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INTRODUCTION |
Moulting and many other physiological processes in insects
are regulated by molting hormones (ecdysteroids) (1). In immature stages, the prothoracic glands are the primary source of ecdysteroids, generally ecdysone in most species. However, it has become apparent that in most Lepidopteran species studied, the major product of the
glands is 3-dehydroecdysone
(3DE),1 together with a
varying proportion of ecdysone (2-4). For instance, the glands of
Manduca sexta and Pieris brassicae secrete in
excess of 95% 3DE, whereas some other Lepidopteran species secrete
only a 1:1 ratio (Leucinia separate) and others
(Bombyx mori) merely traces of 3DE (2-5). Thus, even within
a single order, the ratio of ecdysteroids from the prothoracic glands
are markedly different. The physiological significance of these
differences remains to be explained. Previously, we demonstrated that
the prothoracic glands of last instar larvae of Spodoptera
littoralis primarily secreted 3DE (~82%), with lesser amounts
of ecdysone (~18%) (6). The fact that interconversion of ecdysone
and 3DE by prothoracic glands was not detectable suggested that 3DE is
more likely an independent product of pathways of ecdysteroid
biosynthesis in the glands.
After secretion from the prothoracic glands, the 3DE is reduced to
ecdysone by an NAD(P)H-linked 3DE 3
-reductase in the hemolymph (3,
4, 6, 7). Ecdysone undergoes C-20 hydroxylation in certain peripheral
tissues yielding 20-hydroxyecdysone, which is considered to be the true
molting hormone in most insects (8). Thus, 3DE 3
-reductase-catalyzed
reduction of 3DE to ecdysone is viewed as an important regulatory step
in the production of the molting hormone in Lepidopteran species. 3DE
3
-reductase was demonstrated in the hemolymph and the activity of
the enzyme during the last larval instar reached a peak just preceding
that of the hemolymph ecdysteroid titer, supporting a role of the
enzyme in production of ecdysteroids. It was revealed that the enzyme exhibited maximum activity at low 3DE substrate concentrations, with a
drastic inhibition of activity at higher concentrations (>5
µM), suggesting the existence of an inhibition site for
3DE (6).
The 3
-reductase enzyme was purified from the hemolymph and was shown
to be a monomer with molecular mass of approximately 36 kDa (6). Amino
acid sequences of the NH2 terminus as well as of interior
tryptic peptides of the purified enzyme have been determined. Here, we
report the molecular cloning and characterization of the cDNA
encoding 3DE 3
-reductase of the cotton leafworm, S. littoralis. Conceptual translation and amino acid sequence analysis indicates that 3DE 3
-reductase is closely related to those
of the third superfamily of oxidoreductases.
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EXPERIMENTAL PROCEDURES |
3DE 3
-Reductase Purification and Protein Sequencing--
3DE
3
-reductase from hemolymph of S. littoralis was purified
to homogeneity using a combination of polyethylene glycol 6000 precipitation and successive FPLC fractionation on Mono Q, phenyl Superose (twice), and hydroxyapatite columns, as described previously (6). Purified protein was subjected to SDS-PAGE on 10% gels, electrotransferred to ProBlottTM membrane, and visualized
by Coomassie staining (9). A single band was observed, excised and
sequenced by an automated pulsed liquid-phase sequencer (Applied
Biosystems 471A). The NH2-terminal amino acid sequence thus
determined was as follows, ATIDVPMLKMLNDREMPAIALGTYLGFDKG. To obtain
sequences of the interior region of the enzyme, the purified protein
was resolved by SDS-PAGE as described above, visualized by Coomassie
staining, and the corresponding band was excised and cleaved with
trypsin. The resulting tryptic peptides were purified by
high-performance liquid chromatography and sequenced. The sequences of
four of the resulting peptides were determined. These peptides had the
following sequences: peptide 1, HFDTAAIYNTEAEVGEAIR; peptide 2, FGMDLPGPK; peptide 3, LKEEEIEK; peptide 4, INQFNSNTR.
cDNA Cloning and Sequencing--
A PCR-based cloning
strategy was used to clone a cDNA fragment encoding the region
between the NH2 terminus and an internal peptide sequenced
as described above. Three degenerate primers were synthesized.
