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Proc Natl Acad Sci U S A. Oct 14, 2003; 100(21): 11986–11991.
Published online Oct 6, 2003. doi:  10.1073/pnas.2134232100
PMCID: PMC218700
Agricultural Sciences

Juvenile hormone acid methyltransferase: A key regulatory enzyme for insect metamorphosis


Juvenile hormone (JH) acid methyltransferase (JHAMT) is an enzyme that converts JH acids or inactive precursors of JHs to active JHs at the final step of JH biosynthesis pathway in insects. By fluorescent mRNA differential display, we have cloned a cDNA encoding JHAMT from the corpora allata (CA) of the silkworm, Bombyx mori (BmJHAMT). The BmJHAMT cDNA encodes an ORF of 278 aa with a calculated molecular mass of 32,544 Da. The predicted amino acid sequence contains a conserved S-adenosyl-l-methionine (SAM) binding motif found in the family of SAM-dependent methyltransferases. Purified N-terminal 6×His-tagged recombinant BmJHAMT protein expressed in Escherichia coli catalyzed conversion of farnesoic acid and JH acids I, II, and III to their cognate methyl esters in the presence of SAM, confirming that this cDNA encodes a functional JHAMT. Putative orthologs, DmJHAMT and AgJHAMT, were identified from the genome sequence of the fruit fly Drosophila melanogaster, and a malaria vector, Anopheles gambiae, respectively. Northern blot and quantitative RT-PCR analyses revealed that the BmJHAMT gene was expressed specifically in the CA throughout the third and fourth instar. At the beginning of the last (fifth) instar, the expression level of BmJHAMT declined rapidly and became undetectable by day 4 and remained so until pupation. Correlation of the BmJHAMT gene expression and the JH biosynthetic activity in the CA suggests that the transcriptional suppression of the BmJHAMT gene is crucial for the termination of JH biosynthesis in the CA, which is a prerequisite for the initiation of metamorphosis.

Keywords: Anopheles gambiae, Bombyx mori, Drosophila melanogaster

Juvenile hormones (JHs) are a group of acyclic sesquiterpenoids that regulate many aspects of insect physiology, such as development, reproduction, diapause, and polyphenisms (13). Therefore, the strict regulation of JH titer in insect body is crucial throughout their life. The biosynthetic activity in the corpora allata (CA), the primal site of de novo JH biosynthesis, is generally considered to be a major factor in the regulation of JH titer (4), although the clearance of JH by JH-degrading enzymes in the peripheral tissues also plays a substantial role (5).

The biosynthetic pathway of JHs is divided conventionally into two steps, the early step and the late step (6, 7). In the early step, farnesyl pyrophosphate (FPP), an important intermediate in the biosynthesis of cholesterol and other bioactive terpenoids, is generated via the classical mevalonate pathway, which is common to vertebrates and invertebrates (8). In the late step that is unique to insects or arthropods, FPP is hydrolyzed by a pyrophosphatase to farnesol, then oxidized successively to farnesal and farnesoic acid (FA) by an alcohol dehydrogenase and an aldehyde dehydrogenase, respectively. Finally, FA is converted to active JH (JH III) by C-10,11 epoxidation by a P450 monooxygenase and methylation of the carboxyl group by an S-adenosyl-l-methionine (SAM)-dependent methyltransferase (MTase). Ethyl-branched JHs, JH I and JH II, predominant JHs in lepidopteran insects, are also synthesized via the same pathway, but are obtained by combinations of precursors derived from homomevalonate and mevalonate (9). Because the late-step enzymes are highly specific to insects, they could be excellent targets for selective insect growth regulators; thus, characterization of these molecules is of great importance. However, none of the late-step genes have been cloned to date, apparently because of the lack of vertebrate orthologs and the difficulty in purifying sufficient amounts of these proteins from the extremely small CA.

In the tobacco hornworm, Manduca sexta, periodical surges of ecdysteroid in the presence of hemolymph JH cause larval-larval molts. During the last instar JH disappears from the hemolymph, and a surge of small ecdysteroid titer at the end of the feeding stage induces early metamorphic responses, such as wandering behavior and the pupal commitment of the epidermis (1). Toward the end of the feeding period, the CA of M. sexta stops secreting JH and henceforth secretes only JH acid (JHA) thereafter (1012). This physiological switching observed in the CA during metamorphosis is considered to be caused by the loss of JHA methyltransferase (JHAMT) activity in the tissue (13). A similar disappearance of JHAMT activity in the CA at the beginning of the final instar is suggested to occur in the silkworm, Bombyx mori (14).

