From the Department of Parasitology, National
Yang-Ming University, Taipei 112, Taiwan, the § Department
of Entomology, Michigan State University, East Lansing, Michigan
48824-1115, the ¶ Programs in Genetics and Program in Cell and
Molecular Biology, Michigan State University, East Lansing, Michigan
48824-1115, and the ** Laboratory of Electron Microscopy, Institute of
Cytology of the Russian Academy of Sciences,
194064 St. Petersburg, Russia
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ABSTRACT |
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Here we report identification of a novel member
of the thiol protease superfamily in the yellow fever mosquito,
Aedes aegypti. It is synthesized and secreted as a latent
proenzyme in a sex-, stage-, and tissue-specific manner by the fat
body, an insect metabolic tissue, of female mosquitoes during
vitellogenesis in response to blood feeding. The secreted, hemolymph
form of the enzyme is a large molecule, likely a hexamer, consisting of
44-kDa subunits. The deduced amino acid sequence of this 44-kDa
precursor shares high similarity with cathepsin B but not with other
mammalian cathepsins. We have named this mosquito enzyme vitellogenic
cathepsin B (VCB). VCB decreases to 42 kDa after internalization by
oocytes. In mature yolk bodies, VCB is located in the matrix
surrounding the crystalline yolk protein, vitellin. At the onset of
embryogenesis, VCB is further processed to 33 kDa. The embryo extract
containing the 33-kDa VCB is active toward
benzoyloxycarbonyl-Arg-Arg-para-nitroanilide, a cathepsin B-specific
substrate, and degrades vitellogenin, the vitellin precursor. Both of
these enzymatic activities are prevented by
trans-epoxysuccinyl-L-leucylamido-(4-guanidino)butane
(E-64), a thiol protease inhibitor. Furthermore, addition of the
anti-VCB antibody to the embryonic extract prevented cleavage of
vitellogenin, strongly indicating that the activated VCB is involved in
embryonic degradation of vitellin.
Cathepsin B is a thiol (cysteine) protease with both endopeptidase
and peptidyldipeptidase activities. Due to its broad specificity, cathepsin B plays a key role in intracellular protein catabolism in the
lysosomal system (1). Cathepsin B has been well characterized both
enzymatically and molecularly (2-10). The mammalian cathepsin B has
been implicated in tumor invasion, progression, and metastasis (11-15). Tumor-specific cathepsin B is secreted by malignant cells as
a latent high molecular weight precursor, presumably activated at
cell contacts (16, 17).
In addition, cathepsins B, as well as the related cathepsins L, have
been identified in numerous parasitic protozoa and helminthes, including prevalent pathogens of human and domestic animals (18-26). In the blood-sucking bug, Rhodnius prolixus, cathepsin B is
the major gut proteolytic enzyme (27). In these organisms, cathepsins B
and L are presumably involved in the degradation of host hemoglobin.
In insects and other arthropods, cathepsins B and L also participate in
key developmental processes. In the flesh fly, Sarcophaga peregrina, hemocytes produce the extracellular form of a cathepsin B-like enzyme that participates in decomposition of the larval fat body
during metamorphosis (28-30). Moreover, cathepsins B and L have been
implicated in degradation of yolk proteins during embryonic development
(31-41).
The elucidation of developmental mechanisms in the mosquito is
important because this insect transmits the most devastating of
vector-borne human diseases, including malaria, lymphatic filariasis, Dengue fever, and many others. Little is known, however, about the
process of yolk protein degradation in the mosquito embryo. Previously,
we have found that during vitellogenesis, the female fat body, a
metabolic tissue analogous to the vertebrate liver, synthesizes and
secretes a latent proenzyme of a serine carboxypeptidase which is
homologous to yeast carboxypeptidase Y (42, 43). This 53-kDa proenzyme,
which we named vitellogenic carboxypeptidase (VCP),1 is specifically
accumulated by developing oocytes and deposited in yolk bodies.
Although we have demonstrated that VCP is activated during
embryogenesis, its role remains unknown.
In this paper we report the discovery of an unusual cathepsin-B-like
thiol protease from the mosquito, Aedes aegypti. Similar to
VCP, this enzyme is produced by the fat body of vitellogenic female
mosquitoes in response to blood feeding. The cDNA encoding this
unique mosquito enzyme, which we named vitellogenic cathepsin B (VCB),
exhibits high similarity to vertebrate cathepsin B. VCB is secreted by
the fat body as a latent proenzyme, similar in size to the latent tumor
cathepsin B (44-kDa); it is accumulated by developing oocytes, where it
is stored in yolk bodies. For the embryogenesis, VCB is processed to 33 kDa, similar in size to the activated single chain mammalian cathepsin
B (16, 17). We have demonstrated that the embryo extract containing the
33-kDa VCB is active against
benzoyloxycarbonyl-Arg-Arg-para-nitroanilide (Z-Arg-Arg-pNA), a cathepsin B-specific substrate, and
degrades vitellogenin (Vg), a yolk protein precursor. Both enzymatic
activities are prevented by trans-epoxysuccinyl
-L-leucylamido-(4-guanidino)butane (E-64), a thiol protease
inhibitor. Furthermore, addition of the anti-VCB antibody to the
embryonic extract prevented cleavage of vitellogenin, strongly
indicating that the activated VCB is involved in embryonic degradation
of vitellin.
Reagents--
All analytical grade chemicals and protease
inhibitors were purchased from Sigma and Calbiochem, respectively,
unless stated otherwise. [32P]dATP (3,000 Ci/mmol) and
[35S]dATP were obtained from NEN Life Science Products
Inc., while [35S]methionine (1,120 Ci/mmol) was from ICN
Radiochemicals. DEAE-Sepharose was purchased from Pharmacia Biotech
Inc. Bio-Rad was the source for protein assay reagents, econo-Pac
desalting columns, and molecular weight standards for electrophoresis.
Safety Solve II scintillation mixture was supplied by Research Product
International. Paraformaldehyde, glutaraldehyde, and LR White were
obtained from Polyscience, and protein-A-colloidal gold conjugates from
E-Y Laboratories.
Animals--
Mosquitoes, A. aegypti, were reared as
described previously (42). Three to five days after eclosion adult
females were fed on white rats to initiate vitellogenesis. Mosquitoes
were dissected in TES-buffered Aedes physiological saline
(44) at room temperature.
Protein Preparation and Electrophoresis--
Fat body, ovarian,
and embryonic proteins were prepared as described previously (42) in B3
buffer containing 25 mM TES, pH 7.5, 150 mM
NaCl, 10% glycerol, 10 mM EDTA. Unless otherwise noted,
all solutions contained the following inhibitors: 1 mM 4-(2-aminoethyl)-benzenesulfonylfluoride, HCl (AEBSF)
(Calbiochem), 1 mM phenylmethylsulfonyl fluoride
(PMSF) (Roche Molecular Biochemicals), 5 mM
Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE)
was done using either a 10% or 12% straight gel by the method of
Laemmli (45) in vertical slab gels. Proteins were visualized by
staining with either Coomassie Brilliant Blue R-250 or silver, or were
processed for fluorography.
