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Expression of the Helicoverpa cathepsin b-like proteinase during embryonic development.

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Archives of Insect Biochemistry and Physiology 58:39–46 (2005)
Expression of the Helicoverpa Cathepsin B-Like
Proteinase During Embryonic Development
Xiao-Fan Zhao,1* Xiao-Meng An,1 Jin-Xing Wang,1 Du-Juan Dong,1 Xin-Jun Du,1
Shinji Sueda,2 and Hiroki Kondo2
Cathepsin B-like proteinase from Helicoverpa armigera (HCB) was proposed as being involved in the degradation of yolk
proteins during embryonic development. Recombinant HCB was expressed as a fusion protein with GST in Escherichia coli
BL21 on the basis of its cDNA and purified to homogeneity. The fusion protein was cleaved with thrombin to generate a
soluble protease with a mass of 37 kDa. A polyclonal antiserum against this recombinant protein, raised in the rabbit,
recognized three isoforms of HCB in an ovary homogenate of this insect. Expression of this enzyme during embryonic development was studied using immunoblotting, immunohistochemistry and activity assay. It was found that HCB was expressed
during embryonic development and that its proteolytic activity was detected from embryonic developmental eggs. The fact that
HCB activity is observed in ovaries and developing eggs suggested that the enzyme had already been activated before embryonic development. Immunohistochemistry indicated that the enzyme was located in follicular cells, the sphere of yolk granules, and the fat bodies of female adult. These lines of evidence suggested strongly that HCB takes part in the degradation of
yolk proteins during the development of embryo. Arch. Insect Biochem. Physiol. 58:39–46, 2005. © 2004 Wiley-Liss, Inc.
KEYWORDS: Helicoverpa armigera; cathepsin B-like proteinase; expression; embryonic development
Cathepsin B-like proteinase (EC plays
a vital role in various physiological and pathological processes in mammals, such as the regulation
of cell death (Takuma et al., 2003) and tumor invasion (Eijan et al., 2003). Cathepsin B-like proteinases also are involved in the degradation of yolk
proteins during embryonic development in oviparous animals (Carnevali et al., 1999). Several cathepsin B-like proteinases have been reported from
insects, such as the fruit fly, Drosophila melanogaster
(Adams et al., 2000), the flesh fly, Sarcophaga
peregrina (Takahashi et al., 1993), the mosquito,
Aedes aegypti (Cho et al., 1999) and from the silk
worm, Bombyx mori (Xu and Kawasaki, 2001). Although actions of cysteine proteases and serine proteases in embryonic development are known
(Takahashi et al., 1996), the role of cathepsin B in
embryonic development was implicated only for
A. aegypti. The cathepsin B-like protease from mosquitoes is synthesized and secreted as a latent
proenzyme in a sex-, stage-, and tissue-specific
manner, by the fat body (Cho et al., 1999). The
secreted hemolymph form of the enzyme is a large
molecule, likely a hexamer, consisting of 44-kDa
Department of Biology, School of Life Sciences, Shandong University, Jinan, China
Department of Biochemical Engineering and Science, Kyushu Institute of Technology, Iizuka, Japan
The nucleotide sequence reported in this paper has been submitted to the GenBank with accession number AF222788.
Abbreviations: HCB, cathepsin B-like proteinase from Helicoverpa armigera
Contract grant sponsor: Natural Science Foundation of China; Contract grant numbers: 39870095 and 30330070.
*Correspondence to: Xiao-Fan Zhao, Department of Biology, School of Life Sciences, Shandong University, Jinan 250100, China. E-mail:
Received 10 December 2003; Accepted 6 October 2004
© 2004 Wiley-Liss, Inc.
DOI: 10.1002/arch.20030
Published online in Wiley InterScience (
Zhao et al.
subunits, which is processed to a 33 kDa mature
form at the onset of embryogenesis (Cho et al.,
1999). Another cathepsin B-like cysteine proteinase was purified from ovaries of Helicoverpa armigera and named HCB (Zhao et al., 1998, 2002). It
was demonstrated that the optimum pH of the proteinase is 3–4 and the molecular mass of the mature enzyme is 30 kDa on SDS-PAGE. The cDNA
encoding the pro-HCB was then cloned by RT-PCR
and the identity of the enzyme to cathepsin B from
A. aegypti was 49% (Zhao et al., 2002). It was found
by Northern blot analysis that this proteinase was
transcribed in fat bodies from larvae to adult, and
in ovaries, and the function of the enzyme was
probably regulated on a posttranslational level.