To uncover the molecular events underlying the physiological changes in JH biosynthesis in the CA during metamorphosis, we have used mRNA differential display analysis of the CA of the silkworm, B. mori. Consequently, we have isolated a cDNA that was expressed specifically in the CA during a larval molt but not during metamorphosis. Sequencing and functional analysis using the recombinant protein revealed that it encodes an SAM-dependent JHAMT (BmJHAMT). This report demonstrates the cloning of JHAMT gene in insects. Furthermore, the developmental expression profile of BmJHAMT suggests that the transcriptional suppression of the gene is crucial for the termination of JH biosynthesis in the CA before metamorphosis.

Materials and Methods

Animals. Silkworms, B. mori (Kinshu × Showa F1 hybrid), were reared on an artificial diet (Silkmate, Nihon-Nosan-Kogyo, Japan) at 25°C under 12-h light/12-h dark cycles.

Chemicals. Racemic JH I and JH II were purchased from SciTech, Prague, and racemic JH III was from Sigma. Purified JHs used as standards in RP-HPLC, GC, and GC/MS analysis were prepared by semipreparative RP-HPLC (column: Shiseido ODS UG120, 10 × 250 mm; solvent: 60% CH3CN; flow: 5 ml/min; detector: UV 254 nm), followed by extraction with n-hexane. JHA I, II, and III were synthesized by alkaline hydrolysis from the cognate JHs (15). JHs and JHAs were quantified by spectroscopy (15, 16). FA and methyl farnesoate (MF) were purchased from Echelon Research Laboratories (Salt Lake City). Fatty acids and their methyl esters were purchased from DOOSAN Serdary Research Laboratories (Toronto). All other chemicals were analytical grade purchased from Wako Biochemicals (Osaka).

Fluorescent mRNA Differential Display (FDD) Analysis. FDD analysis was performed with a commercial kit (Takara, Shuzo, Kyoto) (17). The fourth (penultimate) and fifth (last) instar larvae were divided into 12 stages based on the developmental time and various morphological and behavioral markers (18, 19). The corpora cardiaca (CC)-CA complexes were dissected from the animals (30 to 40 animals per stage), and the total RNA was extracted with a RNA extraction kit (RNeasy Mini, Qiagen, Chatsworth, CA) and quantified fluorometorically with ribogreen (Molecular Probes). The total RNAs (100 ng) were reverse-transcribed with a rhodamine-labeled downstream primer (5′-T15GC). Then the first-strand cDNAs were amplified by PCR with the downstream primer and an arbitrary upstream primer (10 mer). Twenty-four upstream primers were used in total. A series of PCR products amplified from the 24-stage cDNAs with each upstream primer were separated contiguously in a denaturing polyacrylamide gel (20 × 60 cm; width × height). After electrophoresis, the gels were scanned with a fluorescence image analyzer (FM-BIO II, Hitachi, Tokyo), and the bands showing various stage-specific expression patterns were excised from the gel. cDNAs extracted from the bands were reamplified by PCR, purified with two steps of agarose gels containing H.A.-Yellow and H.A.-Red (Hanse Analytik, Bremen, Germany), and directly sequenced. About 400 stage-specific bands were sequenced in total. An ≈1.4-kb band (tentatively designated as CA01a) amplified with an upstream primer 5′-GATCATAGCC-3′ was chosen for further analyses.