Nonreducing native polyacrylamide gel electrophoresis was performed
using 3.5-25% acrylamide gradient gels cross-linked with 2.6%
methylene bisacrylamide (46-48). The native PAGE sample buffer contained 125 mM Tris-HCl, pH 6.8, 12% glycerol, and 15 µM bromphenol blue. Gels were run for 18-21 h at 125 volts at 4 °C.
In Vitro Protein Labeling--
The in vitro culture
of fat body and ovaries was as described previously (42). Synthesized
and secreted fat body proteins were radiolabeled by incubating 3 female
or 6 male fat bodies at 27 °C in 50 µl of TES-buffered culture
media modified (49, 50) to contain the appropriate concentration of
[35S]methionine (pulse media). If only pulse media was
needed, fat bodies were incubated for 3 h.
Ovary-synthesized proteins were labeled in vitro by placing
five pairs of ovaries (18 h post-blood meal) into 100 µl of
TES-buffered culture media (51, 52) with [35S]methionine
replacing unlabeled methionine. The ovaries were removed after 2.5 h, rinsed 3 times in APS and frozen at Amino Acid Sequence Analysis--
The proteins, prepared as
described above for antibody production, were electroblotted onto
polyvinylidene difluoride membrane (0.2 µm pore size; Bio-Rad). The
44- and 42-kDa bands were excised from the blots of fat body-secreted
and ovarian protein, respectively. The blots were sent to Harvard
Microchem (Harvard University) for tryptic digestion and amino acid
sequence analysis.
Cloning of VCB cDNA--
The probe used for screening a
cDNA library for VCB was generated by PCR. RNA was isolated from
fat bodies of 24-h post-blood fed female mosquitoes. cDNA was
synthesized from the fat body mRNA and used as the template for
PCR. An antisense degenerate primer was constructed from a portion of
an internal peptide sequence, MADVEDL, obtained from microsequencing a
tryptic fragment of the 44-kDa fat body-secreted VCB. The sequence of
the first primer was:
ATGGC(A/T/G/C)GA(T/C)GT(A/T/G/C)GA(A/G)GA(T/C)(T/C)T. The second primer
was a 17-mer d(T). The resulting PCR product was subcloned into a
pGEM-T vector for sequencing. The confirmed VCB fragment was used to
screen a mosquito vitellogenic fat body cDNA library which was
constructed at the EcoRI site of the DNA Analysis and Alignment--
The putative signal peptide in
the deduced amino acid sequence was determined according to Kyte and
Doolittle (56). The deduced amino acid sequence was analyzed by FastA,
Motifs, and Gap programs of Genetics Computer Group software
(University of Wisconsin, WI).
Isolation of RNA and Northern Blot Hybridization--
Total RNA
was prepared using the guanidine isothiocyanate method (57-59). For
Northern blot analyses, total RNA and RNA markers (Life Technologies)
were separated by electrophoresis in 1.2% agarose formaldehyde gels.
The RNA was transferred to a nitrocellulose membrane and hybridized to
32P-labeled probes. The conditions of hybridization at high
stringency were used according to Cho et al. (60). A 255-bp
EcoRI-HincII fragment located at the 5'-end of
the VCB cDNA was used as a probe.
As a control for RNA loading, the cytoplasmic actin 5C gene of A. aegypti was used to probe the mosquito actin mRNA. Cloning of
mosquito actin cDNA was described previously (61).
In Vitro Transcription and Translation--
For in
vitro transcription and translation, the entire VCB cDNA was
subcloned into pGEM 7Z(+) (Promega) under control of the SP6 promoter.
Plasmid DNA was purified by the Wizard Minipreps DNA Purification
System (Promega). Two micrograms of purified plasmid DNA were used for
transcription and translation in the SP6 TNT Coupled Reticulocyte
Lysate system (Promega). [35S]Methionine was used to
label the translated product. All protocols were followed as described
by the manufacturer.
Antibody Production--
Ovaries dissected from A. aegypti 28 h after blood feeding were processed and subjected
to DEAE-Sepharose anion-exchange chromatography (51). Proteins in the
Vn-free unbound fraction were separated by preparative SDS-PAGE. A band
containing VCB (42 kDa) was excised and electroeluted using an ISCO
model 1750 Electrophoretic Concentrator according to recommendations of
the manufacturer. The elute provided the source of antigen to produce
antibodies to this peptide (VCB-Ab1). Rabbits were injected with
antigen/adjuvant (TiterMax, CytRx) two times at a 1-month interval.
Serum proteins from the immunized rabbit were precipitated with
ammonium sulfate at 35% of saturation and reconstituted at 5 mg/ml.
To avoid possible cross-hybridization with other cathepsins B, an
Escherichia coli-expressed fusion protein, containing the VCB 20-kDa N-terminal region was used for production of another polyclonal antibody (VCB-Ab2). This VCB region contained the signal peptide, the entire propeptide, and an N-terminal portion of the mature
VCB, which was less conserved that the rest of the protein. The
antibody production was conducted as described for VCB-Ab1. Polyclonal
antibodies against VCP and the Vg large subunit as well as monoclonal
antibodies against Vg small subunit were already available (42,
62).
Immunoblots and Immunoprecipitation--
Proteins from SDS-PAGE
and native PAGE gels were transferred to BA-S 83 supported
nitrocellulose (Schleicher and Schuell) as described previously (48).
Immunopositive bands were detected with the ECL Western blotting
detection system (Amersham). Prior to immunoprecipitation, Vn or Vg
were partially removed from ovarian extracts or fat body secretions,
respectively, using a suspension of DEAE-Sepharose CL-6B as described
previously (42). After removal of Vn or Vg, VCB was immunoprecipitated
with anti-VCB antibodies. Protein A-Sepharose was added as a
precipitating agent (42). After immunoprecipitation, the resulting
pellet was washed and used in radioimmunoassay or SDS-PAGE and
fluorography experiments (42).
Immunolabeling--
For immunocytochemical examination, tissues
were fixed with 2% formaldehyde and 0.1% glutaraldehyde in 0.05 M sodium phosphate buffer, pH 7.4, for 45-60 min at room
temperature. The specimens were then blocked with 100 mM
glycine in the same buffer for 1 h at room temperature. For light
immunocytochemistry, cryosections of 8 µm were applied on
poly-L-lysine-coated slides and processed as described by
Raikhel and Lea (63). The results were visualized by using
phase-contrast and epifluorescence microscopy.
For electron microscopy, the tissues were dehydrated in ethanol and
embedded in LR-White resin. The ultra-thin sections were prepared using
an Ultracut E microtome (Reichert-Jung) and mounted on formvar-coated
or uncoated nickel grids. The labeling was carried out on drops of
reagents as described previously (64). Double labeling was performed
according to the two-side method (65, 66). The controls included
substitution of primary antibody by nonimmune rabbit IgG and the
omission of primary antibodies followed by normal staining procedures.
After immunolabeling, the sections were stained with uranyl acetate and
lead citrate and examined with a JEOL 100CX II transmission electron microscope.