Primer N was designed on the basis of a part of the
NH2-terminal amino acid sequence
(5'-GCGAATTCYTNAAYGAYMGNGARATGCC, where Y represents T/C, R
is A/G, M is A/C, N is A/T/C/G); reverse primers A and B were made
according to the sequences of peptide 1 and peptide 2 (primer A,
5'-CAGGATCCACYTCNGCYTCNGTRTTRTA; primer B,
5'-ATGGATCCCCNGGNARRTCCATNCCRAA). An EcoRI
restriction site was built into primer N and a BamHI site
into primer A and primer B for use in cloning; these are underlined in
the primer sequences.
Since the site of synthesis of the reductase enzyme was unknown, total
RNA was extracted using TRIzol (Life Technologies, Inc.) from total
insect tissues (including hemolymph, but with the cuticle, head, and
gut contents removed) dissected from larvae 90 h into the last
larval instar. mRNA from total RNA was isolated using Dynabeads
mRNA Purification Kit (Dynal) (UK) Ltd. First strand cDNA was
reverse transcribed from the mRNA using a 1st Strand
cDNA Synthesis Kit from Boehringer Mannheim with either random
primer p(dN)6 supplied with the kit or QT
adapter primer, 5'-CCATCAGTGCTAGACAGCTAAGCTTGAGCTCGGATCC(T)17 (modified
from Ref. 10). cDNA synthesized with random primer served as
template for the PCR in which the above degenerate primers were used.
PCR reactions were carried out as follows: 1 cycle of 94 °C for 4 min and 4 cycles of 94 °C for 30 s, 37 °C for 45 s,
72 °C for 2 min; followed by 31 cycles of 94 °C for 30 s,
58 °C for 45 s, 72 °C for 2 min. PCR products were analyzed
by electrophoresis on a 1% agarose gel. This revealed that PCR with
primer N and primer B yielded a product of approximately 660 bp, while
PCR with primer N and primer A yielded two products of approximately 380 and 180 bp, respectively. The 660-bp PCR product was gel-purified using HybaidTM DNA Purification Kit (Hybaid) and used as a
template along with primer N and primer A in a second PCR reaction
using the same cycling conditions as described above. This second PCR
only yielded the 180-bp product, implying that peptide 1 is located in
between the NH2 terminus and peptide 2.
PCR products were purified, digested with EcoRI and
BamHI, and subsequently cloned into
EcoRI/BamHI-digested pBluescript vector. Transformants were screened by colony PCR using primers
T3 and T7 (5'-ATTAACCCTCACTAAAG and
5'-AATACGACTCACTATAG, respectively), and those showing the
correct size of inserts were propagated in LB broth containing 100 µg/ml ampicillin and plasmid DNA was purified after 16 h
incubation at 37 °C. Double-stranded DNA sequencing was performed by
the dideoxy termination method using Sequenase Version 2.0 (USBTM, Amersham Pharmacia Biotech).
Rapid Amplification of cDNA 5'- and 3'-Ends (5'- and
3'-RACE)--
5'- and 3'-RACE were carried out to obtain the 5'- and
3'-ends of the cDNA. A 5'-RACE System (Life Technologies, Inc.) was used to amplify the 5' terminus of the message for sequencing. Briefly,
a gene-specific primer 5'-1 (5'-TTCAGCCTCCGTGTTGTAG) was hybridized to
the mRNA prepared as described above and cDNA was synthesized
using Superscript II reverse transcriptase. The RNA was then degraded
with RNase mix (RNase H and RNase T1), and the cDNA was purified by
GlassMax spin cartridge supplied with the kit. A poly(dC) tail was
added to the 3' terminus of the purified cDNA using dCTP and
terminal deoxynucleotidyl transferase, and the cDNA region
corresponding to the 5'-end of the mRNA was amplified by two
successive rounds of PCR using additional gene-specific primers 5'-2
and 5'-3 (5'-TCGAATTCGTGTCTGTACCCGAGGTC and
5'-GCGAATTCTTGCATCACTACATTGCG, respectively, which
incorporated an EcoRI site (underlined)), together with the
anchor primers supplied by the manufacturer. The second round of PCR
yielded a single amplified product of approximately 240 bp. To amplify
the 3'-end, another two gene-specific primers C1 and
C2 (5'-GAAGACCTGATCACGTACG and
5'-GCGAATTCAGGCACCATTGTCATGGGC, respectively, incorporating
an EcoRI site (underlined)) in combination with the adapter
primers Q0 and Q1 (5'-CCATCAGTGCTAGACAGCT and 5'-TAAGCTTGAGCTCGGATCC, respectively, modified from Ref. 10) were used
for the PCR in which QT primed cDNA served as template (see earlier). The nested PCR with primers C2 and
Q1 yielded a product of approximately 750 bp. The 5'- and
3'-RACE products were digested with the appropriate restriction
enzymes, cloned into pBluescript vector, and sequenced across both strands.
