九州大学学術情報リポジトリ
Kyushu University Institutional Repository
カイコ体液中のキモトリプシンインヒビターの生 理・生化学的研究
白井, 孝治
九州大学農学研究科遺伝子資源工学専攻
https://doi.org/10.11501/3130902
出版情報：Kyushu University, 1997, 博士（農学）, 課程博士 バージョン：
権利関係：
Chapter 2
Biosynthesis, secretion and incorporation of CI-8 in the fat body at the 5th instar and during the larval-pupal
metamorphosis
The previous chapter confirmed that the amount of CI-8 in the hemolymph increased steeply at the mid-5th instar, suggesting that the
synthetic rate of CI-8 augtnented. It may be the fat body that synthesizes CI-8, since this organ not only functions as a center of intermediary metabolism but also synthesizes many humoral proteins (Palli and Locke,
1988)
including the 30 k Da proteins (Sakai,1 988)
and SPs (Izumi etal., 1988);
these functions are comparable to those of the vertebrate liver. The 30 k Da proteins are composed of structurally related proteins and occupy a large part of thehumoral proteins (their physiological roles are unknown but interestingly these are partially incorporated into the ovary). The synthetic activities of the 30 k Da proteins are in parallel with the amount of their mRNAs (Sakai,
1988),
whose amounts in the fat body increased in allatectomized larvae (Izumi et
al., 1984).
Also the synthesis rate of SPs accelerates after the disappearance of juvenile hormone at the mid-5th ins tar (Tojo etal., 1981 ).
Immunological analyses after allatectomy (Eguchi et
al., 1986b;
Matsuiand Eguchi,
1991;
Eguchi,1994)
suggested that inhibitor-d (serpin type, partially similar to Cl-8) was synthesized in the larval fat body and that this synthesis was stimulated by the addition of 20-hydroxyecdysone into the medium in which fat body was incubated (it was after 2 days that a difference was recognized between the test and control; this time span seems to be so long50
that the apparent increase of CI activity in the medi urn would be of a
secondary response). On the other hand, the synthesis of SCI is controlled by the amounts of mRNA, and a juvenile hormone analogue, S31183, inhibited the synthesis of mRNA but ecdysteroid did not (Ichikawa and Sasaki, 1991) (in this report the time span between the hormone treatment and the fat body
collection was unclear). It is likely that the disappearance of juvenile hormone stimulated the synthesis of Cis, whereas the ecdysteroid effect seems to be a matter of confusion. In addition, fluctuation is slightly different among the components of humoral Cis in allatectomized silkworm (Eguchi et al.,
1986b), suggesting that the endocrine effect is not uniform.
These situations prompted the author to confirm the organ that
synthesize CI-8. It was the fat body as described below. Also in vivo and in vitro experiments were made to see the effects of ecdysteroid and a juvenile hormone analog on the CI-8 synthesis.
The fat body incorporates again SPs after the onset of spinning. As described above, the fat body seems to change drastically its function at the spinning stage (Tojo et al., 1981 ). After pupation, SPs undergo proteolysis and mainly become amino acid sources for the de novo synthesis of
vitellogenin etc. The amount of CI-8 in the hemolymph also reduced steeply during the larval-pupal metamorphosis. CI-8 would survive for a long period
if the hemolymph has a protease that can make a protease/inhibitor complex.
When chymotrypsin was injected into the hemocoel, a marked amount of CI-8 was detected immunologically for more than 6 hr, kept in the form of complex (Fujii and Shirai, unpublished data). This result is taken to indicate that no endogenous protease that can make complex with CI-8 exists in normal
hemolymph. Thus the rapid reduction of CI-8 at the spinning stage may imply the sequestration into some organ(s). Previously, CI-13' was shown to be tncorporated into the ovary (Aratake et al., 1990) like the 30 k Da proteins.
On the other hand, investigation made in the latter half of the present chapter indicated that CI-8 was incorporated into the fat body like SPs.
The dynamic aspects of CI-8 demonstrated in this chapter suggested that this CI would provide a good investigation system for the elucidation of
endocrine control mechanisms and physiological significance of humoral proteins.
Materials and Methods
Materials
Grace's insect cell culture medium with and without L-methionine and Sf-900 II SFM medium with and without L-methionine and L-cystine were purchased from Gibco BRL Co. Ltd. Fetal bovine serum was obtained from Bio Whittaker Co. Ltd. Penicillin and streptomycin were from Sigma Chern.