Cloning of Full-Length cDNA by RACE. The 5′ and 3′ ends of the CA01a cDNA was obtained by a modified RACE method using a SMART-RACE cDNA amplification kit (Clontech). A template first-strand cDNA was synthesized from the total RNA extracted from the CC-CA complexes of the fourth-instar larvae. Primers were designed based on the sequence of the CA01a cDNA fragment obtained by FDD. The 3′ RACE product amplified with primer F1, 5′-AAGCCGCAGTAAGATGGCGGTGTTG-3′, (Fig. 1) showed a single band (≈1.1 kb) in an agarose gel. It was subcloned into a cloning vector (pCR2.1, Invitrogen) and sequenced. The 3′ end sequence containing poly(A) tail was obtained from several clones. The 5′ RACE product amplified with primer R1, 5′-CAACACCGCCATCTTACTGCGGCTT-3′, showed multiple bands ranging from ≈0.5 to 2.0 kb in an agarose gel. The PCR product was subcloned into pCR2.1 and sequenced. To confirm the 5′ end, a second 5′ RACE assay was carried out by using primer R2, 5′-TGTCCACTGAACTCCACTGTTTCCG-3′, which was designed based on the sequence obtained from the first 5′ RACE product (Fig. 1). The product showed a single band (≈1.3 kb) in an agarose gel. It was subcloned into pCR2.1 and sequenced. Several longest clones contained an identical sequence (1,258 nt), suggesting that it represents the 5′ end sequence of the transcript. To eliminate possible PCR errors occurring during the 5′ RACE procedure, a region containing putative ORF (4–1,053 nt) was amplified from the first-strand cDNA by using a proofreading polymerase (PfuTurbo, Stratagene) with primers F2, 5′-AGACTGTTCGGCGATACCGCAGT-3′, and R3, 5′-CGAAAATCTGGGAAGACAAAGAGAGAGAA-3′. The RT-PCR product was subcloned into a vector pDrive (Qiagen) and sequenced. Finally, the sequences obtained from the initial FDD fragment, the 5′ and 3′ RACE products, and the RT-PCR products were combined to generate a full-length sequence of CA01a (BmJHAMT) (2,890 bp) (Fig. 1).

Fig. 1.
Nucleotide and deduced amino acid sequence of BmJHAMT cDNA. Binding sites for PCR primers are overlined. The nucleotide sequence corresponding to the cDNA fragment obtained by FDD is lowercase. The SAM binding motif (motif I) is underlined. The full nucleotide ...

RNA Blot Analysis. RNA blot analysis was performed essentially as described (20). Digoxigenin (DIG)-labeled cRNA probe specific for BmJHAMT was prepared from pDrive plasmid containing the full CA01a ORF. DIG-labeled cRNA probe for a reference gene was prepared from pBluescript KS(+) containing a 220-bp B. mori rp49 cDNA fragment. Total RNAs (≈1 μg) extracted from 12 tissues dissected from the fourth-instar larvae (0–24 h after head capsule slippage) were separated in a denaturing agarose gel (1%), blotted onto a nylon membrane (Hybond-N+, Amersham Pharmacia), and hybridized with BmJHAMT and rp49 probes simultaneously.

Quantitative RT-PCR (Q-RT-PCR). The BmJHAMT transcript was quantified on a real-time thermal cycler (LightCycler, Roche Diagnostics). Total RNAs were extracted from various tissues and treated with RNase-free DNase I. The RNAs (150 ng) were converted to cDNAs with an oligo(dT)18 primer and Moloney murine leukemia virus reverse transcriptase (Clontech) in 20-μl reaction volume, and the reaction was diluted with MilliQ water (130 μl). Serial dilutions of a pCR2.1 plasmid containing 1- to 1,258-nt BmJHAMT cDNA were used for standards. rp49 was chosen as a reference gene, and serial dilutions of a pCR2.1 plasmid containing 340 bp of rp49 cDNA were used for standards. Primers for BmJHAMT were Q1, 5′-TGGCTGCGACATAAGCGAAGA-3′, and Q2, 5′-CCTTGTTTCAGGTCTGCGGTCAA-3′ (Fig. 1). Primers for rp49 were 5′-CAGGCGGTTCAAGGGTCAATAC-3′ and 5′-TGCTGGGCTCTTTCCACGA-3′. Q-RT-PCR was carried out in 20-μl reaction volume containing 5 μl of template cDNAs (equivalent to 5 ng of total RNAs) or standard cDNAs, 1× QuantiTect SYBR Green PCR premix (Qiagen), and 0.5 μM each primer. PCR conditions were 95°C for 15 min; 1 cycle, followed by 94°C for 15 s; 55°C, 20 s, 72°C, 8 s; 40–50 cycles. After PCR, the absence of unwanted by-products was confirmed by automated melting curve analysis and agarose gel electrophoresis of the products. The molar amounts of BmJHAMT and rp49 transcripts were calculated based on the crossing point analysis, with standard curves generated from the standard cDNAs. BmJHAMT transcript levels were normalized with rp49 transcript levels in the same samples.