Therefore, the transcription of the enzyme is tissue-specific but not gender- or developmentally
specific, which was different from that from mosquito. Besides, it degraded yolk proteins well in
vitro and thus HCB was proposed to be involved
in the degradation of yolk proteins during embryonic development (Zhao et al., 1998, 2002). In
this report, cDNA of HCB was expressed in E. coli
and a specific antiserum was raised against HCB
purified from E. coli. Distribution of the enzyme
in ovaries and in embryonic developmental eggs
was demonstrated for the first time by immunoblotting and immunohistochemistry. Evidence that
the enzyme takes part in the development of the
embryo is presented below.
Experimental Animals
H. armigera was cultured in the laboratory with
an artificial diet made from wheat and soybean at
dark:light as 8:16 h. Moths were reared in a cage,
covered with gauze for egg laying, and fed 5% saccharose for the development of ovaries. During
embryonic development, eggs were harvested by
time interval and used for experiments.
Glutathione sepharose 4B, pGEX-4T-1, and standard marker proteins (phosphorylase b, 94 kDa;
albumin, 67 kDa; ovalbumin, 43; carbonic anhydrase, 30 kDa; trypsin inhibitor, 20.1 kDa; α-lactalbumin, 14.4 kDa) are products of Amersham
Biosciences Company (Buckinghamshire, England).
Goat anti-rabbit IgG-horse radish peroxidase was
purchased from Zhongshan Biotechnique Company (Beijing, China). The Nitro-cellulose membrane was a product of Zhejiang Sijia Biochemical
Plastic Company (Zhejiang, China). 4-Chloro-1Naphthol was from Amresco (Solon). Complete
Freund’s adjuvant and thrombin were produced by
Sigma Chemical Co. (St Louis, MO). The other
chemicals used were of analytical grade.
Expression of Recombinant HCB
A 1,017-bp cDNA encoding the procathepsin Blike proteinase from H. armigera was inserted into
pGEX-4T-1 plasmid in-frame with glutathione Stransferase and the integrity of the construct was
confirmed by DNA sequencing. After transformation of E. coli BL21, the target protein was expressed
as inclusion bodies induced by 1 mM IPTG in LB/
Ampicillin (100 µg/ml) medium. The inclusion
bodies were solubilized with 8 M urea according
to the method described by Kuhelj et al. (1995)
and then refolded in Tris-HCl buffer (0.1 M, pH
8.0, 5 mM EDTA, 5 mM cysteine) for 16 h at a
concentration of 2 mg/ml proteins, with 2 changes
of same buffer. Target protein was purified by affinity chromatography on Glutathione Sepharose
4B and then cleaved with thrombin (10 U/mg protein) at 23°C overnight. HCB was isolated from
the mixture by SDS-PAGE and the N-terminal
amino acids were sequenced by Edman degradation using an Applied Biosystem model 470 protein sequencer.
Preparation of Rabbit Antiserum Against Cathepsin
B-Like Proteinase From H. armigera
About 100 µg recombinant HCB in the gel was
homogenized with 1 ml of complete Freund’s adjuvant and injected into a rabbit on the back, hypodermically, once a week for 3 weeks. Three boost
injections into an ear vessel followed each week.
Archives of Insect Biochemistry and Physiology
Helicoverpa Cathepsin B-Like Proteinase
The titer of the antiserum thus prepared was determined by indirect enzyme-linked immunosorbent assay (ELISA) (Ausubel et al., 1995) with
a homogenate of ovaries from H. armigera as an
antigen (5 µg/ml). This antiserum was used in all
experiments using immunoassays in this report.
anti-rabbit IgG (1:10,000 in TBS) for 1 h, followed
by 3 × 15 min washes in TBST and 1 × 15 min
wash in TBS. The target protein was then visualized
by allowing peroxidase to react with peroxidase
staining reaction mixture (1 ml 4-chloro-1-naphthol in methanol (6 mg/ml), 10 ml TBS, and 6 µl
H2O2) in the dark for 10 min.