Construction of a cDNA Containing an Open Reading Frame and
3'-Noncoding Region--
The DNA sequence containing the entire open
reading frame and 3'-noncoding region was amplified by PCR from the
cDNA synthesized with primer QT as described
above using the following gene-specific primers:
-5'
(5'-GCGAATTCATGTTTCGCGCCAGTTTT) and
-3'
(5'-CGGGATCCTGCAGAGATTGATTTCACATATT). These primers
were designed to incorporate EcoRI, and BamHI and PstI sites (underlined in the primer sequences) into the 5'-
and 3'-ends of the sense strand, respectively. PCR was conducted as follows: 1 cycle of 94 °C for 4 min, and 4 cycles of 94 °C for 30 s, 37 °C for 45 s, 72 °C for 2.5 min, followed by 31 cycles of 94 °C for 30 s, 45 °C for 45 s, 72 °C for
2.5 min. The resulting PCR product was gel-purified, digested with
EcoRI and PstI, ligated into pBluescript vector
that had been previously digested with EcoRI and
PstI, transformed into Escherichia coli strain
DH5
, selected, and sequenced. The sequence of three independent
cDNA clones were compared to detect errors that could have occurred during the reverse-transcription and the PCR amplification.
Northern and Southern Blot Analysis--
Total RNA from various
tissues or whole animals with the gut content removed was isolated
using TRIzol reagent (Life Technologies, Inc.). 10 µg of RNA was
fractionated on a formaldehyde/agarose gel, transferred to Electran®
nylon membrane (BDH), and hybridized with a probe corresponding to the
open reading frame and 3'-noncoding region of the 3
-reductase
cDNA. Probes were radiolabeled by random priming (Boehringer
Mannheim), and loading was normalized by probing with a PstI
fragment of the A3 actin gene from B. mori (a gift from Dr.
A. Mange, Université Claude Bernard Lyon 1) (11) and an 18 S rRNA
fragment from mouse (a gift from Ms Yi-Ping Bao, Liverpool University).
Blot signals were quantified using a Molecular Dynamics densitometer.
Total genomic DNA was prepared from whole animals using Qiagen
Genomic-Tip 500/G (Qiagen), following the manufacturer's
recommendations. 10-µg aliquots of DNA were digested with
EcoRI, SalI, HindIII, PvuII, NcoI as well as EcoRI + SalI, fractionated on a 1% agarose gel, transferred to a
nylon membrane, and hybridized using radiolabeled probes corresponding
to different regions of the 3
-reductase cDNA. Prehybridization
and hybridization were carried out using QuikHyb (Stratagene) under the
conditions recommended by the manufacturer. The blots were washed at
high stringency (0.1 × SSC, 0.1% SDS) and labeled bands
visualized by autoradiography.
Preparation of Antibodies and Western Immunoblot
Analysis--
Polyclonal antibodies against 3DE 3
-reductase were
raised in chickens using the purified protein as antigen. The purified protein was first resolved by SDS-PAGE on a 10% gel, visualized with
0.25 M KCl. The resulting white band was cut out, extracted with 50 mM Tris-HCl buffer, pH 7.2, containing 0.1 mM EDTA, 150 mM NaCl, and used as antigen.