Preparation of Recombinant Protein. A full ORF of CA01a cDNA was amplified from the pCR2.1/CA01a[1–1258] plasmid by using PfuTurbo with primers, F0, 5′-AAACATATGAACAATGCAGATTTATACCGC-3′, and R0, 5′-AAGGATCCAATCACGAAAATCTGGGAAGAC-3′ (Fig. 1). Underlined sequences indicate NdeI (F0) and BamHI (R0) recognition sites added for subcloning. The PCR product was directly subcloned into pDrive and sequenced to check the absence of PCR errors in the insert. The insert was excised with NdeI and BamHI and ligated into an expression vector pET28a(+) (Novagen) that was linearized with the same restriction enzymes, yielding pET28a/CA01a. Escherichia coli strain BL21(DE3) was transformed with the construct and cultured in 5 ml of LB medium containing 50 μg/ml kanamycin at 37°C with shaking at 200 rpm overnight. Then 0.2 ml of the culture medium was transferred to a flask containing 200 ml of the same medium and further incubated under the same conditions. When OD600 of the culture reached ≈1.0, isopropyl β-d-thiogalactoside was added to a final concentration of 0.1 mM and incubated at 20°C with shaking at 150 rpm for 24 h. The bacterial cells were harvested by centrifugation and stored at –30°C. The frozen bacterial pellet was thawed and dispersed in ice-cold Tris·Cl buffer (50 mM, pH 7.5) containing 1 mM PMSF and disrupted by sonication for 5 min by using a sonicator (Bioruptor, CosmoBio, Tokyo). The bacterial lysate was centrifuged (15,000 rpm, 15 min, 4°C), and the supernatant was filtrated through a 0.2-μm filter. Recombinant His-tagged protein was purified from the supernatant by using a HiTrap chelating column (Amersham Pharmacia). Elution buffer containing 300 mM imidazole was exchanged to Tris·Cl (50 mM, pH7.5) with a PD-10 desalting column (Amersham Pharmacia). Glycerol was added to the enzyme solution (final concentration 50%), and the sample was stored at –30°C until use. Quantification and SDS/PAGE analysis of the purified protein were performed as described (21).

Enzyme Assay. Qualitative assay. JHA I, II, and III and FA (50 μg) dissolved in toluene were transferred individually to a siliconized glass tube (10 × 120 mm), and the solvent was evaporated with a N2 stream. The residue was dissolved in 450 μlofTris·Cl buffer (50 mM, pH 7.5) containing 1.1 mM SAM. After the reaction mixtures were preincubated at 25°C for 5 min, the enzymatic reaction was started by the addition of 50 μl of recombinant JHAMT solution (371 ng/μl). For negative controls, only the buffer was added to the same reaction mixtures. After incubation at 25°C for 30 min, the reaction was stopped by the addition of 125 μl of stop solution (methanol/water/concentrated ammonium hydroxide solution, 10:9:1). The reaction mixtures were extracted twice with 1 ml of isooctane. The solvent was evaporated from the extract with a rotary evaporator at 40°C; the residue was dissolved in appropriate solvent and subjected to RP-HPLC, GC, and GC-MS analysis to identify the products as described below.

Quantitative assay. Reaction mixtures (90 μl) containing JHAs, FA, or fatty acids (100 μM) in 50 mM Tris·Cl buffer (pH 7.5) containing 1.1 mM SAM were preincubated for 5 min at 25°C, then 10 μl of recombinant JHAMT solution (124 ng/μl) was added to the reaction mix and incubated at 25°C for 10 min. Under these conditions, the rate of product formation was linear during the assay. The reaction was stopped with 25 μl of the stop solution and extracted with 200 μl of isooctane. After centrifugation, the top phase (100 μl) was transferred to a small vial and dried with a N2 stream. Using a similar extraction procedure, >99% of the JH I is recovered into isooctane phase (22). The residues were dissolved in CH3CN, and the produced methyl esters were quantified by RP-HPLC and GC. Assays were performed in triplicate.

RP-HPLC. RP-HPLC was performed with a Jasco (Tokyo) HPLC system (column: Shiseido ODS UG80, 3 × 150 mm; solvent: 70% CH3CN in H2O, 0.5 ml/min; detection: UV 217 nm).

