residue of the regions shown and indicates their position within the respective protein. The arrows mark putative Zn2+-binding sequences and asterisks indicate amino acid residues relating to
enzymatical activity. DmACER: Drosophila melanogaster Acer (Taylor et al., 1996; AC: X96913), DmANCE: Drosophila melanogaster Ance
(Cornell et al., 1995; AC: U25344),HsACE: Homo sapiens Ace(Ehlers et al., 1989; AC: M26657–1).
Fig. 3. Developmental profile of the BmAcer transcript. Fifteen (wing) or twenty (fat body, mid gut, silk gland) micrograms of total RNA were isolated at various developmental stages during the fifth larval stadium and were Northern blotted and hybridized with a 268 bp cDNA probe. Larvae fed for six days in the fifth larval stadium and then began spinning. Larvae pupated 3.5 days thereafter. The top panel shows the hybridization bands. The bottom panel shows rRNAs stained with ethidium bromide as a control. (A) Wing discs in the fifth larval instar. a: Day 0, b: Day 2, c: Day 4, d: Day 6, e: W1, f: W2, g: W3, h: P0.
1975; Natzle, 1993) that may have been present in some of the larvae at the time of disc isolation, the discs were incubated for 4 h prior to the addition of the hormone. As shown in Fig. 4, the levels of BmAcer mRNAs had a graded response to 20E over a 1000-fold range of con-centrations; theBmAcertranscript was weakly detectable at 1027 M and had a peak at around 1025 M. When
hormonal levels increased to 1024 M, the expression of
BmAcer decreased. These results indicate that BmAcer
expression depends on hormonal concentration and can be ecdysone-inducible.
Wing discs were cultured for various periods with 1025 M 20E. Fig. 5 shows that the BmAcer transcript
Fig. 4. Dose-response profile forBmAcertranscript induction by 20E in vitro. Day 6 wing discs from the fifth instar larvae were incubated in various molar concentrations of 20E for 12 h, then total RNA (15
µg per lane) was then Northern blotted and hybridized with the same cDNA probe as in Fig. 3. Ribosomal RNA (rRNA) was used as a quantitative control. The alphabet on the top of the panel denotes the concentration of 20E. a: 0 M, b: 1028M, c: 1027M, d: 1026M, e:
1025M, f: 1024M.
Fig. 5. Time-course of BmAcer expression in cultured wing discs treated with 1025M 20E. Numbers on the top of the panel denote the
incubation time (h). Total RNA (15µg per lane) was Northern blotted and hybridized with the same cDNA probe as used in Fig. 3. Riboso-mal RNA (rRNA) was used as a quantitative control.
of this gene may constitute a primary or direct response to the presence of the hormone.
To test this hypothesis, a Northern blot was prepared with RNAs extracted from wing discs of the W0 stage incubated in culture medium alone, in medium sup-plemented with cycloheximide (50 µg/ml, CH, Sigma Chemical Co.), or 1025 M 20E, with cycloheximide (50
µg/ml) and 1025 M 20E. Though the accumulation was
slightly inhibited in the presence of CH, the transcript
of theBmAcergene accumulated in the presence of both 20E and cycloheximide (Fig. 6A). In contrast, Urbain
gene accumulation was remarkably inhibited by the addition of CH (Fig. 6B) indicating that, in the absence of protein synthesis, the transcription of the BmAcer
gene in the wing discs is inducible by 20E and may be a primary hormone response.
4. Discussion
Two types of angiotensin-converting enzyme (ACE, peptidyl–dipeptidase) are known in mammal, somatic ACE (sACE), and testicular ACE (tACE). sACE has two active domains, the amino and carboxyl domains, named after their positions relative to the N- and C-termini and each possessing a catalytic site that cleaves dipeptides from the C-terminus of oligopeptides; in contrast, tACE has only one domain (Corvol et al., 1995). ACE is known to have several substrates of small peptide: angi-otensin, bradykinin, enkephalin, substance P, neuro-tensin, and cholescystokinin (Hooper, 1991). These sub-strates are activated after the digestion of the carboxy-terminus.