Enzyme Assay--
For enzymatic assay, embryos, 12-48 h after
egg deposition, were extracted in B3 buffer containing 1 mM
PMSF, 1 mM AEBSF, and 5 µM pepstatin. Each
enzymatic reaction received 38 µg of total protein of embryonic
extract. All enzymatic reactions were performed in duplicate using the
MES buffer, pH 5.5, containing 100 mM MES, 1 mM
PMSF, 1 mM AEBSF, 5 µM pepstatin, and 2.5 mM EDTA. Enzymatic assays were initiated by the addition of
81.1 mM Z-Arg-Arg-pNA (Bachem Biosciences) in
100% methanol to the reaction mixture, yielding a final concentration
of 1.6 mM substrate and 2% methanol. To study the effect
of specific inhibitors, either E-64 or chymostatin were added to the
protein mixtures in the following concentrations: 0.3, 0.6, 1.2, or 2.5 mM; the mixture was incubated for 20 min at 37 °C prior
to the addition of the enzymatic substrate. The control reaction was
preincubated without any inhibitor. Data were collected by measuring
the absorbance at 405 nm with a spectrophotometer after 2.5 h. A
standard curve of pNA was plotted and regressed by the
linear least-squares method. This curve was used to calculate the
amount of pNA liberated by enzymatic activity. In
experiments to determine the optimum pH of the enzyme, a range of pH
from 3.5 to 7.5 was used.
Identification of a Novel Yolk Protein Precursor from the Mosquito
Fat Body--
Previous analysis of proteins synthesized and secreted
by the vitellogenic mosquito fat body has revealed a polypeptide of 44 kDa, in addition to Vg and VCP; furthermore, the presence of a
polypeptide of 42 kDa has been observed among proteins extracted from
vitellogenic ovaries or newly laid eggs (42). Here, polyclonal antibodies were produced against the 42-kDa polypeptide that was gel-purified from the ovarian source (VCB-Ab1). Immunoblot analysis showed that these antibodies recognized not only the 42-kDa ovarian polypeptide but also the single polypeptide of 44 kDa from vitellogenic fat bodies and their secretions, thus confirming the immunological identity of these two polypeptides. The immunoreactive polypeptides were present in neither previtellogenic female fat bodies nor males
(Fig. 1).
Amino acid sequences were obtained by microsequencing tryptic fragments
of the 42-kDa ovarian polypeptide and the 44-kDa polypeptide from
vitellogenic fat body secretions. These sequences were analyzed for
protein similarity. The results indicated that microsequenced fragments
from both 42- and 44-kDa polypeptides shared similarity with human and
other vertebrate cathepsins B (data not shown).
Cloning and Analysis of the cDNA Encoding the 44-kDa Yolk
Protein Precursor, Vitellogenic Cathepsin B--
A 863-bp cDNA
fragment was obtained from the amplification of vitellogenic fat body
RNA by the PCR technique using the degenerate primer derived from the
44-kDa polypeptide sequence and an oligo(dT) primer. Both ends of the
fragment matched the PCR primer sequences, and an open reading frame
showed high similarity to vertebrate cathepsin B (data not shown). This
PCR fragment was subcloned into a pGEM-T vector and was subsequently
used as a probe to screen the cDNA library prepared from the fat
bodies of vitellogenic female mosquitoes.
Several positive clones were isolated from the cDNA library, all of
which measured approximately 1.3 kb and shared identical restriction
maps. Partial sequencing of these clones showed they contained a
sequence identical to that of the PCR fragment. The longest clone was
sequenced from both strands. This cDNA clone of 1,239 bp contained
an open reading frame of 1,158 nucleotides. It had an unusual putative
polyadenylation signal, ATTAAA, reported previously (67, 68), located
20 bp upstream of the poly(A) tail (Fig.
2). The cDNA clone encoded a
prepro-protein of 386 amino acid residues with a predicted size of
43,069 Da. The two sequences determined by direct microsequencing of
the ovarian 42-kDa VCB and the fat body 44-kDa VCB were both found in
its deduced amino acid sequence (Fig. 2). Hydropathy analysis (56) revealed that the protein was hydrophilic, possessing a strongly hydrophobic putative signal peptide of 16 residues (Fig. 2); moreover, the protein was expected to be positively charged with an isoelectric point (pI) of 7.95. However, in light of two potential
N-linked glycosylation sites (69) and 10 potential
phosphorylation sites (70) in the deduced VCB amino acid sequence, the
fully processed protein could differ in its net charge (Fig. 2).
To verify its identity, the 1.3-kb cDNA clone was expressed using a
coupled in vitro transcription/translation system. A single polypeptide of the predicted size, 43 kDa, was immunoprecipitated from
the translation reaction by the VCB-Ab1 antibodies, prepared against
ovarian 42-kDa polypeptide (Fig. 3).
Furthermore, in the presence of canine microsomal membranes, the
expressed polypeptide increased to 44-kDa, identical in size to the
44-kDa VCB secreted by the fat body (Fig. 3). The 1.3-kb cDNA clone
was also used as a probe for Northern hybridization. It hybridized
strongly only to 1.3-kb mRNA from the fat body of vitellogenic
female mosquitoes (see below). Taken together, these data confirm that
the 1.3-kb cDNA encodes the 44-kDa fat body precursor of the
ovarian 42-kDa VCB.
Mosquito 44-kDa Yolk Protein Precursor (VCB) Is a Proenzyme
Homologous to Cathepsin B--
The deduced amino acid sequence of VCB
exhibited high similarity to the family of eukaryotic thiol proteases,
particularly the mammalian cathepsins B and cathepsin B-like proteases
of invertebrates (Fig. 4). However, it
shared only limited similarity to other cathepsins and related
proteases, such as papain (not shown). The multiple alignment of
homologous thiol proteases predicted that a putative start of the
mature VCB occupied position Leu-125 (Figs. 2 and 4). Furthermore,
three active sites characteristic for cathepsin B were conserved in VCB
at positions Cys-150, His-315, and Asn-335 (Figs. 2 and 4). In
addition, from six disulfate bonds typical for thiol proteases (4), the
cysteine positions of five putative bonds were conserved in VCB.
In contrast to mature enzymes (Fig. 4), the pro-peptide portion of VCB
exhibited no significant similarity to those of cathepsins B (data not
shown). Interestingly, however, similar to mammalian cathepsin B, the
mosquito VCB pro-peptide has one putative glycosylation site, while the
cathepsin B-like proteases of other invertebrates have none (Fig.
2).