GC. Samples were dissolved in n-hexane and analyzed with a gas chromatograph (GC-15A, Shimadzu) equipped with a hydrogen flame ionization detector and a capillary column (Omegawax 250, 0.25 mm i.d. × 30 m, 0.25-μm film thickness, Supelco). The carrier gas was helium at a pressure of 1.5 kg/cm2, the injection port temperature was 280°C, and the samples were introduced by split injection. The column oven temperature was programmed at 50°C for 5 min, before being elevated to 260°C at 10°C increase per min and then held for 14 min.

GC-MS. Fractions corresponding to the major peaks observed in RP-HPLC analysis of the qualitative enzyme assays were collected and extracted with isooctane. After evaporation of the solvent with N2 stream, the residues were dissolved in n-hexane and analyzed with a Hewlett–Packard 5890 gas chromatograph coupled to a JEOL Automass 20 mass selective detector at 70-eV ionization, with total ion detection from 100 to 320 atomic mass units. The GC column was a J & W Scientific (Folsom, CA) DB-1, 15 m × 0.25 mm i.d. × 0.25 μm film. The temperature profile was 130°C for 2 min, and then 10°C increase per min to 200°C and held for 5 min. The injector temperature was 250°C and the detector was set at 200°C, with helium carrier gas at a flow rate 1 ml/min.


Cloning and Sequence Analysis of BmJHAMT. When the RNAs extracted from the different stages of CA of B. mori were analyzed by FDD, we found a distinctive band (CA01a) that was present constitutively from the beginning of the fourth instar to the early fifth instar, then disappeared (Fig. 2). Initial blast search for the nucleotide and the deduced amino acid sequences of the cDNA showed no homology. The full-length nucleotide sequence of the cDNA (≈2.9 kb; hereafter designated as BmJHAMT) obtained by a combination of modified 5′ and 3′ RACE revealed a predicted ORF encoding a protein of 278 aa, with a calculated molecular mass of 32,544 kDa. The cDNA has a short 5′ UTR (124 bp) and a relatively long 3′ UTR (≈1.9 kb), which encompasses the initial cDNA sequence (Fig. 1). The conserved domain search on the National Center for Biotechnology Information database revealed that the predicted ORF contains a sequence similar to conserved motifs found in several SAM-dependent MTases. Among functionally characterized MTases, those involved in the biosynthesis of ubiquinone/menaquinone, e.g., UbiE, COQ5 (C-MTases), and COQ3 (O-MTase), showed the highest similarities; however, the overall identities were only 12–13%.

Fig. 2.
FDD analysis of B. mori CA mRNAs. SI, spiracle index (18); HCS, head capsule slippage. Only a top part of a sequencing gel is shown. Arrow indicates a band corresponding to BmJHAMT cDNA.

Tissue- and Stage-Specific Expression of BmJHAMT. The expression of BmJHAMT in various tissues of the fourth-instar larvae 0–24 h after head capsule slippage was examined by Northern blot analysis. A single band was detected only in the CC-CA by using a BmJHAMT-specific probe (Fig. 3). The size of the transcript (≈3 kb) agrees well with the length of the BmJHAMT cDNA (2,890 bp), suggesting that it covers nearly the full-length transcript. To examine the BmJHAMT expression in more detail, we used Q-RT-PCR analysis, because the amount of RNA obtainable from the CC-CA is too small (<10 ng per animal) to perform Northern blot analysis routinely. Consistent with the result of the Northern blot analysis, BmJHAMT mRNA was detected only in the CC-CA of the fourth-instar larvae (Fig. 4A). The CC-CA-specific expression was also observed in day 2 fifth larvae, although the expression level was only ≈2% of that found in the day 2 fourth-instar larvae (Fig. 4A). In contrast, no BmJHAMT mRNA was detected in the CC-CA of the fifth larvae 1 day after spinning (day 6). Instead, at this stage, a small amount of BmJHAMT mRNA (≈1% of day 2 fourth instar) was detected in the testes and ovaries and trace amounts in the epidermis, prothoracic glands, and Malphighian tubules (Fig. 4A). The developmental changes of BmJHAMT expression in the CC-CA were analyzed in more detail by Q-RT-PCR (Fig. 4B). In the CC-CA, BmJHAMT was expressed constitutively throughout the third and fourth instar. However, its expression declined rapidly after the fourth ecdysis and became undetectable by day 4 of the fifth instar and remained so until pupation, except for a trace at gut purge. This developmental expression profile of the BmJHAMT was to the same as that observed for CA01a in the FDD analysis (Fig. 2).