ACEs have been purified and their enzyme activity has been examined in insects (Cornell et al., 1995; Lam-ango et al., 1996; Wijffels et al., 1996). Insect ACEs also have similar activity against mammalian targeting peptide: angiotensin, bradykinin, enkephalin, and other
Fig. 6. Effects of 20E and cycloheximide on theBmAcerandUrbain
gene expression. Northern blot analysis of total RNA (15µg per lane) isolated from wing discs after 8 h in four different culture conditions. (A) Northern blot analysis ofBmAcer. The 268 bp cDNA was used as a probe, and ribosome RNA (rRNA) was used as a quantitative control. a: medium alone, b: cycloheximide (50µg/ml), c: 1025M 20E
only, d: cycloheximide and 1025 M 20E. (B) Northern blot analysis
synthesized peptides (Lamango et al., 1996). Antiboby againstHaematobiaACE reacts with the compound gan-glion in the posterior region of the mid gut and the testis (Wijffels et al., 1996). Relatively higher activity has been observed from head and body soluble fractions from the adult fly Musca domestica (Lamango et al., 1996). Antibody againstMuscaACE has shown immu-noreactives in theLocustacentral nervous system (Isaac et al., 1998), neuropiles, and neurosecretory cells of sev-eral insects (Schoofs et al., 1998). Schoofs et al. (1998) have described the possibility that insect ACE functions in the generation of active peptide hormone. Two differ-ent types of cDNA have been cloned from Drosophila melanogaster, Ance (Cornell et al., 1995) and Acer
(Taylor et al., 1996). In addition, two types of ACEs have shown the stage-specific accumulation of each ACE, and the authors have implied that the Ance
accumulation in the pupal stage correlates with ecdys-teroid levels (Houard et al., 1998).
Imaginal discs of insects are ecdysteroid sensitive and show morphogenetic changes in response to ecdysteroids (Fristrom et al., 1973). Wing discs ofBombyx morishow dramatical morphogenesis before pupation (Kawasaki and Iwashita, 1987) accompanied by the synthesis of several ecdysteroid-inducible polypeptides (Kawasaki, 1998) and ecdysteroid-inducible cDNA clones reported by Chareyre et al. (1993). We isolated BmAcer cDNA (Figs. 1 and 2) from the wing disc cDNA library of Bom-byx mori at the W2 stage, at which time a dramatical morphogenesis in the wing discs was observed. The results of the Northern hybridization (Fig. 3) correlated well with the ecdysteroid titer in the hemolymph of
Bombyx mori(Quan et al., 1998).BmAcerwas inducible by 20E in a dose-dependent manner (Fig. 4). The induc-tion of the BmAcer gene occurred in 2 hr (Fig. 5) and was not inhibited by the addition of CH (Fig. 6A); these results are distinct from those for theUrbain gene (Fig. 6B; Besson et al., 1996), which is induced by 20E addition but is inhibited by CH. These results indicate a direct induction of BmAcer transcription by 20E. The deduced amino acid sequence of BmAcer has a signal peptide sequence and an enzymatically active region (Fig. 1), implying that BmAcer belongs to the early effector gene family. These results suggest that the pro-tein encoded by the BmAcer works ecdysteroid-depen-dently in a short period and correlates with the metamor-phosis of the wing disc.
In contrast, different results were obtained in other organs. No signal for the BmAcer gene was detected in either the mid gut or the silk gland in the fifth larval instar (data not shown). Among the examined organs, the signals of the BmAcer gene in the wing discs were strong and stage-specific (Fig. 3). Though faint signals were observed in the wing tissues in the pupal stage (data not shown), they did not correspond with those of the hemolymph ecdysteroid titer, whose peak is at P3 in
the pupal stage. Moreover, faint signals were observed at V3 and V4 in the fat body in the fifth larval instar when the ecdysteroid titer was at an undetectable level (data not shown). Despite the 20E-inducible results in the wing disc, the transcriptions of the BmAcer gene showed ecdysteroid-independency in other organs, sug-gesting that the transcriptions of the BmAcer gene have organ-specificity.
The function of insect ACE is not clear, and hormone-inducible ACE has not been found in insects thus far. Based on the results of this study, we can conclude that
BmAcerexpression shows stage-specificity and is ecdys-teroid-inducible, which may help us in our understanding of the function of insect ACE. The function of ACE will be demonstrated in future experiments by an examin-ation of specific enzyme activity using purified ACE from tissues or the expression of recombinant BmAcer
gene.
Acknowledgements
This work was supported in part by a grant-in-aid for scientific research (NO. 07660068) from the Ministry of Education, Science and Culture of Japan.
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