Mosquito VCB Is a Fat Body-specific Protein Produced in
Vitellogenic Females in Response to a Blood Meal--
Use of the
entire VCB cDNA clone as a probe in the Northern blot analyses
revealed a 1.3-kb transcript specific to vitellogenic fat bodies;
additionally, weak hybridization to a slightly smaller mRNA band
was observed in all other tested tissues (data not shown). This trace
hybridization was likely due to partial similarity shared by VCB and
other thiol proteases possibly of lysosomal origin. However, the use of
a 208-bp of 5'-end of VCB fragment including the 5'-end noncoding
region and the prepro-portion of VCB as a probe resulted in
hybridization only to the 1.3-kb transcript from vitellogenic fat
bodies (Fig. 5). Thus, Northern blot
analyses confirmed the data we obtained at the protein level and
demonstrated that the VCB gene was expressed exclusively in the fat
body of vitellogenic female mosquitoes (Fig. 5). The 1.3-kb VCB
transcript appeared in the fat body only after blood feeding,
indicating that similarly to yolk protein precursors, it was initiated
in response to a blood meal. The levels of the 1.3-kb VCB transcript reached a peak between 18 and 24 h PBM and then gradually
declined. The message was not detected in the fat bodies at the end of
vitellogenic cycle, 42 and 48 h PBM (Fig.
6).
The secretory activity of the fat body with respect to production of
yolk protein precursors was evaluated by pulse labeling using
[35S]methionine for 1 h and collecting the chase
media secretions for analysis. VCB secretion was monitored by VCB-Ab1
antibodies. As a control, anti-VCP antibodies were used to trace
secretion of this yolk protein precursor. The data from these analyses
showed that the kinetics of VCB secretion by the fat body were similar to those of VCP (Fig. 7). Both yolk
protein precursors were detected at 4 h after initiation of
vitellogenesis by a blood meal. Their synthesis and secretion increased
rapidly to a maximum at 24-h post-blood meal, declined to a very low
level by 36 h, and reached almost background level by 48 h
(Fig. 7).
Western blot analyses of the VCB protein in the fat body, utilizing
either VCB-Ab1 or VCB-Ab2 antibodies, revealed a 44-kDa protein profile
similar to that of secreted VCB, indicating that like yolk protein
precursors, Vg and VCP, VCB was not stored in the fat body after its
synthesis and was immediately secreted into the hemolymph (not shown).
Both VCB-Ab1 and VCB-Ab2 antibodies recognized the same 44-kDa protein
band in fat body preparations at all stages of vitellogenesis when the
VCP gene was expressed (not shown).
VCB Is Localized in Fat Body Cell's Secretory and Oocyte's
Endocytotic Organelles--
Previously (66) we utilized the VCB-Ab1
antibodies (designated there as anti-44KP antibodies) for localization
of VCB. In this work we compared the localization of VCB by VCB-Ab2,
produced against the recombinant VCB, with that by VCB-Ab1, produced
against purified ovarian 42-kDa peptide. Fluorescent
immunohistochemical analyses using either VCB-Ab1 or VCB-Ab2 antibodies
demonstrated that VCB was localized only in the vitellogenic fat body
and ovary after a blood meal (Figs. 8 and
9). In both tissues, the distribution of
VCB was similar to that of VCP (42). Of particular interest was
localization of VCB in the ovary, where it was seen surrounding the
yolk granules (Fig. 9B).
The subcellular distribution of VCB, immunocytochemistry at the
electron microscopic level, was used to localize VCB in the vitellogenic fat body and ovary. In the fat body cells, the
trophocytes, VCB was present in the organelles of the secretory
pathway: the Golgi complex and secretory granules. When double
immunolabeling was performed utilizing antibodies for Vg, VCB, and
protein A-colloidal gold particles of two different sizes, VCB was
co-localized with Vg in these organelles, indicating the simultaneous
processing of these proteins in the trophocyte's secretory system
(Fig. 10).
In vitellogenic ovarian follicles, which consist of the oocyte and
nurse cells surrounded by follicle cells, VCB was present only in the
oocyte's vesicles, endosomes, and yolk bodies (66). Simultaneous
labeling for VCB and Vg demonstrated that these proteins were mixed in
endosomes (66). However, localization of VCB differed dramatically in
mature yolk bodies, where VCB was distributed as a narrow layer on the
surface of the crystalline Vn, a storage form of Vg (Fig.
11). Morphologically, this area of the
yolk body was visible as a non-crystalline matrix separating the
crystalline yolk from the yolk body membrane. Double immunolocalization
showed that in mature yolk bodies, this non-crystalline matrix was free of Vn, while, in contrast, the crystalline yolk was always free of VCB
(66). The distribution of VCB in mature yolk bodies was similar in
oocytes at the peak of endocytosis (24 h post-blood meal) and in those
after termination of yolk accumulation (48 h post-blood meal).
Correlation of Changes in the Native and Subunit Composition of VCB
in the Ovaries and Eggs with Embryonic Development--
The processing
of the 44-kDa hemolymph form of VCB in oocytes and eggs was monitored
by immunoblot analysis and SDS-PAGE. In the oocyte, the internalized
VCB reduced to 42 kDa; it remained unchanged for most of oocyte
development; at 24 h PBM (Fig. 12, lane 1) in oocytes at the peak of protein uptake (51).
However, in fully grown oocytes with completed protein yolk
accumulation and nearly completed choriogenesis (87) at 48 h PBM,
a 37-kDa band appeared which was recognized by either VCP-Ab1 or
VCB-Ab2 antibodies (Fig. 12, lane 2). VCB further diminished
to 33 kDa at the onset of embryogenesis in newly laid eggs (Fig. 12,
lane 3). Immunoblot analyses utilizing either VCB-Ab1 or
VCB-Ab2 showed that VCB was maintained as a 33-kDa polypeptide until
the end of embryogenesis (Figs. 12 and
13). No immunopositive VCB bands were
detected in extracts of newly hatched first instar larvae or later
stages (Fig. 13).
Native PAGE and immunoblot analyses revealed that in the hemolymph, VCB
existed as a high molecular weight molecule: its apparent size varied
slightly depending on gel conditions within an average value of
236 ± 7 kDa (Fig. 15). The VCB native size decreased to 132 ± 4 kDa at the onset of embryonic development in the egg, when VCB was
processed to its 33-kDa form; there was also a minor immunopositive
band of 66 kDa present in the same preparation (Fig.
14).
Mosquito VCB Is Activated in Embryos and Is Involved in Degradation
of the Major Yolk Protein, Vitellin--
The processing of VCB to its
33-kDa form is reminiscent of the proteolytic activation of mammalian
cathepsin B (71). To evaluate whether this VCB processing results in
its enzymatic activation, we used Z-Arg-Arg-pNA, a substrate
exhibiting specificity to cathepsin B (72). Secretions from
vitellogenic fat bodies, containing 44-kDa VCB, did not exhibit
appreciable cathepsin B enzymatic activity (data not shown). In
contrast, protein extracts from embryos containing 33-kDa VCB showed
significant cathepsin B activity (Fig.
15). Enzymatic activity was optimal at
pH 5.5 (data not shown). This enzymatic activity, associated with
embryonic extracts containing the 33-kDa VCB was highly sensitive to
E-64. It showed partial sensitivity to chymostatin (Fig. 15).
Next, we determined whether the processing of VCB to its 33-kDa form
coincided with the beginning of vitellin (Vn) degradation. We applied
immunoblot analysis using monoclonal antibodies against the Vg/Vn small
66-kDa subunit (Fig. 16A)
and polyclonal antibodies against the Vg/Vn large 200-kDa subunit (Fig.