Fig. 3.
Northern blot analysis of BmJHAMT transcript. Total RNAs (≈1 μg) extracted from various tissues of fourth-instar larvae were separated in a denaturing agarose gel, blotted onto a nylon membrane, and hybridized with cRNA probes for BmJHAMT ...
Fig. 4.
Q-RT-PCR analysis of BmJHAMT transcript. BmJHAMT/rp49 indicates the levels of BmJHAMT mRNA normalized to the levels of internal rp49 mRNA. Note that the y axis is a log scale. (A) BmJHAMT mRNA in various tissues. Abbreviations are the same as in Fig. ...

Enzymatic Properties of Recombinant BmJHAMT. Because of the SAM-dependent MTase-like sequence and the CA-specific expression of the cDNA, we speculated that it encodes a functional SAM-dependent MTase involved in JH biosynthesis, namely, JHAMT. To test this hypothesis, we prepared a purified recombinant 6×His-tagged BmJHAMT expressed in E. coli and examined its enzymatic activity. In qualitative enzyme assays, when JHA I, II, and III and FA (50 μg/500 μl) were incubated with the purified recombinant BmJHAMT in the presence of 1 mM SAM, a major peak was observed in each isooctane extract by RP-HPLC (Fig. 5, middle lanes). In contrast, these peaks were not observed in the same reactions that lacked recombinant BmJHAMT (Fig. 5, bottom lanes) and in the reactions with recombinant BmJHAMT but lacking SAM (data not shown). These results indicated that these peaks were indeed the metabolites generated from juvenoid acids (JHAs and FA) and SAM by recombinant BmJHAMT. The retention times of the metabolites of JHAs correspond to those of the cognate standard juvenoid methyl esters (JHs and MF) (Fig. 5, top lanes). The peaks of metabolites of acids and the cognate methyl esters comigrated in the RP-HPLC, when they were coinjected (data not shown). RP-HPLC fractions corresponding to the major peak of the each metabolite were collected and subjected to GC/MS analysis. Total ion chromatogram of the standard juvenoids and the metabolites gave single major peaks. The retention time and major fragment ions at m/z were as follows: JH I, 8.6 min, 163 (100%), 177 (80%), 247 (12%), and 262 (6%); JHA I metabolite, 8.6 min, 163 (100%), 177 (78%), 247 (14%), and 262 (6%); JH II, 7.9 min, 163 (100%), 195 (32%), 220 (14%), and 248 (7%); JHA II metabolite, 7.9 min, 163 (100%), 195 (21%), 220 (19%), and 248 (10%); JH III, 7.0 min, 163 (100%), 189 (45%), 206 (32%), and 234 (16%); JHA III metabolite, 7.0 min, 163 (100%), 189 (54%), 206 (33%), and 234 (18%); MF, 5.9 min, 207 (100%), 175 (71%), 219 (48%), and 250 (22%); and FA metabolite, 5.9 min, 207 (100%), 175 (45%), 219 (59%), and 250 (17%). The correspondence in the retention times and the mass spectra of the standard juvenoids and juvenoid acids metabolites confirmed the generation of juvenoid methyl esters from the cognate juvenoid acids by the recombinant BmJHAMT.

Fig. 5.
RP-HPLC of JHA metabolites generated with recombinant BmJHAMT. Standard JH I (A), JH II (B), JH III (C), and MF (D) are shown in the top lanes. Isooctane-extracted reactions of JHA I (A), JHA II (B), JHA III (C), and FA (D) with recombinant BmJHAMT (+E) ...

At 100 μM juvenoid acids, recombinant BmJHAMT showed the highest activity on JHA I and a similar level of activity on JHA II (Table 1). However, the activity on JHA III and FA were ≈50% and 30% of that on JHA I, respectively. In contrast, under the same conditions no enzymatic activity was detected against the several saturated and unsaturated fatty acids examined (Table 1). These results showed that BmJHAMT encodes a functional SAM-dependent MTase highly specific to juvenoid acids.