Preparation of Homogenates of Eggs
Freshly laid eggs on gauze were harvested in the
morning as a 0-h developmental sample. Other
eggs were harvested every 8 h and homogenized
in 1 ml of buffer A (10 mM Tris-HCl, pH 7.5, 1
mM EDTA, 2 mM 2-mercaptoethanol). The final
concentration of the sample was diluted to 0.5 mg/
ml prior to use. Protein concentration was determined according to Lowry et al. (1951).
Sodium dodecyl Sulfate Polyacrylamide Gel
Electrophoresis (SDS-PAGE) was run following the
method of Laemmli (1970). Gelatin-SDS-PAGE or
in situ hydrolysis was performed by SDS-PAGE on
a gel containing 0.1% gelatin and the sample was
treated with SDS-sample loading buffer without
heating. Proteolytic activity was detected by Coomassie Brilliant Blue R-250 staining after incubating the gel in 2.5% Triton-X 100 for 1 h, followed
by incubation in acetate buffer (pH 3.6, 0.5 M)
for 3 h (Heussen and Dowdel, 1980).
After SDS-PAGE, proteins were transferred onto
a nitrocellulose membrane electrically and the
bands were detected by the immunoblotting assay
described by Sambrook et al. (1989). The membrane was treated as in the following procedures
at room temperature with shaking: blocked in 2%
nonfat dry milk in TBS (10 mM This-HCl, pH 7.5,
150 mM NaCl) for 1 h, incubated in antiserum
against HCB (1:100 in block solution) for 1 h,
washed in TBST (0.1% Tween-20 in TBS) for 3 ×
15 min, incubated in peroxidase-conjugated goatJanuary 2005
Tissues were dissected and fixed in 4% paraformaldehyde in PBS buffer, pH 7.2 at 4°C for 16
h. After being well washed and dehydrated, samples
were imbedded in paraffin wax and sectioned at a
thickness of 8 µm. Sections were treated as in the
following procedures: the sections were dewaxed
and rehydrated into PBS (140 mM NaCl, 10 mM
sodium phosphate, pH 7.4), blocked endogenous
peroxidase by 3% H2O2 in PBS for 30 min at room
temperature, washed for 3 × 10 min in PBS, and
digested in 0.1% trypsin, 0.134% CaCl2 for 1–5
min at room temperature, washed for 3 × 10 min
in PBS, covered with a solution of 1% normal goat
serum and 1% BSA for 10 min at room temperature, incubated with polyclonal antiserum against
HCB (1:100 in PBS) for 1 h at room temperature,
washed for 3 × 10 min in PBS, incubated with horseradish peroxidase-goat anti-rabbit IgG (1:2,000 in
PBS) at 37°C for 1 h, washed for 3 × 10 min in
PBS and 1 × 10 min in TBS. Color was developed
with 4-Chloro-1-Naphthol and H2O2 (0.1 mg/ml
4-Chloro-1-Naphthol, 0.05% H2O2 in TBS). Negative controls were treated as in the above method
simultaneously but with rabbit pre-immune serum
used in place of the antiserum against HCB.
Expression of Recombinant HCB
The cDNA of cathepsin B-like proteinase from
H. armigera (HCB) was cloned into pGEX-4T-1 inframe with glutathione S-transferase (GST). HCB
was then expressed as a fusion protein with GST
in E. coli BL21 with a molecular mass of about 63
kDa upon induction with IPTG. The protein was
Zhao et al.
kept in inclusion bodies and was solubilized with
8 M urea followed by renaturation as detailed in
Materials and Methods. The fusion protein was purified by affinity chromatography on glutathione
sepharose and then cleaved with thrombin into 26and 37 kDa proteins, molecular masses coincident
with those of GST and HCB, respectively (Fig. 1).
The N-terminal amino acid sequence of the 37 kDa
protein was determined as G S P E F M A A S R A
T F V A; the first 5 amino acid residues (underlined) were coincident with those of GST and the
following 10 residues with those of HCB, proving
that the protein was properly expressed.