About 50 µg of the antigen was emulsified with complete Freund's
adjuvant and injected into a chicken. Booster injections were made 12 and 20 days later with 100 µg of the antigen. Extraction of
antibodies from the yolk of the immunized chicken eggs with
polyethylene glycol was carried out following the procedures described
by Gassmann et al. (12).
Western blotting was carried out essentially as described previously
(13). Briefly, hemolymph samples taken at various times during the last
larval instar were separated by SDS-PAGE on a 10% gel in accordance
with the method of Laemmli (14) and electrophoretically transferred to
a nitrocellulose membrane (Schleicher and Schuell) (15). Immunoblotting
was performed with the antibodies at a final dilution of 1:500. Blots
were developed by incubation with an alkaline phosphatase-conjugated
goat anti-chicken IgG at a final dilution of 1:3000, followed by
incubation in 15 ml of alkaline phosphatase color-development buffer
(100 mM NaHCO3, pH 9.8) to which was added 150 µl each of Bio-Rad AP color reagents A and B. Immunoreactive protein
was quantified using a Molecular Dynamics densitometer.
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RESULTS |
Cloning of the cDNA Encoding 3DE 3
-Reductase--
Using a
combination of polyethylene glycol 6000 precipitation and column
chromatography on Mono Q, phenyl Superose, and hydroxyapatite, we
purified 3DE 3
-reductase to homogeneity from Spodoptera
hemolymph. A silver-stained SDS gel revealed that the purified 3DE
3
-reductase consisted of a single protein band of apparent molecular
mass 36 kDa (see Fig. 4. in Ref 6). NH2-terminal and
tryptic peptide amino acid sequences were obtained from this 36-kDa
protein as detailed under "Experimental Procedures." At the time
when these sequence data were obtained, data base searches using FASTA
revealed that these sequences were novel.
A PCR-based cloning strategy, as detailed under "Experimental
Procedures," allowed us to obtain a clone corresponding to the sequence between nucleotides 142-785 in Fig.
1. Gene-specific primers derived from
this sequence were synthesized and used for 5'- and 3'-RACE to obtain
the 5'- and 3'-ends of the clone. 5'-RACE produced, homogeneously, a
cDNA clone of 237 bp that contains two putative translation start
sites at nucleotides 23-25 and 68-70, respectively. On the other
hand, 3'-RACE resulted in heterogeneous cDNA clones. Sequencing
experiments revealed that this resulted from multiple polyadenylation
signals (AATAAA) occurring in the 3'-noncoding region which directs a
poly(A) tail to be added to different sites. The longest cDNA clone
obtained with 3'-RACE was 746 bp in length and contained a stop codon
and three putative polyadenylation signals. Taken together, all the
overlapping cDNAs span a total of 1425 bp. This sequence was
further confirmed by sequencing of a cDNA clone, which was
constructed to contain the open reading frame and 3'-noncoding region,
as detailed under "Experimental Procedures." As shown in Fig. 1,
using the first ATG as the start codon, the full-length 3DE
3
-reductase cDNA encodes a protein of 345 amino acids with a
predicted molecular weight of 39,591. The predicted molecular weight of
mature protein is 37,689, which is similar to its apparent
Mr observed in our SDS-PAGE analysis. The
protein is mildly acidic with an estimated pI of 6.23.

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Fig. 1.
Nucleotide and deduced amino acid sequences
of 3DE 3 -reductase. Nucleotide numbers
are indicated on the right, and amino acid numbers are
indicated on the left. The open reading frame is shown in
uppercase letters, and the 5'- and 3'-noncoding regions are
shown in lowercase letters. The putative polyadenylation
signals are indicated in bold, and the sequence indicated in
italics identifies the part that is missing in another
clone. The predicted signal peptide is double-underlined,
and the amino acid sequences obtained from the NH2 terminus
and four tryptic peptides derived from purified 3DE 3 -reductase are
underlined. Two HindIII sites and a
SalI site are boxed, while the three ATTTA
sequences are boxed and lightly shaded.
