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Novel aspartyl proteinase associated to fat body histolysis during Ceratitis capitata early metamorphosis.

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Archives of Insect Biochemistry and Physiology 57:51–67 (2004)
Novel Aspartyl Proteinase Associated to Fat Body
Histolysis During Ceratitis capitata Early
Metamorphosis
Alejandro Rabossi,1 Veronika Stoka,2 Vida Puizdar,2 Vito Turk,2 and Luis A. Quesada-Allué1*
During larva to adult transition, the larval fat body of the Medfly (Ceratitis capitata) progressively disintegrates to be replaced
by the adult one, after imago ecdysis. Here we show that a temporal correlation exists among the microscopy images of fat
body progressive disintegration, the activation of fat body lysosomes (as judged by acid phosphatase activity), and the activity
of a novel fat body aspartyl proteinase. The enzyme was purified and partially characterized. This proteinase exhibited a wide
range of acid isoforms with isoelectric points from 5.6 to 7.3, an optimum pH of 3.0 for hemoglobin digestion, and was
completely inhibited by pepstatin A. The apparent molecular weight was estimated (42 ± 1 kDa) and the protein was
characterized as N-glycosylated, judging from affinity to Concanavalin A. From the biochemical characteristics, the enzyme
that we called “Early Metamorphosis Aspartyl Proteinase” (EMAP) appears to be similar to mammalian Cathepsin D. However,
the N-terminal sequence of EMAP showed no similarity with any known animal Cathepsins and exhibited an important instability to neutral and alkaline pH. This feature seems to be a peculiar characteristic of insect aspartyl proteinases. The temporal
activity profile of EMAP during metamorphosis correlated well with the microscopy images of fat body cell autolytic death. Our
data support the notion that EMAP is a metamorphosis-specific lysosomal proteinase, mostly expressed during larval fat body
histolysis. Arch. Insect Biochem. Physiol. 57:51–67, 2004. © 2004 Wiley-Liss, Inc.
KEYWORDS: fat body; metamorphosis; histolysis; Medfly; aspartyl proteinase
INTRODUCTION
Insect fat body performs a myriad of metabolic
functions including the homeostatic maintenance
of hemolymph proteins, lipids, and carbohydrates
(Haunerland and Shirk, 1995). As the principal
metabolic-storage tissue, the fat body is structurally organized to provide maximal exposure to the
hemolymph and is well-suited for both absorbing
and releasing metabolites (Keeley, 1985). The fat
body is composed of two or three cell types: the
adipocytes (also called trophocytes) that are the
predominant metabolic-storage cells, the uric acid–
rich urocytes, and, depending of the insect order,
the mycetocytes (Dean et al., 1985). As other mesoderm-derived tissues, fat body cells became differentiated during embryogenesis, apparently as a
sheet of interacting cells located on the inner face
of mesoderm (Bate, 1993). In dipterans, during larval stages, the fat body cells increase their size and
concomitantly increase their politenization.
In higher Diptera, as most of the larval tissues,
the fat body undergoes histolysis during metamorphosis. After pupariation, the larval fat body disintegrates in an anterior to posterior direction
(Fraenkel and Hsiao, 1968). Whitten (1964) re-
Grant sponsors: University of Buenos Aires and YPF S.A.
1
Instituto de Investigaciones Bioquímicas, CONICET and Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires, Buenos Aires, Argentina
2
Department of Biochemistry, Jozef Stefan Institute, University of Ljubljana, Ljubljana, Sloven
Abbreviations used: BPF = before puparium formation; APF = after puparium formation; kDa = kilodaltons; PTU = N-phenylthiourea.
*Correspondence to: Luis A. Quesada-Allué, Instituto de Investigaciones Bioquímicas, CONICET and Facultad de Ciencias Exactas y Naturales, Universidad de
Buenos Aires, Av Patricias Argentinas 435, Buenos Aires 1405, Argentina. E-mail: lualque@iib.uba.ar
Received 13 November 2003; Accepted 28 March 2004
© 2004 Wiley-Liss, Inc.
DOI: 10.1002/arch.20011
Published online in Wiley InterScience (www.interscience.wiley.com)
52
Rabossi et al.
ported in Sarcophaga bullata and Drosophila melanogaster the first step of this disintegration that consists in the loss of the surrounding basement
membrane. This is followed by the dissociation
from a cohesive tissue into individual cells, free
within the haemocel. Kurata et al. (1989, 1990,
1992a,b) and Kobayashi et al. (1991) reported the
role of two pupal-specific hemocyte proteins involved in the dissociation of the flesh fly Sarcophaga
peregrina larval fat body. After pupariation, the
hemocytes specifically express a 200 kDa surface
protein, essential for hemocyte recognition of the
basement membrane of the larval fat body (Kobayashi et al., 1991). After pupal hemocytes bind to
the basement membrane, a 29 kDa hemocyte
cystein-proteinase is excreted, digesting the membrane and starting the dissociation of the fat body
(Kurata et al., 1989, 1990, 1992a,b). A similar molecular weight cystein proteinase has been also isolated during Ceratitis capitata early metamorphosis
(Rabossi, 2001).