Co. Ltd. and kanamycin from Meiji Confectionery Co. Ltd.
e
5S]Methionine(in vitro
cell labeling grade L-e
5s] methionine) was obtained from Amersham Life Science Co. Protein A-Agarose was purchased from Seikagaku Kogyo Co. Ltd. Methoprene (isopropyl (2E, 4E)-11-methoxy-3, 7, 11-trimethyl-2, 4-dodecadienoate) and 20-hydroxyecdysone were kindly awarded from Dr. Y.Utsumi (Laboratory of Agricultural Chemical Research, Otsuka
Pharmaceutical Co. Ltd.) and Dr. 0. Ninagi (National Institute of Sericultural and Entomological Science, Matsumoto), respectively. Poly-L-lysine solution was purchased from Sigma Co., and a Bas 1000 system for autoradiography and Centriplus for ultrafiltration from Fuji Film Co. Ltd. and Amicon Co.
Ltd., respectively. Sf 900 II medium and fetal bovine serum (lot. No.
4M0055)
were purchased from Gibco Co. Ltd. and Bio Whittaker,respectively.
Animals
The cz strain with CI-8 was mainly used. If necessary, the r06 strain which has no CI -8 was applied as indicated.
Preparation of tissue extracts
The tissues were homogenized in one volume (w/v) of the homogenizing buffer (0.1 M Tris-HCl, pH 6.8, containing 0.25 M saccharose, 0.5 mM
dithiothreitol, 1 mM phenylmethylsulfonyl fluoride (PMSF), 1 mM
ethylendiaminetetraacetic acid (EDT A) and 10 mM 3-[ (3-cholamidopropyl)
dimethylammonio ]-1-propanesulfonate in 1.5 ml plastic tubes. The mixture was sonicated on ice and centrifuged at 15,000 x g for 1 hr at 4 °C. After centrifugation, the supernatant was saved at -80°C until used.
In vitro tissue culture and assay for synthesized and secreted CI-8
Larvae were dissected in a clean bench and the fat body, midgut, silk glands, testes and ovaries were taken out with forceps. The tissues were washed with sterilized physiological saline (0.75 % NaCl in distilled water), weighed for wet weight, pre-incubated at 28°C for 1 hr in 400 Jll of sterilized Grace's medium with L-methionine (containing 20 units of penicillin and 20 }lg of streptomycin) to remove contaminating hemolymph as far as possible and then incubated at 28°C for 6 hr in 400 Jll of freshly sterilized Grace's medium without L-methionine (including the above antibiotics) and
e
5S]methionine (1 0 flCi when radioactivity in the medium was measured or 20 JiCi whenradioactivity in the tissues was measured). The mixtures were centrifuged at 800 x g for 5 min.
The supernatant was directly subjected to immunoprecipitation for CI-8,
whereas the tissue pellets were washed twice with physiological saline and homogenized in 3 volumes (w/v) of the homogenizing buffer and centrifuged
at 12,000 x g for 1 hr at 4°C. The supernatant was subjected to immunoprecipitation for CI-8.
If necessary, the media were subjected to native or SDS-PAGE followed by autoradiography.
Jmmunoprecipitation for CI-8
The sample in 1 00 Jll of TBST (Chapter 1) was thoroughly mixed with 10 Jll of the antiserum raised against CI-8 (Chapter 1), allowed to stand at 4°C overnight, mixed with 50 Jll of protein A-Agarose, incubated at room
temperature for 2 hr and centrifuged at 800 x g for 1 min.
The precipitate was washed three times with 200 Jll of TBST, taken up in water and transferred into vials and measured for radioactivity by an Aroka LSClOOO liquid scintillation counter. The values corrected for the controls using normal rabbit serum instead of antiserum were regarded as the ability of CI-8 synthesis of the tissue.
Effects of hormones on the CI-8 synthesis in the fat body
This was examined both by in vivo and in vitro tests using 20-
hydroxyecdysone and methoprene (the latter was omitted in the in vitro test).
In vivo test: 20-hydroxyecdysone (dissolved in physiological saline) was injected into the hemocoel of larvae through the abdominal intersegmental epidermis. Alternatively, methoprene diluted in acetone as a vehicle was
applied topically to the dorsal abdomen of larvae. All larvae were left for 3 hr at 26°C and fat body from each larva was taken into 500 Jll of Grace's insect medium without L-methionine (including the antibiotics) and cultured at 27°C
for 6 hr with 10 JiCi of
ess
]methionine.In vitro test: Fat body was taken out and incubated in Grace's insect medium with hormone at at 27oC for 3 hr, and the tissues were taken into new medium with radioactive methionine and incubate at 27°C for 6 hr as described above.
The media after incubation of both tests were subjected to
immunoprecipitation with anti-CI-8 serum to assess the ability of CI-8 synthesis of the fat body (see above). In controls, larvae were treated with vehicle alone in the in vivo test or the fat body was incubated after the addition of physiological saline instead of hormone in the in vitro test.
Preparation of cryostat specimen and immunological localization of CI-8
Larvae or pupae were cut into round slices (semi whole-mount specimens). The specimens were prefixed for 2 to 4 hr at 4 OC with 2%
paraformaldehyde dissolved in PBS-Sac (0.2 M phosphate-buffered saline, pH 7.4, containing 20% (w/v) sucrose and 130 mM NaCl), washed sufficiently with PBS-Sac overnight with a few exchange of the buffer, embedded in Tissue-TEK (O.C.T. Compound, Miles Inc.) after smeared sufficiently and then frozen in isopentene by dry ice. The frozen specimens were cut at -20°C, mounted on a slide glass coated by poly-L-lysine and stored at -20°C until used.