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Similarity of the Deduced Amino Acid Sequence to Those of the Third
Superfamily of Oxidoreductases--
The deduced amino acid sequence
for the cDNA coding region was compared with all sequences in the
data base Swiss-Prot using FASTA in the GCG package. The cDNA gene
product was found to be most similar to proteins which belong to the
third superfamily of oxidoreductases. 3DE 3
-reductase particularly
resembles mammalian aldose reductases. Among the 20 proteins most
closely matched, six are mammalian aldose reductases, others include a
mammalian alcohol dehydrogenase, a probable mammalian
trans-1,2-dihydrobenzene-1,2-diol dehydrogenase, two mammalian aldose
reductase-related proteins, an amphibian lens protein, a putative
reductase from Leishmania, a mammalian 3
-hydroxysteroid
dehydrogenase, a mammalian chlordecone reductase, a mammalian
trans-1,2-dihydrobenzene-1,2-diol dehydrogenase, a yeast xylose
reductase, a mammalian 3-oxo-5-
-steroid 4-dehydrogenase, a plant
D-sorbitol-6-phosphate dehydrogenase, a hypothetical
37.1-kDa protein in hxt5-cdc12 intergenic region from yeast and a plant 6-deoxychalcone synthase. The amino acid similarity extends across the
entire length of the proteins compared. Fig.
2 shows the comparison of the deduced
amino acid sequence of mature 3DE 3
-reductase to some proteins
corresponding to different subgroups of the third superfamily of
oxidoreductases. It demonstrates that there is 42.1, 41.3, 39.2, 37.6, and 35.4% identity, respectively, between 3DE 3
-reductase and human
alcohol dehydrogenase, rabbit aldose reductase, rat 3-oxo-5-
-steroid
4-dehydrogenase, rat 3
-hydroxysteroid dehydrogenase, and frog
crystallin.

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Fig. 2.
Alignment of the deduced amino acid sequences
of mature 3DE 3 -reductase and some data base
proteins. The deduced mature 3DE 3 -reductase sequence was
compared with all sequences in the Swiss-Prot data base using the FASTA
program in the GCG package. Only the amino acid sequences of some
similar proteins representing different subgroups of the third
superfamily of oxidoreductases are shown in the alignment. Gaps
introduced to maximize alignment are indicated by dots.
Identical amino acids are indicated in shaded boxes. Numbers
on the right refer to the last amino acid residue in each
line of the respective protein sequences. Abbreviations and
accession numbers are as follows: ALDX, alcohol dehydrogenase
(P14550); ALDR, aldose reductase (P15122); 3O5B,
3-oxo5 -steroid 4-dehydrogenase (P31210); DIDH,
3 -hydroxysteroid dehydrogenase (P23457); CRO, Rho crystallin
(P17264). The alignment was constructed by use of the PILEUP
program.
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Tissue Distribution and Developmental Expression of 3DE
3
-Reductase mRNA--
As demonstrated in Fig.
3A, a cDNA probe
representing the protein coding and 3'-noncoding regions of 3DE
3
-reductase detected a major transcript of approximately 1.4 kilobases in a variety of tissues prepared from larvae 78 h into
the last larval instar. The size of this major transcript is consistent
with that of the cDNA obtained. The highest expression of 3DE
3
-reductase is detected in Malpighian tubules, followed by midgut
and fat body, with low levels expressed in hemocyte. No detectable
expression was found in the central nervous system (data not shown).
Less expression is detected in Malpighian tubules and hemocytes
dissected at 46 h into the last larval instar, but no expression
was found in all four tissues from early instar larvae. It is
noticeable that RNAs at approximately 2.6 and 3.6 kb are also
recognized in fat body by this probe but at lower intensity (Fig.
3A). It remains to be determined whether these larger RNAs
represent pre-mRNAs for 3DE 3
-reductase or other mRNAs with
significant sequence similarity to 3
-reductase, although the former
seems most probable due to the high stringency used. The signals
detected by the actin probe used for normalization are substantially
lower in the fat body than those in other tissues (Fig. 3B).
rRNA probe was therefore used to further verify the equal loading (Fig.