Specificity of Rabbit Antiserum Against HCB
A polyclonal antiserum against HCB was raised
in the rabbit using recombinant HCB. The titer of
the antiserum was determined as 1:5,000 by indirect ELISA for a homogenate of ovaries from H.
armigera as an antigen. The specificity of the antiserum was tested by an immunoblotting assay using purified HCB from female fat bodies and
ovaries and extract of ovaries from H. armigera. The
results revealed 1–3 immunostaining bands from
purified HCB and from the ovaries (Fig. 2A). To
Fig. 1. Purification of recombinant HCB from transformed
E. coli BL21. Lanes 1–7: Standard proteins, non-induction,
induction expression, supernatant after sonicating, pellet
after sonicating, purified fusion protein GST-HCB by affinity column of glutathione sepharose 4B, and cleaved fusion protein, respectively. 10% gel; 30-µg proteins per lane.
GST: glutathione S-transferase.
Fig. 2. Assay of specificity of the antiserum against HCB.
A: Immunoblotting to show the specificity of the antiserum. Lanes 1,2: Ovaries of H. armigera and purified HCB
from fat bodies and ovaries of female adult. 10% gel; 20µg proteins per lane. B: SDS-PAGE showing the isoforms
of purified HCB. Lanes 1,2: Standard proteins and purified HCB from ovaries of H. armigera (HCB) on SDS-PAGE.
Lanes 3,4: Activity of HCB and inhibition of the activity
of HCB on gelatin-SDS-PAGE. 12.5% gel.
demonstrate the specificity of anti-HCB to these
multibands, purified HCB was examined by SDSPAGE. There were 1–3 near bands purified HCB
with molecular masses ranging from 29 to 31 kDa,
all of which were active as protease and their
activities could be inhibited by E-64 (N- [N- (1,
3-trans-carboxyoxiran-2-carbonyl)-L-leucyl]-agmatine), a specific inhibitor of cysteine proteinases,
suggesting that these 3 bands were derived from
the same protein (Fig. 2B). Besides, the N-terminal amino acid sequences of band 1 and 2 of HCB
from top were sequenced and they were identical: APEAFDPRDKWPNXP, sequence coincident
with that of the N-terminal sequence of mature
HCB (Zhao et al., 2002). The same antiserum recognized all of the 3 HCB bands by immunostaining, further confirming that they shared an
overlapping epitope(s). Despite the anomaly, it
is certain that the antiserum recognized specifically HCB and its derivatives.
Archives of Insect Biochemistry and Physiology
Helicoverpa Cathepsin B-Like Proteinase
Expression of HCB During Embryonic Development
Expression of Helicoverpa cathepsin B-like proteinase during development of the embryo was
demonstrated by immunoblotting and in situ hydrolysis assay by gelatin-SDS-PAGE. The quantity
of yolk proteins from embryonic eggs declined with
the developmental time from 0 to 24 h (Fig. 3A).
By association, HCB was detected by immunoblotting assay from embryonic eggs at 0, 8, 16, and
24 h, too (Fig. 3B). The activity of HCB paralleled
the results of the immunoblotting assay and it
tended to decline with a decrease in yolk proteins
and with the progress (time) of embryonic development (Fig. 3C) until it disappeared completely
before hatching of the larvae (data not shown).
located around the membranes of follicular cells
and sphere of yolk granules (Fig. 4C).
Location of HCB in tissues was investigated by
immunohistochemistry using fat bodies of female
adults and ovaries. Strong signals were detected
from both fat bodies and ovaries (Fig. 4A,B). Follicular cells, oocytes, and yolk granules were positively stained. Intensive staining in ovary was
There have been many reports on the multiform
of cathepsin B, which normally includes preprocathepsin B, procathepsin B, several intermediate
forms of cathepsin B, and mature isoforms during
maturation of this enzyme. For example, the cathepsin B-like protease from mosquitoes is synthesized
and secreted as a large latent proenzyme, likely a
hexamer, consisting of 44 kDa subunits, which is
processed to a 33 kDa mature enzyme (Cho et al.,
1999). The cysteine proteinase from S. zeamais also
consisted of some bands (Matsumoto et al., 1997).
Three isoforms of mature cathepsin B were recently
purified from bovine kidney: a 31 kDa single-chain
form and a double-chain form with molecular
masses of 23.4 and 25 kDa (Sentandreu et al.,
2003). Compared with the above description, the
3 HCB bands at 31, 30, and 29 kDa were likely
mature active isoforms. This conclusion was supported by several lines of evidence including an
activity assay, inhibition test, N-terminal amino
acid sequencing, and immunostaining. Although
Fig. 3. Expression of HCB during embryonic development.