It is accepted that in cyclorrhaphous Diptera the
number of fat body cells decrease gradually during metamorphosis until 3 days after adult ecdysis,
when no more larval fat body cells can be found
(Bodenstein, 1950; Richard et al, 1993). The lysis
of these cells is stimulated by ecdysteroids through
the formation of lysosomal protein granules (Dean,
1978; Keeley, 1985). Van Pelt-Verkuil (1979) detected a rise in the acid phosphatase activity (a lysosome marker) in protein granules of Calliphora
erytrocephala larval fat body, starting at the end of
the larval feeding period, in response to treatment
with 20-OH ecdysone. However, the role of lysosomes during histolysis is not completely understood. As far as we know, not a single proteolytic
enzyme directly related with histolytic processes
has been isolated from Diptera.
Former studies on the metamorphosis of the
Mediterranean fruit fly, Ceratitis capitata, allowed
us to define with high precision the onset, duration and end of pre-pupal, pupal, and pharate adult
stages (Rabossi et al., 1992), as well as of several
developmental programs like pupariation and pupation (Rabossi et al., 1991). We also established
that a temporal correlation exists between the
changes in overall lysosomal proteolytic activity
and the above events, demonstrating that proteolysis can be taken as an indicator of histolysis progression (Rabossi et al., 2000). It is important to
note that the peak of overall lysosomal proteolytic
activity occurs more than 30 h after gut proteinases activities completely disappeared (Rabossi et
al., 2000).
In this study, we report the purification and preliminary characterization of an aspartyl proteinase
that is present and active in the larval fat body of
the Medfly during metamorphosis. We show that
a temporal correlation exists between the changes
in fat body overall lysosomal activity (as indicated
by acid phosphatase activity) and the novel aspartyl proteinase activity, which in turn is correlated
with histolytic processes assessed by histological
studies.
MATERIALS AND METHODS
Insects
Wild type Ceratitis capitata, of the strain “INTA
Arg 17,” were reared on carrot-based food as described by Quesada-Allué et al. (1994). Larval and
adult flies were maintained at 23°C and 50–70%
relative humidity, with a 16L:8D photoperiod. All
the results below refer to time-dependent events
occurring during a standard life cycle under these
culture conditions (Rabossi et al., 1992). The age
of larva III is expressed in hours before puparium
formation (BPF). Age within the puparium is expressed in hours after puparium formation (APF),
starting at the definitive immobilization of the
third instar larva, previously defined as “Zero time”
(Rabossi et al., 1991).
Chemicals
All the reagents and solvents were of the highest
purity available. Bovine hemoglobin (Hb), bovine
serum albumin (BSA), p-nitrophenyl phosphate,
pepstatin A, E-64, pepstatin-agarose, and FolinCiocalteau reagent were from Sigma Chemical Co.
(St. Louis, MO).
Archives of Insect Biochemistry and Physiology
Aspartyl Proteinase in C. capitata
Fat Body Isolation
Before dissection under a binocular microscope,
the insects were thoroughly washed with distilled
water. To dissect larval fat body (20 and 8 h BPF)
and pre-pupal fat body (0, 2030 and 40 h APF),
the body was cut off between the second and third
anterior segments in a drop of insect Ringer’s-like
solution (146 mM NaCl, 3.4 mM KCl, 2.7 mM
CaCl2, 0.5 mM NaHCO3, pH 7.2) (Quesada-Allué,
1978) containing 20% sucrose and a crystal of PTU.
In these conditions, fat body cells float until 20:30
h APF and sink down at 40 h APF. The internal
contents were squeezed out by applying pressure
to the body wall. The fat body was carefully freed
from other organs, washed twice with Ringer’s solution (without sucrose), and then frozen at –70°C.
Samples from 40-h APF were washed several times.
This eliminates most of hemocytes and cell debris
from the anterior fat body. Pupal fat body dissection (72, 98, 120, and 144 h APF) was carried out
in insects previously removed from the puparium
as above and maintained in a drop of Ringer’s solution. After several washes, most of the remaining material is posterior fat body.
Fat Body and Whole Body Cell-Free Extracts
Extracts from staged insects at different points
during the life cycle were prepared by homogenizing the flies or dissected tissue with 0.1 M sodium
acetate buffer, pH 4.4. After 20 strokes in a Teflonglass tissue grinder, the homogenates were centrifuged at 12,000g for 20 min at 4°C and the crude
supernatant used as source of enzymes.
Proteolytic Activity
Acid and aspartyl proteinases activities were
measured as described by Anson (1939) with few
modifications (Smith and Turk, 1974). Briefly, the
substrate was 2% hemoglobin, prepared in distilled
water, and denatured by adjusting to pH 3.5 with
272 mM acetic acid, 4 mM (NH4)2SO4. The reaction mixture, containing 0.05 to 0.2 ml of enzymatic extract and 1.2 ml of 2% acid-denatured
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53
hemoglobin (pH 3.5), was incubated for 30 min
at 37°C. The reaction was stopped by addition of
2.4 ml of 0.33 M trichloroacetic acid. After mixing
and 30 min precipitation at room temperature, the
liberated peptides in the soluble phase were recovered by filtration through Whatman 3 MM filter
paper. Two milliliters of the filtrate were assayed
by adding 4 ml of 0.5 M NaOH and 1 ml of diluted 1:3 Folin-Ciocalteau reagent. After 5 min stabilization, absorbance at 750 nm was measured
in a Gilford Response II spectrophotometer. Blanks
were prepared by substrate incubation with 2.4 ml
of 0.33 M trichloroacetic acid prior to addition of
the enzyme. One unit activity was defined as the
amount of enzymatic protein able to generate identical color intensity as 1 milliequivalent of Tyrosine
in the above standard conditions (Anson, 1939).