3C). Some apparent degradation of rRNA is observed in
midgut, Malpighian tubules, and hemocytes, which may be due to the fact
that 28 S ribosomal RNA in insects frequently occurs as two equal-sized
fragments due to RNase activity within certain cells (16). This
activity seems least in the fat body and the total amount of
hybridization to the rRNA probe seems comparable with the other
samples.

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Fig. 3.
Northern blot analysis of the tissue
distribution and expression of 3DE 3 -reductase
mRNA. 10 µg of total RNA from various tissues
(top) at different times (bottom) within the last
larval instar was used. The blots were hybridized with a
32P-labeled 3DE 3 -reductase cDNA probe that
corresponded to the protein coding and 3'-noncoding regions
(A). The same blots were stripped and re-probed with actin
(B) and rRNA (C) probes, respectively. The probes
used are indicated on the right, and the positions of RNA
size markers are shown on the left.
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Northern analysis of total RNA isolated from whole animals from
different developmental stages of the last larval instar revealed that
the 3DE 3
-reductase mRNA is only expressed in the second half of
the instar (Fig. 4). The mRNA starts
to be detected at 66 h into the last larval instar and increases
in intensity, as normalized to actin and rRNA, to 90 h with an
apparent decrease at 96 h before increasing to its highest levels
observed at 114 h.

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Fig. 4.
Northern blot analysis of the expression of
3DE 3 -reductase mRNA during the last
larval instar of the cotton leafworm S. littoralis. 10 µg of total RNA from a whole larva at
various time points within the last larval instar as indicated was
used. The blots were hybridized with a 32P-labeled 3DE
3 -reductase cDNA probe containing the protein coding and
3'-noncoding regions (A), followed by stripping and
re-probing with actin (B) and rRNA (C) probes,
respectively, as indicated on the right side of each panel.
Northern blot signals were quantified using a Molecular Dynamics
densitometer, and values (with ranges) of two separate experiments were
normalized with actin (open column) and rRNA (shaded
column) respectively (D). The dashed line
illustrates the fluctuation of the enzyme activity during the
instar.
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Southern Blot Analysis of Genomic DNA--
Genomic DNA was
prepared from whole animals and digested with various restriction
enzymes, of which SalI, HindIII, and
NcoI sites are found in the 3
-reductase cDNA, whereas
EcoRI and PvuII sites are not found in the
cDNA. The resultant DNA fragments were analyzed by Southern blot
using probes representing different regions of the cDNA. As shown
in Fig. 5A, usually two bands
were observed in all of the digested samples, when the blot was
subjected to hybridization with a probe representing the coding and
3'-noncoding regions of the 3
-reductase cDNA. However, when the
same blot was re-probed with a cDNA fragment that corresponded to
the NH2-terminal region to the first HindIII
site in the cDNA, essentially only one band was detected except for
the restriction digestion with PvuII, where two bands were
observed (Fig. 5B). Again, a probe that represents the
region from the SalI site to the poly(A) site detected only
one band in each case (Fig. 5C). The analysis displayed in
Fig. 5 is most simply interpreted if the 3
-reductase is encoded by a
single copy gene in the Spodoptera genome and if this gene has at least one intron that contains EcoRI,
PvuII, and SalI sites.

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Fig. 5.
Southern blot analysis of genomic DNA of the
cotton leafworm, S. littoralis. 10 µg of
genomic DNA from whole animals was digested with various restriction
enzymes as indicated on the top of each panel. The blot was
hybridized with 32P-labeled cDNA corresponding to: the
entire coding region and 3'-noncoding region (bases 1-1386 in Fig. 1)
(A), a region from the start codon to the first
HindIII site (bases 1-319) (B), and a region
from the SalI site to the 3'-end (bases 809-1386)
(C). The sizes of DNA marker are indicated on the
left.