16B). The immunoblot analysis of Vn in the ovary and embryos
of the same stages as in Fig. 12 showed that clear signs of Vn cleavage
were evident only in embryos, coinciding with the processing of VCB to
the 33-kDa form. Interestingly that the large Vn subunit exhibited a
degradation pattern earlier than the small Vn subunit (Fig. 16,
A and B, lanes 3 and 4). However, both
Vn subunits were completely degraded by the end of embryonic
development (Fig. 16, lanes 6).
To ascertain whether the cathepsin B activity detected in mosquito
embryos is linked to Vn degradation, purified Vg, labeled with
[35S]methionine was incubated with embryonic protein
extracts containing the 33-kDa VCB (Fig.
17). The results of the SDS-PAGE
analyses showed that under these conditions, Vg was degraded.
Importantly, Vg degradation was entirely inhibited by addition of E-64
to the protein mixture (Fig. 17, lane 3); however, protease
inhibitors specific to serine, aspartic, and metalloproteinases were
not effective in inhibiting Vg degradation (Fig. 17, lanes 1, 2, and 4). In the next experiment,
[35S]methionine-labeled Vg was incubated with embryonic
protein extracts containing the 33-kDa VCB in the presence of
increasing concentrations of VCB-Ab1 antibodies (Fig.
18, lanes 4-6). Control
samples were incubated with the same amounts of either preimmune IgG
(Fig. 18, lanes 7-9) or bovine serum albumin (Fig. 18,
lanes 10-12). At the concentration of 0.5 µg/µl, the
specific anti-VCB antibodies almost entirely inhibited cleavage of Vg
(Fig. 18, lane 6). The same amounts of preimmune IgG in the
solution had negligible effect of Vg degradation (Fig. 18, lane
9), while bovine serum albumin had no effect (Fig. 18, lane
12).
To test whether or not pro-VCB is processed and activated under acidic
conditions, as are mammalian cathepsins B (7, 73), the secretory 44-kDa
and ovarian 42-kDa forms of VCB were incubated under different acidic
pH conditions (4.0 and 5.5). After treatment with acidic pH, the 44-kDa
pro-VCB neither catalyzed the substrate Z-Arg-Arg-pNA (Fig.
19A, line 1) nor degraded
[35S]methionine-labeled Vg (not shown). Immunoblot
analysis showed that the 44-kDa pro-VCB was processed to a 35-kDa
peptide but not to a 33-kDa one (Fig. 19B, lanes 3 and
4). Unlike the 44-kDa pro-VCB, the ovarian extract,
containing the 42-kDa VCB, exhibited cathepsin B activity after
treatment with acidic pH (Fig. 19A). The immunoblot revealed
that VCB was processed to a 33-kDa form (Fig. 19B, lane 5).
VCB processing was equally efficient at pH 4.0 and 5.5 and unaffected
by the presence of protease inhibitors specific to serine, aspartic,
and metalloproteinases (Fig. 19B).
In this paper, we report an unusual form of cathepsin B-like thiol
protease from the mosquito, A. aegypti. Cloning and analysis of the cDNA encoding VCB demonstrated its high similarity to
mammalian cathepsins B and invertebrate cathepsin B-like proteases, but not to other related cathepsins. The structural analysis of the deduced
amino acid VCB sequence suggests that the folding and activity of this
enzyme are likely similar to cathepsins B: the predicted start of
mature VCB at Leu-125 was similar to mammalian cathepsins B; moreover,
the active sites and cysteine positions of five putative disulfide
bonds were conserved in VCB (Fig. 4).
The mosquito VCB was also observed to resemble mammalian cathepsins B
in its size. It was secreted as a proenzyme of 44 kDa similar in
size to the latent pro-cathepsin B secreted by malignant cells (71).
Moreover, the 33-kDa embryonic form of VCB exhibited the same size as
the active single chain cathepsin B (71).
The presence of the 33-kDa embryonic form of VCB was shown to correlate
with activity characteristic of cathepsin B (72). This activity was
highly expressed with the cathepsin-B-specific substrate,
Z-Arg-Arg-pNA; it was sensitive to E-64, but considerably less to chymostatin.
The unique feature that we observed for this mosquito cathepsin-B-like
enzyme was its exclusive synthesis and secretion by the fat body of
vitellogenic female mosquitoes as a latent, high molecular size
precursor consisting of five or six subunits with a molecular mass of
44 kDa. Its synthesis in the female fat body is initiated by blood
feeding, and the kinetics of its secretion by the vitellogenic fat body
are similar to those of the yolk protein precursors, Vg (59) and VCP
(42, 43).
We established the link between the 44-kDa fat body-secreted hemolymph
VCB and the 42-kDa ovarian VCB by using the anti-42-kDa VCB (VCB-Ab1)
in immunoblotting analyses. The anti-VCB-specific antibodies, generated
against the recombinant protein (VCB-Ab2), verified the identity of 44 and 42 kDa, and other processed peptides as well. Despite the large
amounts of VCB present in the ovary, neither its mRNA nor synthesis
were detected there. Thus, the fat body is the only source of the
44-kDa polypeptide as a precursor of the ovarian 42-kDa polypeptide,
which suggests that its accumulation in the ovary occurs by
endocytosis, similar to other yolk protein precursors (73). VCB
immunolocalization in the ovary confirmed that this yolk protein
precursor is internalized in developing oocytes via the endocytotic
pathway. Interestingly, VCB is segregated from crystalline Vn in mature
yolk bodies, the accumulative endocytotic organelle of the oocyte,
being present only in the non-crystalline matrix surrounding Vn. In
this matrix of mature yolk bodies, VCB is mixed with VCP (66). It is,
therefore, likely that the presence of both these proenzymes in the
matrix surrounding crystalline Vn enhances their rapid activation at
the onset of embryonic development, the point at which yolk bodies
undergo acidification. The latter event has been documented for both
insects and vertebrates (74, 75).
The fat body-secreted hemolymph form of VCB is stable latent proenzyme
which unlike mammalian cathepsins B (7, 76), cannot be activated by
acidic pH alone. Here, the reduction of the hemolymph pro-VCB to 35 kDa
(not 33-kDa) after treatment with acidic pH was not sufficient for
enzyme activation. Clearly, an additional step or steps are required
for the activation of the latent hemolymph pro-VCB. In our experiments,
pro-VCB did not activate to the 33-kDa peptide itself in an in
vitro transcription/translation system (Fig. 3), as has been
reported for Schistosoma mansoni cathepsin-B-like cysteine
protease (77). This stability of the hemolymph pro-VCB as a latent
proenzyme prior to its internalization by developing oocytes is
physiologically important. It may be additionally enhanced by the
presence of the glycosylation site in its proenzyme portion.