The cooled slide glass was soaked into ice-cold absolute acetone for 30 min to fix the tissues and to remove lipid. Subsequent processed were done at room temperature. The slide glass was dried and covered with 0.5% periodic acid solution for 10 min to inhibit endogenous peroxidase. Then the specimens were washed with PBST (Chapter 1) three times for 5 min, blocked with the blocking buffer (normal goat antibody diluted 1/200 with PBST containing 0.02% (w/v) NaN3), washed briefly and soaked in the first solution (antiserum
for CI-8 diluted I
/200
with PBST containing 1% bovine serum albumin, o.02o/o NaN1) for10
to 15 min. After washing three times with PBST for5
min, the specimens were immersed in the second solution (peroxidase-labeled anti rabbit IgG) goat IgG diluted
1/200
with PBST containing 1% bovine serum albumin for10
min, washed three times with PBST for5
min andtreated with the staining solution
(30
ml of0
.1
M sodium phosphate buffer, pH 7.4 containing10
mg of3,3
'-diaminobenzidine,20
mg of imidazole and 4 to5
Jll of 30% H202) for
2
hr or overnight. After the color developed sufficiently, nuclei were stained with hematoxyline. Then the specimens were observed for the localization of CI-8 using optical microscope.Preparation of fat body specimens from different body regions and culture to assay the ability for CI -8 synthesis
Two types of fat body, discriminated as peripheral fat body near the epidermis (eFB) and perivisceral fat body near the gut (gFB), were roughly separated after cutting larvae from the back side. The midgut, silk glands and
other organs were removed with forceps. First, gFB was gently raked off.
Then, eFB was raked carefully around the skin. All specimens were sufficiently rinsed in physiological saline.
To examine the ability of CI-8 synthesis, the eFB and gFB specimens were cultured in vitro with radioactive methionine as detailed above. Aliquots
(5
to 10 Jll) of the media, which contained newly synthesized proteins secreted from the fat body, were spotted onto filter paper tips(5
x5
mm). These were allowed to dry at room temperature, washed five times for5
min withsufficient amounts of
5o/o
TCA solution, drained and put into vials to which liquid scintillation cocktail (Aquasol2)
was added. The vials were left at room te mperature overnight and measured for the radioactivity.Also the tissue themselves of eFB and gFB after culture were extracted
as described above and processed for the counting of radioactivity for newly synthesized proteins.
Other aliquots of the media were subjected to native PAGE and the gels were washed with a fixing solution (methanol/acetic acid/water in the ratio 4 :
1 : 5) to fix proteins and to removing un-incorporated [35S]methionine, dried using a gel drier and then autoradiographed with Kodak X-Omat films.
Incubation of gFB in hemolymph using different strains
Hemolymph was collected from the cz strain (with CI-8), centrifuged to remove hemocytes and mixed with glutathione (reduced form, 0.6 mg/ml) to avoid melanization. Using the prepared hemolymph, gFB dissected from the r06 strain (without CI-8) was incubated at 26°C for 12 hr with a 1.5 ml plastic tube covered with mineral oil to reduce oxidation. Then, the tissue was
washed sufficiently with physiological saline containing 0.05% Triton X-1 00 and homogenized as described above; the homogenate was centrifuged and an aliquot of the supernatant was subjected to native PAGE followed by
zymogram analysis for Cis. Control gFB without incubation (0 hr) was analyzed in parallel.
Isolation of protein granules from gFB
Protein granules were isolated from gFB by the method essentially according to Tojo et al. ( 1978). Fat body was homogenized in 10 volumes (w/v) of 0.3 M sodium-potassium phosphate buffer containing 0.01 M MgC12, pH 6.8, by 15 to 20 strokes in a homogenizer. The homogenate was
centrifuged at 600 x g for 10 min. The pellet was re-suspended in the above buffer and filtered through four layers of cheese-cloth, which retained nuclei.
The filtrate was layered onto 10 ml of 1.8 M sucrose made up in the same buffer and centrifuged at 600 x g for 10 min to sediment urate granules.
Protein granules remaining in the interface was taken, diluted with the same buffer, layered onto a discontinuous gradient containing 7 ml each of 1.2, 1.5 and 1.8 M sucrose in the same buffer and centrifuged at 22,500 x g for 20 min. Protein granules were retained partly at the interface between 1.2 and
1.5 M sucrose layers and 1nainly between 1.5 and 1.8 M sucrose layers. Both were collected, washed, extracted as above and used for further experiments.