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 |
DISCUSSION |
Using a reverse transcription-PCR-based cloning strategy,
employing degenerate primers designed on the basis of the partial amino
acid sequences of fragments of 3DE 3
-reductase, together with 3'-
and 5'-RACE, the complete cDNA encoding the enzyme was isolated and
sequenced. The predicted amino acid sequence of the cDNA contained
the NH2-terminal sequence of the enzyme as well as the four
internal peptides obtained from tryptic digestion of the band
corresponding to the Mr 36,000 polypeptide,
confirming that we had cloned the 3DE 3
-reductase cDNA. The
presence of 3DE 3
-reductase enzyme in hemolymph is due to secretion
from tissues that synthesize it. The first 17 predicted amino acids appear to constitute a signal peptide, since the sequence shares significant characteristics with the signal sequences of some eukaryotic secretory proteins (17): (i) the 17 amino acids are within
the typical range of 13-36 residues of signal peptides; (ii) a
positively charged residue, arginine, is found in the amino-terminal part of the signal; (iii) it contains a highly hydrophobic stretch of
11 residues; (iv) a serine residue at the end of the signal has a small
neutral side chain that is recognized by signal peptidase for cleavage.
Three putative polyadenylation signals were found in the 3'-noncoding
region of the cDNA, suggesting alternative polyadenylation sites
may exist, as the cleavage and polyadenylation specificity factor may
bind to any one of these three sequences (18). Indeed, during
sequencing following 3'-RACE, we found that one cDNA clone showed a
shorter 3'-noncoding region, with the poly(A) tail attaching 14 nucleotides downstream of the second AATAAA sequence (Fig. 1).
Furthermore, three ATTTA sequences were found in the 3'-untranslated region (3'-UTR) of the 3
-reductase cDNA, all located in between the second and the third polyadenylation signals, with two occurring in
the sequence which is missing in the shorter 3
-reductase cDNA (Fig. 1). In fact, the 3'-untranslated region has been increasingly recognized as one of the important sequences which may contain distinct
elements involved in regulating mRNA degradation (19). A most well
studied example is the AU-rich element (ARE) found on many unstable
mRNAs. The sequence consensus for the AREs is loosely defined as
AUUUA repeated once or several times within the 3'-UTR. This
pentanucleotide is often found within an AU-rich region of the
mRNA. It seems to be clear that (i) both nuclear and cytoplasmic
proteins can specifically bind to the ARE, (ii) the binding activity of
some of those proteins appears to increase or decrease in response to
the changes in cellular metabolism that lead to alterations in the
stability of ARE-containing mRNA, and (iii) ARE activity can be
mediated by a complex of proteins as in the case of a 20 S complex
(19). We were interested in addressing the question as to whether the
alternative polyadenylation, using AATAAA signals at different
locations in the 3
-reductase cDNA, is related to the regulation
of mRNA stability and what the role of the AREs in the 3' UTR is
likely to be. For this, total RNA from fat body, midgut, and Malpighian
tubules from 78 h last larval instar larvae was subjected to
Northern analysis using probes corresponding, respectively, to
nucleotides 1072-1176, 1177-1281, and 1282-1386 of the cDNA (see
Fig. 1). Because all three probes gave identical signals, the AREs in
the 3'-UTR do not seem to contribute to the regulation of the mRNA
stability in the tissues examined (data not shown).
It has long been recognized that oxidoreductases, dependent upon
nicotinamide coenzymes, can be divided into two extended superfamilies,
according to their structural type and catalytic mechanism (20). The
enzymes of one superfamily contain catalytically important zinc, many
utilizing primary alcohols as substrates, though use of secondary
alcohols and other activities are also noticed (21-24). A structurally
noteworthy feature of these proteins is the highly conserved cysteine
residues, involved in zinc binding, scattered within the first half of
the amino acid sequences. Enzymes of the other superfamily generally do
not contain any catalytically active metal atom, have shorter subunits,
and frequently but not always use large secondary alcohols as
substrates (25-28). Recently, a third superfamily has been proposed
(29, 30). Enzymes of this superfamily are not homologous to those of
the two superfamilies mentioned above and their polypeptide chain
lengths generally lie between those of the two superfamilies. They
contain no metal atom and their substrates include primary and
secondary alcohols. Sequence data base searching revealed that the
predicted amino acid sequence of 3DE 3
-reductase is most similar to
the proteins that belong to the third superfamily of oxidoreductases
(30). The existence of only one cysteine residue (in position 192, see Fig. 1) in the predicted amino acid sequence of the mature protein indicated that the 3
-reductase of S. littoralis contains
no zinc. Motif searching (MOTIF at http://www.motif.genome.ad.jp/) with the predicted amino acid sequence of 3
-reductase uncovered the three
consensus patterns present in aldo/keto reductases (residues 68-85,
163-180, 277-292, Fig. 1), which are specific to this family of
proteins. In addition, its molecular weight (37,689/subunit) is in the
same range as found for these proteins. Hence, we conclude that 3DE
3
-reductase is a new member of the third superfamily of oxidoreductases.
