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

Abstract

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 6xHis-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.

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 sequence has been deposited in the GenBank database (accession no. AB113578).

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.

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 and rp49 simultaneously. Br, brain; PG, prothoracic glands; FB, fat body; MG, midgut; Ep, epidermis; Mu, muscle; MT, Malpighian tubule; SiG, silk gland; SaG, salivary gland; Ts, testis; Ov, ovary.

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. 3. The normalized BmJHAMT mRNA levels in the CC-CA of day 2 fourth-instar larvae were set as 100%. The template RNAs of CC-CA, PG, Br, SaG, Ov, and Ts were obtained from 20–40 animals. Others were from one to three animals. (B) Developmental changes of BmJHAMT mRNA in the CC-CA. Total RNAs extracted from the CC-CA of 20–40 animals were pooled in each stage and used for template. The value of the 48-h fourth-instar larvae was set as 100%.

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) are shown in the middle lanes and without recombinant BmJHAMT (–E, negative control) are shown in the bottom lanes. Arrows indicate standard JHs (top lanes) and enzymatically produced JHs (middle lanes). Asterisks indicate unidentified peaks originated from CH₃CN used for sample preparation.