Preparation and solubilization of cell membranes from the fat body
Fat body was homogenized in five volumes (w/v) of ice-cold 20 mM Tris-HCl buffer, pH 8.0, containing 150 mM NaCl, 1 mM CaC12, 0.1 mM phenylmethylsulfonyl fluoride and 1 mM 2-mercaptoethanol using potter-type homogenizer. The homogenate was centrifuged at 800 x g for 10 min at 4 OC to remove cell debris and nuclei. The supernatant was centrifuged at 18,000 x g for 1 hr. The pellet was taken up in the same buffer and centrifuged again at
18,000 x g for 1 hr. Then the pellet (membrane fraction) was solubilized with one volume of SDS-PAGE preparation buffer (without sulfhydryl reducing reagent) at room temperature for 4 hr and stored at -80°C until used.
Preparation of recombinant CI-8 (CI-8S) using baculovirus expression system
CI-8 eDNA, Spodopterafrugiperda cell line (Sf 21), Autograpfa californica nuclear polyhedorosis virus (NPV) (AcRP23 LacZ: recombinant baculovirus) and vector (pAcYM1) were kindly given by Mr. Yakiyama (Institute of Genetic Resources, Faculty of Agriculture, Kyushu University).
CI-8 eDNA was incorporated into the virus and expressed according to previously published methods (Matsuura, 1994 ). The resulting recombinant CI-8 named CI-8S was purified as described in Chapter 1. The final
preparation as well as the recombinant virus (NPV/CI-8) was stocked at -80°C until used.
58
Formation of complex with a-chymotrypsin
CI-8 or purified CI-8S ( 4.2 }lg) was incubated with bovine [[t
chymotrypsin ( 1.2 and 2.4 )Ag) in sodium phosphate buffer, pH
7.5,at
30°Cfor
10
min. The mixture was subjected to native PAGE followed by Western blotting analysis using the anti-CI-8 serum.
Preparation of 35S-labeled CI -8S
Using
35mm tissue culture dishes, Sf 21 cells were incubated at 27°C for
1hr in
1.5ml of Sf
900II insect cell culture medium containing
1 Oo/ofetal bovine serum (about
1 x 106cells/well). After washed sufficiently with the same medium without serum, the cells were incubated at 27°C for
3days with NPV/CI-8 at a multiplicity of about
10p.f.u./cell in the mixed Sf
900II media
(300Jll of complete and
900Jll of incomplete lacking L-methionine and L
cystine) containing
200JACi e 5S]methionine. The medium containing labeled CI-8S was dialyzed against buffer A (see Chapter
1)and loaded onto a DEAE
Toyopearl column (as described in Chapter 1). After the break-through fractions were eluted, CI-8S fractions eluted by buffer A containing
0.3M NaCl were collected, concentrated by ultrafiltration with Centriplus™, dialyzed against physiological saline containing 0.02% (w/v) of NaN3 and diluted to
1,000cpm/ JAI using a washing buffer (200 mM Tris-HCl buffer, pH
7.0,
containing
150mM NaCl, 8 mM CaC12•2H20,
0.1 o/oTriton
X-1 00, 0.5 o/obovine serum albumin and
0.05o/oNaN3).
Detection of CI-8S receptor by ligand blotting
The fat body membranes prepared and solubilized as above were separated by SDS-PAGE. The gels were blotted onto PVDF membranes, which were immersed in a blocking buffer (5% bovine serum albumin) at room temperature for
1.5hr, then in the washing (see the above section)
59
buffer at room temperature briefly and finally in the medium containing 35S
labeled CI-8S ( 1,000 cpm/ Jtl of the washing buffer) at 4 OC for 2 days.
Subsequently, the membranes were rinsed five times in the washing buffer each for 10 min with gentle shaking, put into polypropylene bags and subjected to autoradiography by using a Bas 1000 system to detect radioactive bands.
Pursuit of 35S-labeled CI-8S in the fat body after injection
CI-8S labeled with
ess
]methionine as above was injected into body cavity at various stages of development at a dose of about 25,000 cpm/ 25 Jll per individual. After kept for 24 hr at room temperature, the animals were sacrificed for gFBs. Then gFBs were homogenized in three volumes (w/v) of the homogenating buffer (see above) and centrifuged at 15,000 x g for 1 hr at 4°C. The supernatant was separated by SDS-PAGE and the gels were blotted to PVDF membranes. Radioactive protein bands were detected byfluorography (see the previous section).
Other procedures
Conditions for native and SDS-PAGE, zymogram, ELISA etc. were as described in Chapter 1.
Results
Identification of the tissue which synthesizes humoral CI -8 and developmental changes in synthetic ability
The ability of CI-8 synthesis was examined by in vitro culture for 6 hr With
e
5S]methionine followed by immunoprecipitation, using six major larvaltissues from male and female larvae on day 4 of the 5th ins tar and one day after the onset of spinning (Fig. 2-1 ). Hardly any positive signs were given by the midgut, posterior silk glands, testes and ovaries. On the other hand, the fat
xl,OOO
3
2 ('Q >
a.. ('Q
e
c.0 1
0
FB MG SG TS male
• 5-4 medium
• 5-4 tissue
� 5-1 medium
II 5-1 tissue
FB MG SG OV female
Fig. 2-1 . The synthetic ability of CI-8 in various tissues tested in vitro.