Northern analysis has revealed that the mRNA for 3
-reductase has
a wide tissue distribution with considerable variability in the
abundance of the mRNA among the tissues examined. It is not clear
at present which tissue is the major source for the 3
-reductase
activity in the hemolymph or whether all the tissues containing
3
-reductase mRNA contribute to the enzyme activity in hemolymph.
The cytosolic enzymes reversibly interconverting ecdysone and 3DE
(ecdysone oxidase and 3DE 3
-reductase) have been reported to be
widely distributed, even in tissues that do not reduce 3DE to
3-epiecdysone (31, 32). If such tissue 3DE 3
-reductase is closely
related to the hemolymph 3
-reductase, the wide distribution of
transcripts in the Northern blots could be explained by hybridization
of the probe to mRNA corresponding to such tissue 3
-reductase.
Sakurai and co-workers (33) examined the tissue distribution of the
3
-reductase in B. mori at the protein level, as well as
the level of enzyme activity, and they too found a wide tissue
distribution. The absence of the 3
-reductase protein in central
nervous system in B. mori is consistent with our observation
that 3
-reductase mRNA is not detectable in that tissue from
S. littoralis. However, the fact that Malpighian tubules and
midgut from B. mori do not contain detectable enzyme protein seems to be surprising, since we found that the mRNA for
3
-reductase is abundantly transcribed in these tissues from S. littoralis. Preliminary results of Western blotting with tissues
dissected from S. littoralis at 90 h into the last
instar revealed a similar distribution pattern as that of B. mori, with 3
-reductase being detected in hemolymph as well as
hemocytes, but little in midgut and none in Malpighian tubules (data
not shown). It seems, therefore, that although the mRNA is
abundantly transcribed in Malpighian tubules and midgut, none or only
little is translated into the protein in these tissues. This
observation may indicate that selective translation of mRNA is
indeed an important step in regulation of expression of the gene
encoding 3DE 3
-reductase.
Northern analysis also revealed that the mRNA for the enzyme is
only expressed in the second half of the last larval instar. The
mRNA starts to be detected 48-66 h into the last larval instar, at
which time the actual enzyme activity is just about to appear (6). The
increase in expression can be seen from 72 to 90 h, which
coincides well with the sharp rise in the enzyme activity. The small
decline in the expression between 90 and 96 h is also coincident
with the decrease in the enzyme activity at this time. The second
increase in the mRNA level observed between 102 to 114 h is
presumably responsible for the small rise in the enzyme activity at the
end of the instar. It appears evident, therefore, that the change in
the activity of 3
-reductase is, at least in part, regulated by gene
expression at the transcriptional level, although, as mentioned above,
post-transcriptional regulation may also be as important in the
expression of 3DE 3
-reductase in certain tissues.
Southern analysis suggest that 3
-reductase is encoded by a single
copy gene in the genome of S. littoralis. Although the details of the organization of the gene are far from clear, it is
predicted from the results of Southern analysis that the gene encoding
3
-reductase has at least one intron that contains EcoRI, SalI, and PvuII sites. It remains to be
determined whether the existence of two larger RNAs detected in fat
body by a probe representing the entire coding region and 3'-noncoding
region of the cDNA is due to the possible occurrence of
intermediates in the process of mRNA splicing.
The availability of the cDNA clones encoding 3DE 3
-reductase
should facilitate studies of genomic organization, regulation of
expression of the gene, structure-function relationships of the enzyme,
and its involvement in regulation of the production of ecdysteroids.