When 20 µM pepstatin A was used as specific
inhibitor, it was preincubated with 200 µl of enzymatic extract at 4°C, after which the reaction was
triggered by addition of the hemoglobin and the
mixture incubated at 37°C as above.
Acid Phosphatase Activity
Acid phosphatase was assayed in 0.2 M sodium
acetate buffer, pH 4.5, containing 3 mM p-nitrophenyl phosphate and 0.025 ml of fat body supernatant solution, in a final volume of 0.5 ml.
After incubation at 37°C for 30 or 60 min, 0.3 ml
of 6 M KOH was added to stop the reaction. Blanks
used to correct for background absorbance at 410
nm were prepared by substrate incubation with 0.3
ml of 6 M KOH, prior to addition of the enzyme.
One unit of enzyme activity is defined as the
equivalent of 1 µmol of p-nitrophenyl phosphate
hydrolyzed per minute.
Purification of Medfly Aspartyl Proteinase.
All the isolation procedures were carried out at
4°C unless otherwise stated. Frozen in liquid N2
40–45 h APF pupae (60 g) were homogenized in
a Sorvall Omni-mixer, with 120 ml of 0.1 M sodium acetate buffer, pH 5.0, containing 1 mM
EDTA and 0.3 M NaCl (buffer A). After homog-
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Rabossi et al.
enization, the pH of the extract was 6.3. The homogenate was then centrifuged at 12,000g for 10
min at 4°C and the supernatant was adjusted to
pH 4.2 with glacial acetic acid and again centrifuged at 12,000g at 4°C during 30 min. After removal of denatured proteins, the acidic supernatant
was fractionated by salting out with 30–70%
(NH4)2SO4. The precipitated proteins were resuspended in 15 ml of buffer A and dialyzed against
5 L of the same buffer A for at least 2 h at 4°C.
The insoluble material was removed by 5-min centrifugation and discarded.
After dialysis, the soluble material was applied
to a refrigerated column (135 × 3 cm) of Sephacryl
S-200 (Pharmacia, Upsala, Sweden) previously
equilibrated with buffer A and chromatographed
at a flow rate of 33.6 ml/h. The collected fractions
were assayed using 2% hemoglobin as substrate,
and three peaks showing proteolytic activity could
be detected (see Fig. 5A). The active fractions of the
second peak were combined, and then simultaneously concentrated and dialyzed against 10 mM
MES, pH 5.8, containing 10% ethanol (Buffer B),
by ultrafiltration using Amicon YM-5 membrane.
The enzymatic material was chromatographed on
a Mono S (HR 5/5) column of Pharmacia FPLC
system, and a linear gradient (0–0.5 M; 30 ml) of
NaCl in Buffer B was applied. The flow rate was 1
ml/min. The active fractions were combined and
concentrated to 0.5 ml with Centricon-30 at 800g
at 4°C. This material was chromatographed on a
Superose 12 column of Pharmacia FPLC system
equilibrated with 0.4 M NaCl in Buffer B, at a flow
rate of 0.5 ml/min.
Pepstatin A-Agarose Affinity Chromatography
Affinity chromatography was carried out as
previously described (Smith and Turk, 1974).
Pepstatin A-agarose was obtained from Sigma
Chemical Co. (St. Louis, Mo) and routinely maintained in 0.1 M Tris-HCl, 1M NaCl, 1 mM EDTA,
pH 8.6 (buffer C) at 4°C. Before packing the column, the resin was equilibrated in batch with
buffer D (0.1M sodium acetate, 1 M NaCl, 1 mM
EDTA, pH 3,5), then in buffer C and again with
buffer D. Each equilibration step was repeated
three times. The resin equilibrated at pH 3.5 was
incubated with the sample during 45 min at room
temperature. Afterwards, the resin was packed on
a column and washed twice with buffer D. The
elution was carried out with 0.1M sodium acetate,
1 M NaCl, 1 mM EDTA, pH 5.7, and then with
buffer C (pH 8.6) or only with buffer C.
Proteolytic activity was determined as described
above. In some cases, the column fractions were
immediately dialyzed against buffer D during 45
min at 4°C, and the enzymatic activity was measured.
Electrophoresis
Discontinuous SDS/polyacrylamide-gel electrophoresis was performed as described by Laemmli
(1970) in a vertical slab apparatus. Separating gels
of 10% acrylamide in 0.375 M Tris-HCl, pH 8.6
with 0,1% SDS were used. Proteins were visualized with Coomassie brilliant blue (CBB R-250).
Discontinuous, non-dissociating polyacrylamidegel electrophoresis was carried out according to
Hames (1987) in a 10% acrylamide gel at pH 4.3.
The pH value of the concentrating gel was 6.8 and
the running buffer was 4.5. The samples (homogenates of isolated fat body in 0.1 M sodium acetate buffer, pH 4.4) were diluted 5-fold with the
same buffer containing 10% glycerol and methylene green. Electrophoresis was carried out at 100 V
during 80 min at 4°C. Then, the gel was incubated
30 min at 28°C with 2% acid denatured hemoglobin
(pH 3.5), and transferred to an incubation tray containing 5 mM 2-mercaptoethanol in 0.1 M sodium
acetate buffer, pH 4.4, during 60 min at 37°C. The
reaction was stopped with the staining solution of
0.8% CBB R-250 in 7% acetic acid. After overnight
tinction, the proteolytic activity was visualized as
a clear area void of the blue stained protein.