The fat body, midgut, testes and ovaries (denoted by FB, MG, SG, TS and OV, respectively) were taken from larvae (the cz strain) on day 4 of the 5th instar (5-4) or on day 1 of spinning (S-1) and cultured in Grace's medium containing [35S]methionine at 28°C for 6 hr. Then the medium and tissue were separately subjected to immumoprecipitation using anti
CI-8 seru1n and counted for radioactivity. The values are recalculated to cpm per larva.
body gave marked radioactivities in the media. The value was much larger in feeding larvae than in spinning larvae. The radioactivities in tissues after culture were very low even in feeding larvae. These results indicate that CI -8
is synthesized in the fat body and rapidly secreted into the medium.
Changes in radioactivity of the fat body culture system prepared during
the period from 5th instar to pupal stage were observed (Fig. 2-2). In male
5th instar larvae, the value was larger on day 4 than on day 2, whereas no significant difference was seen between the two stages in female. The labels in tissues were again negligible. Spinning larvae and day 2 pupae exhibited much smaller titers than feeding larvae, indicating that the synthetic ability was deteriorated after the onset of spinning.
x1,000 15
m 10 • Cl-8 in medium male
>
II
� Cl-8 in medium female
m
�
� Cl-8 in fat body malec. 0
5
0
5-2 5-4 S-1 P-2
Stage
Fig. 2-2. Developmental changes in synthetic ability of CI-8 in the fat body.
Specimens were taken on day 2 or 4 of the 5th in star (5-2 and 5-4, respectively), on day 1 of spinning (S-1) or day 2 of the pupal stage (P-2).
The cz strain was used and males and females were separately analyzed. For other details see the legend to Fig. 2-1.
62
Effects of ecdysteroid and juvenile hormone analogue on the CI-8 synthetic activity.
Effects of excess doses of 20-hydroxyecdysone or methoprene on CI-8 synthesis in the fat body of larvae on day 3 of the 5th in star were examined by in vivo and in vitro tests (Fig. 2-3A and B, respectively).
In the in vivo test, 5 }lg of Ecd (in 25 Jll physiological saline) was
injected into each larva, or 1 }lg of methoprene (in 10 Jll acetone) was topically given to each larva, and, after kept at 26°C for 3 hr, the larvae were dissected and the fat body was incubated (at 27°C for 3 hr) in Grace's medium (500 Jll) containing [35S]methionine. The medium was subjected to
immunoprecipitation using anti-CI-8 serum and the value was taken to indicate the CI-8 synthetic ability of the fat body. It was reduced to 33% by the
ecdysteroid treatment compared to the sham operated control. On the other hand, methoprene did not affect the labeling.
In the in vitro test, the fat body was taken out of fresh larvae and
incubated at 27°C for 3 hr in Grace's medium containing 20-hydroxyecdysone (5 Jlg/500 Jll) and
e
5S]methionine. CI-8 secreted from the fat body was about 70o/o of the control incubated without the horomone.All results clearly indicated that the synthesis of CI-8 in the fat body was inhibited by 20-hydroxyecdysone but not by methoprene at least under the conditions applied in the present study. The inhibition by Ecd may occur
quickly, since the effects were seen within the treatment period of 3 hr in both in vivo and in vitro tests.
Changes in amount and localization of CI-8 in the fat body during development
Zymogram analysis indicated that the fat body extracts consistently exhibited a band at the position of CI-8 during the period from the mid-5th
mstar to the mid-pupal stage (Fig. 2-4 ). In addition, there was a CI band at
slightly upper part of CI-8 in the larval fat body but this band disappeared in the pupal specimens and instead a new CI band appeared closely under the CI-8
band. The latter component will be referred to again.
When the changes in amount of CI-8 in fat body extracts were assayed
by ELISA using the anti-CI-8 serum, there were two peaks (Fig. 2-5); the first one was on day 4 (male) or 5 (female) of the 5th instar and the second one was on day 3 of the pupal stage (both sexes). There after the amount of CI-8
decreased slowly as the pupal-adult development progressed.