After treatment with acidic pH, the ovarian extract containing the
42-kDa VCB exhibited enhanced cathepsin B activity. Significantly, under these conditions, most of the ovarian 42-kDa VCB was processed to
the 33-kDa polypeptide. Although the nature of this processing event is
unclear, the removal of 2 kDa from each VCB subunit apparently renders
pro-VCB capable of activation by acidic pH after the onset of embryonic
development. Although the activation reactions were performed using
crude extracts, it is unlikely that the proteolytic processing of VCB
by treatment with acidic pH was a function of another protease, as both
the 44-kDa hemolymph and the 42-kDa ovarian VCB were processed to 35 and 33 kDa in the presence of protease inhibitors. Autocatalytic
cleavage under acidic conditions, which has been reported for mammalian
cathepsins B (7, 76), may be a possible mechanism for the activation of
the 42-kDa VCB in the mosquito embryo.
The estimation of native molecular mass suggests that in the hemolymph,
VCB exists as a hexamer or a pentamer of 44-kDa subunits (Fig. 14). A
hexameric structure is characteristic for some insect hemolymph
proteins (78). The feature of VCB may also increase its stability in
the hemolymph; alternatively, it may be important for the recognition
of VCB by oocyte receptors. Here, we found that at the onset of
embryonic development, when VCB was processed to the active 33-kDa
form, its native size was reduced to 132 kDa, which likely corresponded
to a tetramer of 33-kDa subunits. The presence of a minor 66-kDa band
indicated that at least some of the 33-kDa VCB were in dimer form (Fig.
14).
Our experiments utilizing 35S-labeled Vg as a substrate
suggest that VCB likely plays a key role in the degradation of Vn, the major yolk protein in mosquito embryos. The embryonic extract containing the 33-kDa VCB displayed high activity to Vg digestion, and
this degradation was blocked by E-64. Moreover, the specific anti-VCB
antibodies almost entirely inhibited cleavage of Vg by the embryonic
extract containing the 33-kDa VCB, strongly suggesting that VCB is the
key enzyme in embryonic degradation of Vn in the mosquito embryo.
Proteases similar to cathepsins B and L have been shown to be active
during embryonic development in a number of insects and other
arthropods (31-40). Best studied is the cathepsin L-like cysteine
protease from the silkworm, Bombyx mori (35-38): it is produced as a latent proenzyme of 47 kDa and processed under acidic conditions to a 39-kDa form, the active enzyme present in embryos. In
addition to exhibiting enzymatic properties characteristic of cathepsin
L-like cysteine proteases, this Bombyx protease shows high
sequence similarity to mammalian cathepsin L. Importantly, the direct
action of Bombyx cathepsin L-like cysteine protease on
vitellin has been demonstrated. The cDNA encoding the proenzyme of
this Bombyx cathepsin L-like cysteine protease has been
cloned from the ovarian cDNA library. Furthermore, immunological
analyses have shown that it is produced in the ovary by follicular
cells and is then deposited in developing oocytes (35-38).
In Drosophila, cathepsin B-like protease is presumed to be
maternally produced; however, its precise origin is not known (32). Previously, we demonstrated that mosquito VCP, one of the enzymes present in the yolk bodies, is secreted by the fat body (42; 43). Furthermore, immunological analyses have implicated the mosquito female
fat body in the production of VCB (44KP) (66). For Blatella germanica, an immunocytochemical study has suggested that the fat
body and follicular epithelium are the sources of the
vitellin-processing protease (79). In this study, however, we provided
solid molecular and biochemical proofs that the mosquito embryonic
cathepsin B-like protease that we describe here is produced exclusively
by the fat body as a yolk protein precursor.
In oviparous animals, extraovarian tissues play an important role in
egg maturation by producing yolk protein precursors, which serve as a
major nutritional source for developing embryos. The liver of oviparous
vertebrates and the fat body of insects both produce large quantities
of yolk protein precursors, which are subsequently internalized by
developing oocytes and deposited in yolk bodies. The most abundant of
these yolk protein precursors is Vg, a large glycophospholipoprotein
conserved throughout evolution of invertebrate and vertebrate oviparous
animals (80-83). In Lepidopteran insects, the fat body additionally
produces a smaller yolk protein precursor, either microvitellogenin or
the 30-kDa yolk protein (84-86).
In the mosquito, however, the proenzymes VCB and VCP are produced by
the fat body as yolk protein precursors. The discovery that enzymes
involved in the embryonic degradation of yolk proteins are produced as
precursors by an extraovarian tissue is a new biological phenomenon
previously unknown in insects or other oviparous animals. Moreover, the
finding that one of these enzymes is a cathepsin B sheds light on yet
another important mode of the utilization of this key protease.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
-amino-n-caproic acid, 1 mM benzamidine, 10 mM EDTA, 10 µg/ml aprotinin, and 2 µg/ml each antipain,
leupeptin, pepstatin (Roche Molecular Biochemicals), and chymostatin.
Fat body-secreted proteins were collected in TES-buffered
Aedes physiological saline supplemented with aprotinin, to
which the remainder of the inhibitors was added upon harvesting.
80 °C.
Zap II vector
(Stratagene). The conditions for reverse transcription, PCR
amplification, and cDNA cloning were previously described (53, 54).
The method of enzymatic sequencing utilized was that reported by Sanger
et al. (55).
RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Presence of VCB in tissues of the mosquito,
A. aegypti. A, immunoblot analysis of
mosquito tissue extracts using polyclonal antibodies to the ovarian
42-kDa form of VCB (VCB-Ab1). B, the same using preimmune
serum. Lane 1, whole male; lane 2,
previtellogenic female fat body; lane 3, vitellogenic female
fat body, 20 h PBM; lane 4, in vitro
secretion from vitellogenic fat bodies, 20 h PBM; lane
5, ovary, 24 h PBM. Notice the increased mobility of VCB in
the ovary. Extracts of ovary or vitellogenic fat body and in
vitro secretions were first treated with DEAE-Sepharose CL-6B to
reduce the amount of Vn or Vg present before separation by SDS-PAGE and
transfer to nitrocellulose. SDS-PAGE was performed in a 12% gel under
reducing conditions. The molecular standards in order of decreasing
molecular mass (in kDa) were phosphorylase b, bovine serum
albumin, ovalbumin, carbonic anhydrase, and soybean trypsin inhibitor
(Bio-Rad).
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Fig. 2.
Nucleotide and deduced amino acid sequences
of vitellogenic cathepsin B from the mosquito, A. aegypti. The deduced amino acid sequence of the
prepro-enzyme is displayed in capital letters. Both
nucleotides and deduced amino acids are numbered. A putative
signal peptide is marked by a thick underline. A potential
pro-enzyme cleavage site is indicated by a solid triangle.
Predicted N-linked glycosylation sites (N) are
marked by squares. Potential phosphorylated serines
(S) and threonines (T) are circled.
Amino acids corresponding to the thiol protease conserved domains are
boxed, and their catalytic residues are in reverse phases.
The deduced amino acid sequences matching that obtained by direct
peptide sequencing of 44-kDa peptide secreted from the fat body is
underlined by a double line, and to that of
42-kDa ovarian protein is underlined by a dashed
line. A polyadenylation signal ATTAAA is in bold.
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Fig. 3.