Table 1.
Enzymatic activity of recombinant BmJHAMT with JHAs and fatty acids


JHAMT is an enzyme that transfers the methyl group of SAM to the carboxyl group of JHAs at the final step of JH biosynthesis. Although the occurrence of JHAMT in the CA has long been recognized by radiochemical assays (23, 24), neither the protein nor its gene has been isolated from any insect. In this study, we have isolated by FDD a cDNA that encodes an SAM-dependent MTase from the CA of B. mori. The expression of the gene was highly specific to the larval CA. Furthermore, the recombinant protein expressed from the cDNA converted JHA I, II, and III and FA to their cognate JH methyl esters in the presence of SAM. These results unequivocally demonstrate that the cDNA encodes a functional JHAMT that is involved in JH biosynthesis in the CA.

SAM-dependent MTases comprise a structurally conserved superfamily (a seven-stranded α/β fold), whose members include MTases of DNA, RNA, proteins, lipids, polysaccharides, and a wide variety of small molecules (25). Although the overall similarity of amino acid sequences among family members is often very low, several conserved motifs are recognized. In particular, motif I, involved in SAM binding, is conserved among all family members: (V/I/L)(L/V)(D/E)(V/I)G(G/C)G(T/P)G (26) and more general consensus, hh(D/E)hGXGXG, where h represents a hydrophobic residue (27). The presence of motif I (VIDLGCADG) indicates that BmJHAMT is a member of SAM-dependent MTases. Putative insect MTases with substantial similarity to BmJHAMT (35–37%) were found in Drosophila melanogaster (CG17330), Anopheles gambiae (EAA09331), and M. sexta (AAF16712) (Fig. 6, which is published as supporting information on the PNAS web site, www.pnas.org). These genes also contain an apparent SAM binding motif. In a preliminary experiment, recombinant protein expressed in E. coli from the D. melanogaster homolog converted JHAs to the cognate JHs in the presence of SAM (unpublished data), indicating that the D. melanogaster homolog indeed encodes a functional JHAMT (DmJHAMT). The relatively high similarity (46%) of the A. gambiae homolog to the DmJHAMT suggests that it also encodes a functional JHAMT (AgJHAMT). In addition, the absence of another apparent homolog in the entire genome of both D. melanogaster and A. gambiae supports the hypothesis that these genes encode functional JHAMTs, which should be present in all insects. Thus, BmJHAMT and the two dipteran homologs represent a novel subfamily of SAM-dependent MTases that specifically methylate JHAs. Whether the M. sexta homolog also encodes a JHAMT is inconclusive at present. The overall identity between BmJHAMT and the M. sexta EST clone (35%) is similar to the value between BmJHAMT and dipteran homologs. Considering the fact that the evolutionary distance between B. mori and M. sexta is much smaller than for the two dipterans, this value seems to be low for a lepidopteran JHAMT ortholog. Because the M. sexta EST clone was found in a male antennal cDNA library (28), it may encode a MTase metabolizing odorant molecules, such as sex pheromones or plant volatiles. Further analysis of the enzymatic properties of M. sexta homolog using recombinant protein is needed to resolve its specificity.

The cloning of a crustacean FA O-methyltransferase (FAMet), which converts FA to MF, a putative crustacean JH (29), has been reported (30, 31). Because FAMet lacks the signature SAM binding motif (motif I), it may represent a novel SAM-dependent MTase (30). Although FAMet shows a low overall similarity to BmJHAMT (11%), the presence of an apparent FAMet homolog in the D. melanogaster (CG10527; 41%) indicates that the crustacean FAMet is not an ortholog of the insect JHAMTs reported in this study. Although MF production is specific to the mandibular organ (MO) (32, 33), a counterpart of the insect CA, the distribution of FAMet mRNA and protein is rather widespread (30, 31); therefore, another FAMet, a JHAMT ortholog, the mRNA and protein of which are likely to be localized primarily in the MO, may exist in the Crustacea.

By Northern blot and Q-RT-PCR analysis, we showed that BmJHAMT was specifically expressed in the CC-CA during the third and fourth instar (Fig. 4). Furthermore, BmJHAMT mRNA was detected in the CC-CA of first- and second-instar larvae by Q-RT-PCR (data not shown). When the CC and the CA of day 2 fourth-instar larvae were analyzed separately, the BmJHAMT transcript was detected predominantly in the CA and only trace (<0.1%) in the CC (data not shown). These results suggest that BmJHAMT is expressed primarily in the CA from the first to the penultimate instar stage.