When inhibitors were assayed, they were added
to the extracts and incubated 20 min at 4°C, prior
to electrophoresis. Alternatively, the inhibitors were
present in the incubation buffer.
To determine the isoelectric point, 60 ng of the
purified aspartyl proteinase enzyme (see above)
Archives of Insect Biochemistry and Physiology
Aspartyl Proteinase in C. capitata
were applied to a precasted IEF gel (Pharmacia
PhastSystem apparatus). The Broad pI Kit (pH 3.5
to 9.3) of Pharmacia was employed. After electrophoresis, the gel was stained with CBB R-250.
Optimum pH and Stability
Acid denatured hemoglobin 2% was used as
substrate to determine the optimun pH. The reaction buffers were 0.1 M phosphate-citrate buffer
pH 2.0 to 7.0 and 0.1 M tris-HCl buffer pH 8.0 to
9.6. The stability of the proteinase was determined
by pre-incubation of the enzyme with the above
buffers of different pH for 20 min at room temperature, prior to proteinase assay with acid denatured hemoglobin at pH 3.5.
Protein Determination
Protein concentration was routinely determined
by the method of Lowry et al (1951), with BSA as
standard. During the enzyme purification, it was estimated by measurement of absorbance at 280 nm.
Detection of DNA Cleavage in Adipocytes
Adipocytes of different developmental times
were isolated as described above and solubilized
in Buffer 10 mM EDTA, 1 M NaCl, 1% (w/v) SDS,
100 mM Tris-HCl (pH 9.0) containing proteinase
K (1 mg/ml), and incubated at 50°C for 4 h. After
two extractions with 1:1 (v/v) phenol:chloroformisoamylic alcohol (24:1), the nucleic acids were
precipitated with 1 vol. ethanol and 0.1 vol. 3M
sodium acetate (pH 5.0). After centrifugation and
washing with 70% ethanol, the DNA samples were
resuspended in TE and digested with RNAse A
(1mg/ml) at 37°C for 2 h.
DNA samples were applied to 1.4% agarose gels
and visualized with ethidium bromide (0.1 µg/ml).
Histological Studies
Different developmental stages were analyzed,
from 20 h BPF to 120 h APF. Batches of not less
than 10 insects of the same age were fixed in Bouin
fixative during 24 h at room temperature. The fixed
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insects were transversally cut between the 4th to 5th
segment and fixed again in Bouin for another 24 h.
Then, the insects were washed twice with distilled
water and subjected to dehydration with the classical alcohols series, after which they were embedded in paraffin and cut. Sections of 5 to 10 µM were
stained with hematoxylin- eosin (H-E). Color images corresponding to Figures 1 and 2 can be viewed
online at www.leloir.org.ar/fil/fatbody/fatbody.htm
Amino Terminal Sequence
Samples of the Early Metamorphosis Aspartyl
Proteinase (EMAP) were sequenced with an Applied Biosystems (Foster City, CA) liquid phase
sequenator model 475A. Phenylthiohydantoin derivatives were identified on line with the 120A
HPLC. The reduced and alkylated protein was hydrolyzed with 6 M HCl at 110°C for 24 and 72 h.
Amino acid compositions were determined by using a Beckman (Fullerton, CA) 118CL analyzer, by
post-column fluorescence detection after reaction
with o-phthaldialdehyde.
RESULTS
As in other cyclorraphous dipterans, fat body
cells of C. capitata are arranged in a structurally
defined network (Fig. 1A). During larval development, the adipocytes increase in size progressively,
reaching the terminal maturity at the end of the
3rd larval stage (Fig. 1A, 20 h BPF).
Histological Changes During Early Metamorphosis
During the Ceratitis capitata larva to adult transition, the fat body changes dramatically. Figure
1B shows that in the anterior region (segments 1
to 4), the fat body cells sheet starts to dissociate
2030 h APF, exactly at the time of apolysis, and that
the liberated adipocytes remain free in the hemolymph. This process was fully completed in the anterior part of the body at around 48 h APF (not
shown, see Fig. 8).
The posterior fat body (from the abdominal region) started to dissociate later, at 40 h APF. In this
56
Rabossi et al.
Fig. 1. Time-dependent changes in Ceratitis fat body
stained with H-E (see original color images online at
www.leloir.org.ar/fil/fatbody/fatbody.htm. A: Characteristic network of cells in the last instar larva fat body (20 h
BPF) (longitudinal cross, 400×). B: Fat body starts to dis-
sociate in the anterior region during larval-pupal apolysis
(2030 h APF) (longitudinal cross, 400×). C: Lysed adipocytes in the anterior region (72 h APF) (longitudinal cross,
400×). D: Hemocyte (arrow) bound to adipocyte from the
anterior region (2030 h APF) (transversal cross, 1,000×).
region, cell liberation takes more time and therefore the network structure can still be observed at
the end of the pupal stage (144 h APF, not shown).
After dissociation, the lysis of the adipocytes of
the anterior region was completed between 48 to
72 h APF (Fig. 1C) whereas in the posterior region, this process starts at 72 h APF (not shown).
In correlation with the progress of fat body dissociation, differentially timed in both regions, we
detected the increasing presence of hemocytes apparently associated to the basal membrane surrounding the adipocytes (arrow in Fig. 1D).