Analysis of
[35S]methionine-labeled products of in vitro
expression of a mosquito VCB cDNA by a coupled transcription
and translation system (Promega). Lane 1, the 1.3-kb
VCB fragment subcloned in pGEM 7Z(+) were transcribed and translated
in vitro. Lane 2, the same reaction conducted in
the presence of canine microsomal membranes (Promega). Translation
products were analyzed by SDS-PAGE in 12.5% gels under reducing
conditions and fluorography. The molecular standards are as described
in the legend to Fig. 1.
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Fig. 4.
Alignment of the deduced amino acid sequence
of mosquito VCB with other thiol proteases. Asterisks
mark the consensus residues of enzyme active sites (*). Conserved amino
acids are marked by black boxes. Paired numbers
under the sequences denote the locations of cysteine residues that form
a disulfide bond. Abbreviations are: AaVCB, A. aegypti VCB; HsCtB, human cathepsin B, Homo
sapiens (3); RnCtB, rat cathepsin B, Rattus
norvegicus (3); BtCtB, bovine cathepsin B, Bos
taurus (87); MmCtB, mouse cathepsin B, Mus
musculus (3); Sp29K, 29-kDa protease of the blowfly,
S. peregrina (30); SjCtB, trematode cathepsin B,
Schistosoma japonicum (25); SmCyP, trematode
cysteine protease, S. mansoni (20); HcCy1,
nematode gut thiol protease 1, Hemonchus contortus (21);
HcCy2, nematode thiol protease 2, H. contortus
(23); CeCy1, nematode gut-specific cysteine protease,
Caenorhabditis elegans (88). The descent rank of similarity
to mosquito VCB was determined by Initn index of FastA
(Genetics Computer Group software).
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Fig. 5.
Northern blot analysis of gene expression of
mosquito VCB. Twenty micrograms of total RNA from different sexes,
tissues, and stages were separated by a 1.2% agarose gel and stained
with ethidium bromide (A). After photography, the gel was
transferred to a nitrocellulose membrane and hybridized with a
32P-labeled 208-bp 5'-end fragment of the VCB cDNA,
including the noncoding region and the prepro-portion of VCB
(B). Lane 1, whole male; lane 2,
vitellogenic female midgut, 24 h PBM; lane 3,
vitellogenic female ovary, 24 h PBM; lane 4,
pre-vitellogenic female fat body; lane 5, vitellogenic
female fat body, 24 h PBM; lane 6, post-vitellogenic
female fat body, 48 h PBM. RNA markers (Bio-Lab) are shown to the
left in A.
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Fig. 6.
VCB RNA kinetics in the mosquito fat
body. For each time point, total RNA (20 µg) extracted from the
fat body was resolved in a 1.2% formaldehyde gel. A, the
ethidium bromide stained gel. B, Northern hybridization with
a 32P-labeled 208-bp VCB specific probe. Lane 1,
pre-vitellogenic female fat body; lanes 2-9, vitellogenic
female fat bodies: lane 2, 6 h PBM; lane 3,
12 h PBM; lane 4, 18 h PBM; lane 5,
24 h PBM; lane 6, 30 h PBM; lane 7,
36 h PBM; lane 8, 42 h PBM; lane 9,
48 h PBM. RNA markers (Bio-Lab) are shown to the left
in A.
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Fig. 7.
Time course of VCB secretion by fat bodies of
female mosquitoes. Abdominal wall with adhering fat body of
vitellogenic females at various stages of vitellogenesis, initiated by
a blood meal, were incubated in culture medium in the presence of
[35S]methionine for 1 h and chased for 1 h in
isotope-free medium. Chase media samples were immunoprecipitated using
polyclonal antibodies to 42-kDa VCB (VCB-Ab1) or to VCP and analyzed as
described under "Materials and Methods." Data are expressed as
counts/min of [35S]methionine-labeled protein per hour
from three fat bodies.
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Fig. 8.
Immunohistochemical localization of VCB in
vitellogenic fat body, 18 h PBM. A, a
phase-contrast image of the fat body. B, an
immunofluorescent image of the same fat body stained with VCB-Ab2
polyclonal antibodies followed by fluorescein-conjugated goat
anti-rabbit IgG. Magnification, × 400.
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Fig. 9.
Immunohistochemical localization of VCB in
the ovary, 18 h PBM. A, phase-contrast image of
the ovarian follicle. B, an immunofluorescent image of the
same ovarian follicle stained with VCB-Ab2 polyclonal antibodies
followed by fluorescein-conjugated goat anti-rabbit IgG.
Arrowheads show the VCB staining pattern as a rim around
yolk bodies. Magnification, × 400.
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Fig. 10.
Co-localization of VCB and Vg in the Golgi
complex (GC) and secretory granules
(SG) of the fat body cell from a vitellogenic female
mosquito (18 h PBM). VCB was localized using VCB-Ab1 antibodies
and protein A-colloidal gold (15 nm); Vg was localized using polyclonal
antibodies to the large Vg subunit and protein A-colloidal gold (10 nm). Magnification, × 100,000.
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Fig. 11.
Immunolocalization of VCB in the mature yolk
body of a developing oocyte (18 h PBM). VCB-Ab1 polyclonal
antibodies followed by protein A-colloidal gold (15 nm). Note that VCB,
marked with 15-nm colloidal gold particles, is localized in the
non-crystalline matrix of the yolk body. Magnification, × 50,000.
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Fig. 12.
Processing of VCB in the ovary and
embryos. Lane 1, ovaries at the peak of Vg uptake,
24 h PBM; lane 2, ovaries with eggs nearly complete in
their development, 48 h PBM; lane 3, 0-3 h
post-oviposition eggs at the onset of embryonic development; lane
4, 48-h post-oviposition eggs during mid-embryogenesis; lane
5, 96-h post-oviposition eggs at the end of embryogenesis;
lane 6, newly hatched first instar larva. Proteins were
resolved by SDS-PAGE under reducing conditions in 12% gel. Proteins
transferred onto a nitrocellulose membrane were probed by VCB-Ab1
antibodies. The molecular mass standards are as described in the legend
to Fig. 1.
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Fig. 13.
Western blot analysis of VCB in developing
embryos. Lane 1, 0-12 h post-oviposition eggs;
lane 2, 24-36 h post-oviposition eggs; lane 3,
48-60 h post-oviposition eggs; lane 4, 72-84 h
post-oviposition eggs; lane 5, first instar larvae;
lane 6, pupae. Protein extracts from oviposited eggs,
larvae, and pupae were resolved with 10% SDS-PAGE, then subjected to
Western blot hybridization using VCB-Ab2. The molecular mass standards
are as described in the legend to Fig. 1.
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Fig. 14.
Native molecular mass of hemolymph and
embryonic forms of VCB. Lane 1, in vitro
secretion from vitellogenic fat bodies 20 h PBM; lane
2, 48-hr post-oviposition eggs during mid-embryogenesis. Proteins
were separated in a 3.5-25% gradient gel and transferred to
nitrocellulose membrane. The blot was processed using VCB-Ab1
antibodies. The molecular standards in order of decreasing molecular
mass (in kDa) were thyroglobulin, ferritin, catalase, lactate
dehydrogenase, and bovine serum albumin (Pharmacia).