CI -8 was localized on cryostat sections of the larval or pupal body by in situ staining with anti-CI-8 serum. Although the larval fat body tissues can be
classified into four types by their shape, spindle-like, small spheroidal, strand
like and massive (Fig. 2-6; Seki et al.,
1978),
stained substances were detected in all types of fat body from larvae on day 5 of the 5th instar (Figs. 2-7), and the intensity seemed to be uniform all over the tissue (Fig. 2-8, this illustrates the features of spindle-like fat body and massive fat body). Moreover, no difference in intensity are seen among different regions of the body cavity in 5th instar larvae. At the spinning stage, however, signal was detected more clearly in the fat body located near the midgut (gFB) than in that located around the epidermis ( eFB) (Fig. 2-9). In the latter, cells were less stained with anti-CI-8 serum. Namely, the distribution showed a bias to eFB after the onset of spinning. Detailed inspection of gFB (Fig. 2-10)
indicated that the stained substances exist in the internal granules (probably the protein granules) well-observed at this stage. In day 3 pupae, the fat body tissues becomeuniform in shape, and also the staining intensity was evenly distributed (Fig. 2-
11).
Although the bunch-like basement membranes of pupal fat body tissues looked fragile and some of the fat body tissues were isolated in hemolymph, signals were kept in the granules (Fig. 2-11 ). These observations suggested that CI-8 was stored in the form of protein granules in gFB.(A) (B)
x1 ,000
X 1,000 10.0
10.0
7.5
(U 7.5
> C'CS
... >
(U I..
�
c. 0 5.0�
C'CS 0. 5.00
2.5 2.5
0 0
Cont Ecd JH Cont Ecd
Fig. 2-3. The effects of hormones on the CI-8 synthesis in the fat body.
(A)
Larvae (the cz strain) on day 3 of the 5th ins tar were eachinjected with 5 Jlg of 20-hydroxyecdysone (Ecd) or topically given 1 Jlg of methoprene
(JH),
kept for 3 hr and sacrificed for the fat body, which was incubated with radioactive methionine as detailed in Fig. 2-1.(B)
Untreated larvae at the same age were sacrificed for the fat body, which was incubated with radioactive methionine plus 5 }lg/500 Jll of Ecd for 3 hr. For other details see the legend to Fig. 2-1. The radioactivities of the media are indicated. Controls received the vehicle (water or acetone) alone.
Cl-8
Fig.
2-4.
Zymogram of CI after native PAGE( 10%)
of fat body extracts during development.Arrow indicates the position of (�1-8. For abscissa see the legend to Fig.
1-22.
66
x100
C) E
0
7
3
Male FB --()-- Female FB
4M 5-1 5-2 5-3 5-4 5-5 s-1 s-2 P-1 P-2 P-3 P-4 P-5 P-6 P-7 Stage
Fig. 2-5. Developmental changes in the fat body content of CI-8 as measured by ELISA.
The results are indicated in ng per 10 mg tissue. For abscissa see the legend to Fig. 1-22.
c
•
•
•
•
.,
• • ....,#
� .... -: • -
.'.f!/1_ ·_
• -� .. . �- .... . .... � .
... . ,..,- ' .. .,. __ ... .
�·;__·-�
.�.:-·.�···�·�- .
, -:. :-· ·.
•.,. �
.
, . ....D
Fig. 2-6. Semi -schematic drawing of the fat body showing various shapes.
A, spindle-like fat body; B, small spheroidal fat body; C, strand-like fat body; D, massive fat body.
(A)
(B)
(A)
(B)
Fig. 2-7. Immunohistochemical localization of CI -8 in the fat body from a larva on day 3 of the 5th ins tar.
Left: the larva was cut to prepare semi whole-mount sections which were immunostained using anti-CI-8 serum (A).
A control section received normal serum (B). Right: the results are schematically drawn. FB, MG, E and M indicate the fat body, midgut, epidermis and muscles, respectively. Nuclei were counterstained with hematoxylin. Scale bar, 200 J-L m.
en c.o
(A) (A)
(B)
-
Spindle-like Massive
Fig. 2-8. Immunohistochemical localization of CI-8 in the fat body from a larva on day 5 of the 5th instar.
(A) lmmunostained using anti-CI-8 serum. (B) A control section received normal serum. Results with spindle-like fat body (left) and massive fat body (right) are seen. Scale bar, 50 1-L m . For other details see
t.he \egend t.o Fig. 2-7.
0 f'...
(A)
(B)
(A) r-1 ..,.I"!"
.�- _P"'!'_.,.._ 'r""' __ .,._ - _P"'!'_ .... _ """'� · _.,._ .,.._ ""'· ·"""'· """'· """·. ·.,...· """· ............ . -. -.""" .... . --. -. -. . -. -. -.
�
�9.�Eil
�(B)
�
: : : �gFB
�-<;K><:.b..-A.J. ( x_ . � '"
"' �
Fig. 2-9. Immunohistochemical localization of CI-8 in the fat body from a larva on day 2 of spinning.
Left: Immunostained using anti-CI-8 serum (A). A control section received normal serum (B). Right: the results are
schematically drawn. eFB and gFB stand for the peripheral fat body and the perivisceral fat body, respectively (for details see
text). T and M denote the tracheae and muscles, respecti_vely.