Figure 2A–C shows the changes in the morphology of anterior larval adipocytes during early metamorphosis. Twenty hours BPF, the adipocytes
forming a net (Fig. 1A) showed a well-defined
plasma membrane (Fig. 2A, solid white arrow). Fat
vesicles of different sizes filled most of the cytoplasm and in most cases appeared connected by
short bridges (Fig. 2A,B, dashed black arrows). The
20-h BPF fat vesicles presented well-defined smooth
borders (Fig. 2A). At this time, the nucleus was big
with prominent nucleolus in a central position, intensively blue-stained by H-E and surrounded by
the chromatin (Fig. 2A, dashed white arrow).
From the onset of metamorphosis (0 h) up to
around 20 h APF, there were no significant visible
changes in the morphology of anterior and posterior adipocytes. When the anterior fat body network started to dissociate at 2030 h APF, a number
Archives of Insect Biochemistry and Physiology
Fig. 2. Cell death of Ceratitis larval fat body. The adipocytes were stained with H-E (see original color
images online at www.leloir.org.ar/fil/fatbody/fatbody.htm. A–C: Transversal cross of anterior adipocytes,
of respectively, 20 h BPF, 2030 h APF and 22 h APF (1,000×). D–F: Transversal cross of posterior adipocytes
of respectively, 48, 72, and 98 h APF (1,000×). Solid white arrows: plasma membrane. Dashed white
arrows: nucleolus. Dashed black arrows: fat vesicles bridges. Stars indicate protein granules.
Aspartyl Proteinase in C. capitata
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Rabossi et al.
of cell morphological changes start to occur. The
fat vesicles membrane seems less defined (Fig. 2B)
and the connection between vesicles appears to be
longer than in late larvae III (Fig. 2B, dashed black
arrows). However, the plasma membrane shows a
well-defined border (Fig. 2B, solid white arrow)
and the nucleolus still maintains its integrity (Fig.
2B, dashed white arrow). Two hours after dissociation (22 h APF), in a number of the free cells,
the nuclear membrane and nucleolus could not
be visualized anymore and the chromatin seemed
to be free in the cytoplasm (Fig. 2C, dashed white
arrow). The plasma membrane started to be less
defined and difficult to recognize (Fig. 2C, solid
white arrow). The fat vesicles membrane seemed
also less defined. At 48 h APF, most of the anterior adipocytes showed degraded nuclei and, as
mentioned above, the anterior fat body was completely degraded between 48–72 h APF (Fig. 1C).
In the abdominal region, the degradation during early metamorphosis starts later and seems to
attain a significant proportion of the cells but not
all of them, beginning 72 h APF and continuing
at least up to the end of the pupal stage 144 h
APF. After the onset of pupariation (0h), the
adipocytes of Ceratitis showed a few small vesicles
surrounding the plasma membrane that, judging
from tinging, seemed to be enriched in protein
(not shown). In the posterior fat body starting at
2030 h APF up to 48 h APF, the number and size of
these granules increased while remaining apparently located in the peripheral cortex of the plasma
membrane (Fig. 2D, stars). The cortical protein
granules were very evident at 72 h APF and the
average size increased (Fig. 2E, stars). Starting at
72 h APF, the plasma membrane and the nucleus
of many of the posterior adipocytes started to disintegrate and at 98 h APF most of the granules
could be found homogeneously distributed in the
cell cytoplasm (Fig. 2F, stars). In the abdominal
region, the adipocyte disintegration was dominant
since 98 h APF (not shown).
Biochemical Changes During Early Metamorphosis
The destruction of the nucleus integrity represents a clear commitment to death. We analyzed
the rate of internucleosomal DNA degradation in
Fig. 3. Detection of internucleosomal DNA degradation of Medfly fat body during early metamorphosis. The DNA samples (see Materials and
Methods) were electrophoresed in a 1.4% agarose gel and stained with ethidium bromide.
Right panel: 144 h APF DNA was digested with
0.1 µg/µl of DNAse during 2 h at 37°C. Lanes
H and L: High and low molecular weight markers (see Materials and Methods).
Archives of Insect Biochemistry and Physiology
Aspartyl Proteinase in C. capitata
the fat body during early metamorphosis. This was
attained by immediate digestion of proteinase K
of the lysed cells followed by phenol extraction
(Kaufmann et al, 2000). Figure 3 shows a conventional 1.4% agarose gel electrophoresis where DNA
clearly appears fragmented during the last hours
of the pupal stage (98 and 144 h APF). This result
is in agreement with the beginning of the adipocytes lysis in the abdominal region (the most important in number of fat body cells, Fig. 2F).
In order to determine if the vesicles enriched
in protein observed in the abdominal region (Fig.
2D–F) were granules associated with fat body ly-
59
sosomal activity, as previously reported by Keeley
(1985), we determined the overall activity of acid
phosphatase, a lysosomal marker. Figure 4A shows
that the fat body lysosomal acid phosphatase activity increased 2.7-fold from 44 h BPF to 98 h
APF. On the other hand, the activity of fat body
acid proteinases was analyzed in polyacrylamide
gels, which showed a well-defined proteolytic band,
evident after 0 h (Fig. 4B), whereas only a background undefined shadow was present in the late
larva III (Fig. 4B, 8 h BPF). As a result of the physiological, non-synchronized cell dissociation and
disintegration processes, at 40 h APF the isolated
Fig. 4. Correlation between
lysosomal activities. A: Changes
in fat body acid phosphatase
during Medfly metamorphosis
(see Materials and Methods).