Importantly, BmJHAMT mRNA level declined rapidly in the CA early in the final larval instar and completely disappeared before spinning. The CA of M. sexta also lose JHAMT activity at the end of the feeding stage during the last instar (10, 13). Similarly, although not directly analyzed, B. mori CA are thought to lose JHAMT activity during the early part of the last instar (14). Thus, developmental changes of JHAMT activity and the BmJHAMT mRNA level in the CA agree quite well. Therefore, we propose that the loss of JHAMT activity in the CA is caused by the termination of synthesis of new JHAMT protein due to the loss of JHAMT mRNA and a steady-state turnover of JHAMT protein, although the inactivation of JHAMT enzyme without a loss of the protein cannot be completely eliminated at present.

The loss of FAMet activity in the CA during the second half of the last larval instar was also observed in the cockroach, Diploptera punctata (34), suggesting that JHAMT is a rather universal regulatory protein in JH biosynthesis during insect metamorphosis. It is thus of interest to see whether the transcriptional suppression of JHAMT gene at the onset of metamorphosis is a common phenomenon in insects. Furthermore, because the transcriptional suppression of BmJHAMT gene is one of the earliest responses in metamorphosis, the analysis of regulatory mechanisms of BmJHAMT gene in the CA may provide clues to better understanding of factors triggering insect metamorphosis.

In the late fifth instar, no BmJHAMT transcript was detected in the CA, but low amounts were detected in the testes and ovaries and trace in other peripheral tissues, such as epidermis (Fig. 4A) and wing discs (data not shown), suggesting that the BmJHAMT gene is under complex tissue-dependent and developmental stage-dependent regulation. In M. sexta, the JH titer in the hemolymph peaks during the prepupal period (35), when the CA produces only JHA. This JH peak is derived from the conversion of JHA to JH by JHAMT present in the imaginal discs (36) and possibly other tissues. Similarly, a significant increase of JH titer was observed in the hemolymph of B. mori during the prepupal period (14), when the CA does not synthesize JH (37), suggesting the presence of JHAMT activity in some peripheral tissues at this stage. Although the levels of the BmJHAMT mRNA found in the peripheral tissues were much lower than that found in the fourth instar CA, the extreme large volume of these tissues compared with the CA indicates that a considerable amount of BmJHAMT protein could be produced by these tissues. Therefore, it is most likely that the BmJHAMT protein present in the peripheral tissues converts JHA to JH at the prepupal stage.

Finally, the discovery of JHAMT gene has practical importance for pest management. Because the JHAMT is an enzyme highly specific to insects, JHAMT inhibitors are expected to be excellent candidates for safe insect growth regulators (IGR). Such IGRs could, via premature reduction of JH titer, induce a precocious lethal metamorphosis and, accordingly, a reduction in the larval feeding stage, and also prevent reproduction in adult stage (38). Moderate similarity of B. mori and D. melanogaster JHAMT peptide sequences enables to clone by homology JHAMT genes from insect pests of agricultural and medical importance. Recombinant JHAMT proteins of these insects could then be used for in vitro screening of their inhibitors and the elucidation of their tertiary structures, which would facilitate computer-assisted design of JHAMT inhibitors.

Supplementary Material

Supporting Figure:


We thank Drs. Jun Takatsuka and Yoichi Kawazu for initial analysis of fluorescent differential display; Dr. Teruyuki Niimi for providing B. mori rp49 cDNA; Dr. Ichiro Honda for helping with GC/MS analysis of JHs; and Prof. Lynn M. Riddiford and Dr. Hubert Wojtasek for critical comments on the manuscript. This work was supported in part by a grant from the Ministry of Agriculture, Forestry and Fisheries of Japan (Bio-Design program). K.I. was supported by the Naito Foundation and is a Domestic Research Fellow of the Japan Society for the Promotion of Science.


This paper was submitted directly (Track II) to the PNAS office.

Abbreviations: JH, juvenile hormone; JHA, JH acid; JHAMT, JHA methyltransferase; CA, corpora allata; FA, farnesoic acid; FAMet, FA O-methyltransferase; SAM, S-adenosyl-l-methionine; MF, methyl farnesoate; FDD, fluorescent mRNA differential display; CC, corpora cardiaca; Q-RT-PCR, quantitative RT-PCR; MTase, methyltransferase.

Data deposition: The sequence reported in this paper has been deposited in the GenBank database (accession no. AB113578).


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