Scale bar, 200 /l m. For other details see the legend t:<> F1g. 2 7_
r-
"
Sequestration of hemolymph CI-8 into gFB as demonstrated by using different
strains
The above notion, together with the stnall sign of CI-8 synthesis in the
fat
body after the onset of spinning (see Fig.2-2),
indicated the possibility that CI-8 is sequestered into gFB. This inference was also supported by theobservation that the CI-8 concentration in the hemolymph decreased while that in the fat body increased after the spinning stage (cf. Figs.
1-24
and2-5).
Toconfirm the issue, gFB specimens having no CI-8 (from the r06 strain with Cls-4, 7 and
13
in the hemolymph) were prepared on day1
of spinning and incubated for12
hr in the hemolymph having CI-8 (from the cz strain with Cls-3, 8 and13'
in the hemolymph) prepared on the same age. The zymogram for the r06 gFB after incubation showed the CI-8 band (Fig.2-12,
lane3),
whereas the control incubated for 0 hr and the untreated r06 gFB did not
(lanes
4 and5,
respectively). Therefore, CI-8 in the r06 gFB must be or
igin
ated from the cz hemolymph. 'These results clearly evidenced the migration of CI-8 from hemolymph into the fat body.Fig. 2-10. Detailed inspection of the gFB region from a larva on day 2 of spinning.
(A) Immunostained using anti -CI -8 serum. Protein granules are stained strongly. (B) A control section received normal serum. Scale bar, 50 11m. For other details see the legend to
Figs. 2-7 and 2-9.
(A)
(B)
Fig. 2-11. Immunohistochen1ical localization of CI-8 in the fat body from a day 3 pupa.
(A)
Immunostained using anti-CI-8 serum.(B) A
control sections received normal serum. Scale bar, 200 Jim.
For other details see the legend to Fig. 2-7.
74
1 2
blood gFB cz
3 4 5 6
treatment 12hr Ohr
gFB r06
blood
Fig. 2-12. Demonstration of transfer of CI-8 frotn hemolymph to the fat body using heterologous strains.
The eFB specimen from day 1 spinning larvae of the cz strain (with CI-3, 8 and
13')
was incubated at 26oC for 12 hr in the hemolymph specimen from the r06 strain (with CI-4, 7 and 13) at the same age, extracted and separated by native PAGE (10o/o) followed by zymogram analysis. Lanes 1 and 2, fresh hemolymph specimens of the cz and r06 strains, respectively, asCI markers; Lanes 2 and 5, the extracts of non treated gFB from the cz and r06 strains, respectively, ascontrols; Lanes 3 and 4, the extracts of incubated gFB from the cz strain (Lane 4 was another control incubated for 0 hr).
?5
Comparison of synthetic ability between eFB and gFB
To compare the ability of CI-8 synthesis between eFB and gFB, these were prepared from the cz strain at different stages and cultured with
e
ss]methionine, and the media were subjected to immunoprecipitation. The specimens on day 4 of the 5th in star gave larger values than those on day 1 after the onset of spinning, and eFB was tnore than 5 times higher than gFB (Fig. 2-13A). Also the autoradiography (after native PAGE) of the medium in which eFB or gFB was incubated (Fig. 2-13B) showed that a larger amount of proteins are synthesized in eFB than in gFB in feeding larvae. The author concluded that eFB plays a leading role in the biosynthesis of CI-8.Properties of recombinant CI-8 prepared by baculovirus expression system
A recombinant CI-8 (named CI-8 ), derived from CI-8 eDNA using a baculovirus (NPV) expression system as described in Materials and Methods, was characterized in comparison with CI-8, to test whether this artificial CI-8 was able to serve as an experimental too] or not. Zymogram analysis and protein staining after native PAGE indicated that CI-8S and CI-8 gave one activity band and one protein band at the same position (Fig. 2-14A; the lanes for "CI-8S protein" and "control protein" received the media in which
recombinant NPV and non-recombinant NPV, respectively, were incubated;
the former gave the band but the latter did not). Therefore, the band detected in the CI-8S lane was concluded to be the product of CI-8 eDNA. Although showing a small gap in native PAGE (see Fig. 2-14A), CI-8S co-migrated with CI-8 on SDS-PAGE (Fig. 2-15), indicating that it was the same in molecular weight as CI-8 (42,000). CI-8S reacted with anti-CI-8 serum and ConA upon the immunoblotting analysis and the ConA-peroxidase blotting analysis,
respectively (Fig. 2-16). The specific activity of CI -8S ( 1.08 units/ )Jg) was comparable to that of CI-8 (1.10 units/�g) (Fig. 2-14B). Finally, to assess the
76
(A)
:l (I) -(I) a E
0 ...
E Q.
u 2000
1000
0
5-4 S-1
stage
(B)
5-4 S-1
gFB eFB gFB eFB
Fig. 2-13. Difference in CI-8 synthetic ability between eFB and gFB tested in vitro.