A: SDS-PAGE showing the degradation of yolk proteins during embryonic development. Lanes 1–5: Homogenates of
eggs developed 0, 8, 16, 24 h after laying and protein
marker, respectively. B: Immunoblotting to show the expression of HCB. Lanes 1–4: Same as A (lanes 1–4). C:
Gelatin-SDS-PAGE showing the proteolytic activity from
the embryonic developmental eggs. Lane 1: Extract of female adult fat bodies. Lane 2: Extract of ovaries. Lanes
3–6: Same as A (lanes 1–4). 10% gel; 20-µg proteins per
lane. Vn H: vitellin heavy chain; Vn L: vitellin light chain;
N: unknown yolk protein.
Location of HCB in Ovaries and Fat Bodies of
Adult Females
January 2005
Zhao et al.
Fig. 4. Immunohistochemistry showing the location of
HCB in fat body and ovary. A: Fat body of female adult.
B: Ovary. a,b: Negative controls correlated to A and B,
60 × 10 amplification under microscope for A, a, B, b.
C: Square in B magnified (1 × 4.5). D: Square in b magnified (1 × 4.5). Fc: follicular cells; Oo: oocyte; Yg: yolk
the difference among these isoforms is not clear,
the bands on immunoblotting were not from the
non-specificity of the antiserum but from the different isoforms of the enzyme. Besides mature
isoforms, cathepsin B may exist as precursor and
intermediate, which might also lead to different
immunostained bands. Aoki et al. (2002) reported
a precursor of cathepsin B-like enzyme with a molecular mass of 60 kDa from mackerel, which con-
verted to matured cathepsin B (molecular mass of
23 kDa), and intermediate forms of 40 and 38 kDa
were detected during the conversion. In addition,
the migration of polypeptides in SDS-PAGE can
be effected by many factors such as glycosylation,
phosphorylation, or unusual amino acid composition (Hodgkinson and Steffen, 1997). Slower
mobility immunostained HCB bands were observed in the immunoblotting assay, compared
Archives of Insect Biochemistry and Physiology
Helicoverpa Cathepsin B-Like Proteinase
with gelatin-SDS-PAGE, which probably resulted
from the recognition of antibody to the precursor,
proenzyme, or intermediate of this enzyme. Theoretical analysis indicated that there were possible
N-glycosylation sites on amino acids 130 and 177,
an O-glycosylation site on 51, and phosphorylation sites at Ser-6, Thr-2, and Tyr-4 (Zhao et al.,
2002), which might also result in the mobility
variation of this enzyme. These possibilities need
to be clarified in future work.
Immunohistochemistry further revealed the location of HCB in fat bodies and ovaries with the
former stained more intensely, suggesting that they
were the major tissues for distribution of the enzyme. That HCB was expressed in fat bodies, membranes of follicular cells, oocytes, and spheres of
yolk granules was consistent with the results from
Northern blot analysis, which suggested that fat
bodies, ovaries including follicular cells, and oocytes synthesized HCB (Zhao et al., 2002). Because
HCB is also synthesized in fat bodies, there thus is
a possibility that, in vivo, HCB is secreted into
hemolymph and internalized by oocytes along
with vitellogenin, as happened in cathepsin L from
B. mori (Yamamoto et al., 1994) and cathepsin B
from A. aegypti (Cho et al., 1999).
Previous studies had already demonstrated that
HCB is synthesized in fat bodies and ovaries and
degrades yolk proteins in vitro. The activity of this
enzyme was detected from fat bodies of adults and
ovaries but was not detected from larval fat bodies
(Zhao et al., 2002). This recent study offered further evidence that HCB is distributed in embryo
developing eggs and its activity is associated with
the degradation of yolk proteins during embryo
development. The enzyme appeared in ovaries and
continued in developing eggs, and then declined
in 24 h along with the degradation of yolk proteins. On the basis of these lines of evidence, it is
concluded that HCB plays an important role in the
degradation of yolk proteins during embryonic development of this insect. The fact that fat bodies
of adults decomposed suggests that HCB might be
involved also in the degradation of fat bodies after metamorphosis of adult as that in S. peregrina
(Takahashi et al., 1993).
January 2005
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