B: Fat body proteinase activity in PAGE gels during early
Medfly metamorphosis. After
incubation with 2% acid denatured hemoglobin, the reaction was stopped and stained
with CBB R-250. C: Inhibition
of Proteinase activity on gels.
Fat body extracts were pre-incubated with (lanes 1–5) or
without (lane c) inhibitors
during 20 min at 4°C. Lanes:
(1) 1 mM PMSF, (2) 5 mM
EDTA, (3) 2 mM of 1,10 Phenantroline, (4) 10 µM E-64,
and (5) 10 µM Pepstatin A.
Controls: (lane 6) propanol
and (lane 7) methanol.
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Rabossi et al.
fat body fraction contained the full posterior fat
body starting to dissociate and the fully dissociated anterior fat body (undergoing disintegration).
At 72 h APF, the fat body isolates contained posterior cells almost exclusively. In agreement with the
acid phosphatase profile, the acid proteinase activity from the fat body increased from low levels
at 0 and 2030 h APF up to 40–45 h APF and remained at a high level at least until 98 h APF (Fig.
4B). This proteinase activity behaved as an aspartyl proteinase since it was insensitive to several proteinase inhibitors (Fig. 4C, lanes 1–4) and only
pepstatin A was able to inhibit the substrate degradation (Fig. 4C, lane 5).
Purification of Medfly Aspartyl Proteinase
Our aim was to correlate presumptive histolysis specific proteinases with the progress of fat body
disintegration, as judged by microscopic images.
However, it was difficult to obtain sufficient fat
bodies from synchronized pupae by dissection. In
particular, dissociated fat body (after 20 h APF)
was difficult to isolate. Mass preparations of free
adipocytes based on cell density failed due to contamination with other cellular material. Therefore,
we decided to purify proteinases from whole synchronized 40–45 h APF pupae, since the procedures were fast and at that stage the larval anterior
muscles and intestine were already histolyzed, and
only abdominal muscles and fat body remained
functional. Due to the scarcity of the material, a
protocol allowing the simultaneous purification of
the different proteinases present in the cell-free extracts was followed. The aspartyl proteinase from
40–45 h APF pupae was first purified by an acid
precipitation step followed by salting out with 30–
70% ammonium sulfate. The soluble material obtained after dialysis was applied to a Sephacryl
S-200 gel filtration column. The profile of hemoglobin proteolysis at pH 3.5 by the eluted protein
shows three main peaks of proteolytic activity (Fig.
5A). Only the pooled fractions 67–75, containing
the most active peak of activity (Fig. 5A, peak II),
was completely inhibited by pepstatin A (99% inhibition), thus suggesting that this material con-
tained most of the pupal aspartic proteinase previously detected in the gels.
The other two activity peaks, peak I (fractions
50–60) and peak III (fractions 80–90), were not
inhibited by pepstatin A but the activity was completely abolished by E-475, a cystein proteinase inhibitor (97 and 99% inhibition, respectively)
(Rabossi, 2001).
The material from peak II was concentrated (see
Materials and Methods) and the buffer and pH
changed (from 4.5 to 5.8), after which it was applied to a cation exchange Mono S column FPLC.
Preliminary experiments that were carried out at
pH 4.5 showed that most of the material was degraded probably due to autocatalysis, as previously
reported in other aspartyl proteinases (Lah and
Turk, 1982). Figure 5B shows that Ceratitis aspartyl proteinase activity eluted between 0.075 and
0.12 M in a linear gradient of 0–0.5 M NaCl. The
fractions containing aspartyl proteinase activity
were pooled, dialyzed, concentrated, and applied
to a gel filtration Superose 12 column FPLC. The
peak of activity behaved as a single polypeptide
band in SDS-PAGE and was named “Early Metamorphosis Aspartyl Proteinase” (EMAP). In reducing conditions, the apparent molecular weight of
the EMAP in an SDS-PAGE was 42 ± 1 kDa (inset
in Fig. 5B). The same result was obtained under
non-reducing conditions (not shown).
Main Characteristics of C. capitata Aspartyl
Proteinase EMAP
The isoelectric point (pI) of EMAP was determined by isoelectric focusing and showed a wide
range of isoforms. The most important showed pIs
of 5.60, 5.90, 6.20, 6.55, and 7.35 (Fig. 6A).
The optimum pH of EMAP was 3.0 as shown
in Figure 6B, using hemoglobin as substrate. The
activity decreased after pH 3.5 and became undetectable after pH 5.5. We tested the effect of pH on
the stability of the enzyme at 23°C, by pre-incubation of the enzyme for 20 min at different pH, followed by a switch to the standard reaction at pH
3.5 (see Materials and Methods). We found that,
apparently, the stability of the enzyme was higher
Archives of Insect Biochemistry and Physiology
Aspartyl Proteinase in C. capitata
Fig. 5. A: Separation of Medfly proteinases by gel filtration. A 30–70% (NH4)2SO4 fraction of 40–45 h APF (pupal) extract was applied to a Sephacryl S-200® column.
The flow rate was 33.6 ml/h. Fractions of 5 ml each were
collected and their activity tested against 2% hemoglobin
(solid triangles). Open diamonds: total protein. B: Cation-exchange chromatography of Peak II was performed
October 2004
61
on a Mono S-FPLC column. Proteins were eluted with a
linear gradient of 0–0.5 M NaCl. The flow rate was 1 ml/
min. Activity tested against 2% hemoglobin (solid triangles). Inset: EMAP apparent MW estimation on 10%
SDS/PAGE, under reducing conditions. The gel was stained
with CBB-R250. Mw St: molecular weight standards.