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Fig. 15.
Differential effect of inhibitors on
enzymatic activity of VCB. The embryonic extract from 12- to 48-h
post-oviposition eggs, containing 33-kDa VCB was examined for enzymatic
activity with Z-Arg-Arg-pNA as described under "Materials
and Methods." The reaction shown was performed for 60 min at pH 5.5. The inhibitors, E-64 and chymostatin, were added to the protein mixture
at the final concentration indicated on the axis and preincubated for
20 min at 37 °C prior to the addition of the enzymatic substrate.
The activity was expressed as micromolar pNA released from
the substrate Z-Arg-Arg-pNA per µg of protein.
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Fig. 16.
Vn degradation during mosquito embryonic
development. A, immunoblot analysis using monoclonal
antibodies against the Vg/Vn small 66-kDa subunit; B, using
polyclonal antibodies against the Vg/Vn large 200-kDa subunit.
Lanes 1, ovaries at the peak of Vg uptake, 24 h PBM;
lanes 2, ovaries with eggs nearly complete in their
development, 48 h PBM; lanes 3, 0-3 h post-oviposition
eggs at the onset of embryonic development; lanes 4, 48-h
post-oviposition eggs during mid-embryogenesis; lanes 5,
96-h post-oviposition eggs at the end of embryogenesis; lanes
6, newly hatched first instar larva. The molecular standards in
order of decreasing molecular mass (in kDa) were myosin,
-galactosidase, phosphorylase b, bovine serum albumin,
ovalbumin, carbonic anhydrase, and soybean trypsin inhibitor
(Bio-Rad).
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Fig. 17.
Degradation of vitellogenin by embryonic
extract (12 to 48 h post-oviposition eggs) containing 33-kDa
VCB. Purified [35S]methionine-labeled Vg was
incubated with the embryonic extract at pH 4.0 and resolved by SDS-PAGE
in a 10% gel under reducing conditions. Lane 1, the
reaction was performed in the presence of serine protease inhibitors, 1 mM PMSF and 1 mM AEBSF; lane 2, in
the presence of 5 µM pepstatin, an inhibitor of aspartic
proteases; lane 3, in the presence of 2.5 µM
E-64, an inhibitor of thiol proteases; lane 4, PMSF, AEBSF,
and pepstatin; lane 5, all listed protease inhibitors;
lane 6, incubation of Vg without the embryonic extract, at
pH 4.0; lane 7, Vg sample without any treatment.
VgL, 200-kDa large Vg subunit; VgS, 66-kDa small
Vg subunit. The molecular mass standards are as described in the legend
to Fig. 16.
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Fig. 18.
Degradation of vitellogenin by embryonic
extract (12-48 h post-oviposition eggs) containing 33-kDa VCB.
Purified [35S]methionine-labeled Vg was incubated with
the embryonic extract at pH 5.35 in the presence of 1 mM
PMSF, 1 mM AEBSF, 5 µM pepstatin, and 2.5 mM EDTA. Proteins were resolved by SDS-PAGE in a 10% gel
under reducing conditions. Lane 1, Vg sample without any
treatment; lane 2, incubation of Vg in thee presence of 2.5 µM E-64, an inhibitor of thiol proteases; lane
3, incubation of Vg without the embryonic extract, at pH 5.35;
lanes 4-6, incubation of Vg in the presence of increasing
concentrations of VCB-Ab1 antibodies; lanes 7-9, preimmune
rabbit IgG; lanes 10-12, bovine serum albumin;
concentrations of VCB-Ab1, IgG, and bovine serum albumin in each of
thee lanes were 0.125, 0.25, and 0.5 µg/µl, respectively. The
molecular mass standards are as described in the legend to Fig.
1.
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Fig. 19.
Activation of mosquito VCB under acidic
conditions. A, protein extracts supplemented with
protease inhibitors (1 mM PMSF, 1 mM AEBSF, 5 µM pepstatin, and 2.5 mM EDTA) were incubated
for the indicated times under the acidic (pH 4. 0 or 5.5) or control
conditions (pH 7.5). Enzymatic activity was measured using
Z-Arg-Arg-pNA as substrate. Activity is expressed as in Fig.
15. Line 1, fat body secretions; line 2,
embryonic extracts from ovaries after the termination of yolk
accumulation, 48 h PBM; line 3, protein extract from
embryos, 12-48 h oviposition eggs. B, immunoblot analysis
of activated VCB peptides. Immunoblot was performed as described in the
legend to Fig. 1. Lane 1, fat body secretions, under the
control conditions at pH 7.5; lane 2, embryonic extract
under the control conditions; lane 3, fat body secretions
under the acidic conditions at pH 4.0; lane 4, fat body
secretions under acidic condition at pH 5.5, corresponds to the
enzymatic reaction (line 1) in A; lane
5, protein extract from vitellogenic ovaries under acidic
conditions at pH 5.5, corresponds to the enzymatic reaction (line
2) in A; lane 6, protein extract from embryos, 12-48 h
post-oviposition eggs, under acidic conditions at pH 5.5, corresponds
to the enzymatic reaction (line 3) in A.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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ACKNOWLEDGEMENTS |
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We thank Drs. T. Sappington, K. Deitsch, and A. Biran for critical reading of the manuscript.
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FOOTNOTES |
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* This work was supported by National Institutes of Health Grants AI-24716 and AI-32154 (to A. S. R.) and by National Science Council (Taiwan) 86-2314-B-010-065 (to W-L. C.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) AF127592.
Present address: Novartis Seeds, 3054 Cornwallis Rd., Research
Triangle Park, NC 27709-2257.
To whom correspondence should be addressed: Dept. of
Entomology, S-150 Plant Biology Bldg., Michigan State University, East Lansing, MI 48824-1115. Tel.: 517-353-7144; Fax: 517-353-3396; E-mail:
araikhel{at}pilot.msu.edu.
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ABBREVIATIONS |
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The abbreviations used are: VCP, vitellogenic carboxypeptidase; AEBSF, 4-(2-aminoethyl)-benzenesulfonylfluoride; E-64, trans-epoxysuccinyl-L-leucylamido-(4-guanidino)butane; kb, kilobase pair(s); PBM, post-blood meal; PCR, polymerase chain reaction; PMSF, phenylmethylsulfonyl fluoride; PAGE, polyacrylamide gel electrophoresis; TES, 2-{[tris-1,1-bis(hydroxymethyl)ethyl]-amino}-ethanesulfonic acid; 20E, 20-hydroxyecdysone; VCB, vitellogenic cathepsin B; VCB-Ab1, polyclonal antibodies produced against ovarian 42-kDa VCB; VCB-Ab2, polyclonal antibodies produced against the recombinatly produced VCB; Vg, vitellogenin; Vn, vitellin; MES, 4-morpholineethanesulfonic acid; pNA, p-nitroanalide; bp, base pair(s).
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