62
Rabossi et al.
Fig. 6. A: EMAP Isoelectrofocusing. The arrows indicate
the main isoforms. B: Solid squares: Optimum pH.
Superose 12® Peak II (Fig. 5A) with EMAP activity was
assayed directly at different pH’s on 2% hemoglobin. Open
circles: pH stability of EMAP. The extract was preincubated
at different pH’s for 20 min at room temperature before
measuring the activity (see Materials and Methods).
at pH 5.0 (Fig. 6) (after incubation at pH 4.0 and
6.0, <40% of the activity was retained, Fig. 6).
In an attempt to isolate EMAP rapidly, minimizing enzyme degradation and increasing purity
and recovery, we performed affinity chromatography on pepstatin-agarose columns, starting from a
crude 30–70% ammonium sulfate fraction. This
column is currently employed in mammalian systems, and the conditions for the aspartyl proteinases purification are usually pH 3.5 for binding
and washing the column and pH 8.6 for elution
(Kregar et al., 1977). When we performed the chromatography in these conditions, the activity of the
eluate was undetectable (Fig. 7). This seems to
agree with the rapid instability of EMAP registered
at above pH 5.0. However, eluting at pH 8.6 and
lowering the pH by immediate dialysis against sodium acetate buffer to pH 4.0, provided a substantial recovery of the enzyme activity (Fig. 7).
lon membrane and processed to analyze the Nterminal sequence (see Materials and Methods).
Table 1 shows the obtained sequence of the first
six amino acids. The N-terminal of EMAP (see
Table 1) does not show homology with mammalian (Yamamoto et al., 1979) or mosquito (Cho
and Raikel, 1992) aspartic proteinases (the latter
is the only aspartyl proteinase previously sequenced in insects). However, the proline in EMAP
position 2 seems to indicate a highly conserved
position among aspartyl proteinases.
N-Terminal Sequencing of EMAP
The scarce material purified close to homogeneity in a SDS-PAGE gel was transferred to a ny-
DISCUSSION
Taking advantage of our previous studies on the
timing of different events during Medfly metamorphosis (Rabossi et al., 1991, 1992, 2000), in particular during the larva to pupa transition, we have
focused our interest in the disintegration and histolysis of the fat body. Although many features of
larval fat body disintegration were observed in
other dipterans, here we have been able to correlate morphological and biochemical events using
the same material. Well-timed histological images
Archives of Insect Biochemistry and Physiology
Aspartyl Proteinase in C. capitata
63
Fig. 7. Pepstatin-agarose chromatography of the 30–70%
ammonium sulfate fraction from 40–45 h APF pupa. Flow
rate was 15 ml/h. Solid squares: Activity against 2% he-
moglobin (see Materials and Methods). Solid triangles:
Activity of fractions after immediate dialysis against 0.1
M sodium acetate, pH 4.0 buffer. Open triangles: A280 nm.
(Fig. 1) showed that in C. capitata, as previously
reported in other flies by Fraenkel and Hsiao
(1969) and Whitten (1964), the fat body histolysis occurs in a sequential way, starting first in the
anterior part of the larval body (segments 1 to 4).
Starting exactly at the moment of apolysis
(20:30 h APF), the Ceratitis larval anterior fat body
loses first its net-like structure, apparently due to
the action of hemocytes as shown in Figure 1D.
Then, the adipocytes freely suspended in the
hemolymph initiate a programmed cell death path-
way. As early as 22 h APF, a small proportion of
the cells show dramatic changes in the nucleus
structure, including the absence of visible nucleolus and nuclear membrane. These adipocytes have
smaller fat vesicles with less defined membranes.
Forty-eight hours APF, these changes are visible in
most of the anterior adipocytes and at around 72
h APF all the anterior cells appear lysed. Therefore, the whole histolytic process in adipocytes of
the anterior part of the body occurs in around 50
h since the definitive immobilization of the larva
(see Fig. 8).
Starting later, a similar sequence of events affects the posterior part of the fat body. This starts
to dissociate at 40 h APF, in coincidence with the
pre-pupal to pupa transition, as demonstrated by
the deposition of the first pupal cuticle material,
(see Boccaccio and Quesada-Allué, 1993). The first
posterior adipocytes undergoing disintegration are
detected at around 72 h APF, when almost all the
anterior ones have disappeared (see above and Fig.
TABLE 1. N-Terminal Amino Acid Sequence of EMAP From C. capitata and
Comparison With Aspartyl Proteinases From Mammals, the Only Insect
Aspartyl Proteinase Known Sequence (of Mosquito) (Cho et al, 1992) and
the Rhizomucor miehei Mucorpepsin (Rawlings and Barret, 1995)
EMAP C. capitata
I
P
N
F
M
G
—
—
—
—
Aspartic-P mosquito
Cathepsin D pig
Cathepsin D human
Cathepsin E human
Mucorpepsin fungal
G
G
G
Q
I
P
P
P
S
P
V
I
I
A
V
P
P
P
K
S
E
E
E
E
I
P
V
V
P
G
L
L
L
L
T
S
K
K
I
P
N
N
N
N
G
Y
Y
Y
Y
Q
Conserved amino acids in bold.
October 2004
64
Rabossi et al.
Fig. 8. Correlation between main developmental events
during Ceratitis capitata metamorphosis (letters) and fat
body histolysis (numbers). Age within the puparium is
expressed in hours after puparium formation (APF) starting at “Zero time” (Rabossi et al., 1991). (a) “Zero time” of
metamorphosis; (b) larval–pupal apolysis (2030 h APF); (c)
cryptocephalic pupa, beginning of the pupal stage (40 h
APF); (d) head eversion (48 h APF); (e) definitive body
proportions (72 h APF); (f) beginning of the pharate adult
stage (144 h APF); (g) imago ecdysis (370 h APF). Anterior
(top) and Posterior (bottom) fat body events are indicated:
(1) dissociation of fat body adipocytes; (2) nuclear lysis;
(3) beginning of cellular lysis; (4) end of cellular lysis.
8). The histolytic process in the posterior part is
much slower since at the end of the pupal stage,
144 h APF, a significant part of the fat body still
remains apparently unaffected. At the moment of
adult ecdysis, remnants of the larval fat body still
persist in the adult abdomen. This is similar to the
described behavior of posterior fat body in Drosophila, where a portion of posterior adipocytes persists until 3 days after adult ecdysis (Bodenstein,
1950). Therefore, in Ceratitis capitata, fat body degradation is a sequential process where cell death is
induced differentially along the axe of the body in
an anterior to posterior way. The bases for the different timing in the regulation of disintegration in
this and other insects are currently unknown but
probably reflect temporal differences in ecdysteroid
levels along the body axe. Emery et al. (1994) described the 20-OH-ecdysone-dependent expression
of Broad complex transcription factors during Drosophila metamorphosis.
The activity profile of previously unknown lysosomal proteases is fully coincident with the histological images in Figures 1 and 2. In particular,
we have partially characterized an aspartyl proteinase designated Early Metamorphosis Aspartyl Proteinase (EMAP) having an activity profile similar
to the lysosomal marker acid phosphatase. Nucleases activity, as judged by DNA fragmentation,
partially overlaps with EMAP maximum activity
during the pupal stage (Fig. 3). EMAP is a protein
with an apparent MW of 42 ± 1 kDa as other
Cathepsin D–like proteinases in different organisms and is apparently an N-glycosylated high mannose glycoprotein, judging from the affinity for the
lectin Con-A (not shown). EMAP was co-purified
together with two other cystein proteinases that will
be described elsewhere. It is very likely that autolysis of EMAP occurred, especially when subjected to acid pH during co-purification (see
Materials and Methods). Judging from the optiArchives of Insect Biochemistry and Physiology
Aspartyl Proteinase in C. capitata
mum pH (3.0) for hemoglobin digestion, isoform
heterogeneity, and sensitivity to pepstatin A, EMAP
share similarities with mammalian Cathepsin D
(Barret, 1977). The exhibited instability of EMAP
to neutral and alkaline pH (Fig. 7B) is also a characteristic of two previously reported insect aspartyl proteinases, from Aldrichina and mosquito
(Kawamura et al., 1987; Cho et al., 1991). When
performing affinity chromatography in pepstatinagarose at pH 8.6, we were able to overcome the
inactivating effect of high pH, subjecting the eluates to immediate dialysis at pH 4.0. The instability to neutral and alkaline pH seems to be a
distinctive characteristic of insect aspartic proteinases absent in mammalian ones. Moreover, ethanol was the EMAP stabilizing agent of choice,
showing a difference with mammalian Cathepsins
D. The N-terminal sequence of EMAP showed no
similarity with any other known Cathepsins, mammalian or mosquito (Table 1). According to databanks, only the fungal mucorpepsin, belonging to
the A1 group of aspartic proteinases (Rawlings and
Barret, 1995) shares 3 of the 6 amino acids in the
N-terminus of EMAP. The eukaryotic aspartic proteinases in A1 group are endopeptidases of the digestive
tract such as pepsin and chymosin, lysosomal enzymes such as Cathepsin D, and enzymes involved
in post-transcriptional processing such as renin.
It is noteworthy that up to 40 h APF, the dissected fat body was cleaned of other cells and cellular debris and, therefore, the measured enzymatic
activity shown in Figure 4 B is expected to correspond to that tissue. Due to technical difficulties
to obtain antisera (death of animals), we have been
unable to obtain antibodies against EMAP. Therefore, direct proof of cellular and sub-cellular location of the enzyme is not available yet.
No EMAP activity was detected in the digestive
system of both young larvae III and adults. Our
data show that EMAP is a novel histolysis-specific
lysosomal proteinase, which seems expressed in the
fat body. The profile of activity during metamorphosis correlates with the programmed cell death
of adipocytes. Deiss et al. (1996) proposed that
Cathepsin D mediates a regulated type of programmed cell death in HeLa and U937 cells, initiOctober 2004
65
ated by cytokines like IFN-γ , TNF-α, as well as the
ligand to the FAS/APO-1 receptor. Efforts are underway to identify the gene coding for this protein
and its temporal expression, as well as to confirm
the precise cellular and intracellular localization
of EMAP.
ACKNOWLEDGMENTS
This work was supported by grants from the
University of Buenos Aires and YPF S.A. LQA is a
career investigator of the CONICET (Argentina).
We thank Drs. Martha Grinfeld and Anka Ritonja
for their invaluable assistance.
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