Experimental conditions were the same as those described in Figs.
2-1 to 2-3 except that eFB and gFB specimens isolated from day
4
of the 5th ins tar{5-4)
or on day 1 of spinning (S-1) were used. Only the results of the media are shown (recalculated to the tissue weight basis). (A)Immunoprecipitation with anti-CI-8 serum. (B) The media
(5
#1 for5-4
and25
�tl for S-1) separated by native PAGE (10%) and autoradiographed.ability to form a complex, CI-8S or CI-8 was subjected to SDS-PAGE followed by Western blotting with the anti -CI -8 serum before and after incubated with a-chymotrypsin at 26°C for 1Om in (Fig. 2-17). After incubation, the band of CI-8S or CI-8 was reduced and a band with a
molecular weight of
59,000
appeared. This value was in agreement with the expected size for the CI -8/ a-chymotrypsin complex. The author supposes that77
( ) (B)
-
0
....
0.80 :::L en
Cis
;!::::
c.:
::s 0.4
protein CI-8S
Fig. 2-14. Comparison of CI-8S (recombinant Cl-8) with CI-8 (Exp.
1).
CI-8S was synthesized by a baculovirus expression system using recombinant virus for CI-8 (NPV /CI-8) and purified. (A) Analysis by native PAGE
(10%)
followed by zymogram and protein staining with Coomassie Brilliant Blue. The medium in which NPV/CI-8 wasincubated gave the same results as purified CI-8S. (B) Specific activity.
78
L.M.M.
Cl-8 CI-BS
Fig.
2-15.
Comparison of CI-8S with CI-8 (Exp.2).
SDS-PAGE
(7.4%)
followed by protein staining with Coomassie Brilliant Blue. Molecular weight markers (L.M.M.) were also run in parallel. Each CI was electrophored in two lanes in different amount.79
{A)
1 2 3
{B)
�... ,. .
. . .
. . 't.Af•.Jlr: �.
. �-.·
Fig. 2-16. Comparison ofCI-8S with CI-8 (Exp.
3).
Native PAGE (10�)) followed by Western blotting analysis using anti-CI-8 serum (A) and by lectin blotting with ConA-peroxidase.
CI-8S 8 I+
+ehy chy Cl-8
Complex-
molecular weight
-94,000
-67,000
Cl-8- ---·1-42,000
partial digested- 1 ... -•• .- ...
Cl-8?
-29,000
-14,400
-front
Fig.
2-17.
Comparison of CI-8S with CI-8 (Exp.4).
CI-8S and larval hemolymph (the cz strain, on day 5 of the 5th instar) were separately incubated with a -chymotrypsin
(CI-8S+chy and Bl+chy, respectively) and separated by SDS-PAGE
(10%);
the gel was subjected to Western blotting using the anti-CI-8 serum. The amount of a-chymotrypsin was varied(2.4
and1.2
jlgfor left and right lanes, respectively). Untreated CI-8 was run as a marker (the rightmost lane). The molecular weights deduced from the markers were drawn along the right margin.
81
CI-8S as well as CI-8 can make a cotnplex with the enzyme. Although some uncertainty still exists (e.g., the different mobility in native PAGE), it will be concluded that CI-8S is very close to CI-8 in nature. Consequently, some of the subsequent experiments will be done using CI-8S instead of natural CI-8.
Detection of receptor for CI-8S on the fat body at various stages of development by ligand assay
Cell membrane fractions, prepared from eFB and gFB and solubilized as described in Materials and Methods, were subjected to SDS-PAGE and the gels were blotted onto PVDF membranes, which were incubated with 35S-labeled CI-8S (Fig. 2-18). No labeled bands were seen in eFB and gFB from larvae on day 4 of the 5th instar. However, a label was present at a molecular weight of about 70,000 in the case of gFB frotn anin1als on day 1 of spinning and on days
1 and 3 after pupation. In the lane of eFB from spinning larvae, a faint label was detected at the same position as for gFB. These results suggested that the fat body, in particular the gFB fraction, has a receptor for CI-8 at and after the spinning stage.
Detection of CI -8 in the protein granules from pupal gFB
To confirm the notion that CI -8 was accumulated in the protein granules of gFB, the contents of the isolated granules from day 3 pupae were analyzed by SDS-PAGE (Fig. 2-19). Although the major components were SP2 etc.
judging from their mobilities, a band for Cl-8 co-migrating with active band was detected in the granule extracts. In addition, closely below the position of CI-8, a CI band similar to that detected in the fat body extract from pupae ( cf.
Fig. 2-4, see also the following section) was revealed in the same lane.
5-4 S-1 P-1 P-3 gFBeFB gFBeFB gFB FB
Fig. 2-18. Detection of CI -8S receptor in fat body by ligand assay using 35S-labeled CI-8S.
The membrane fractions of gFB and eFB (see text) were
prepared from animals on day 4 of the 5th in star, on day 1 of spinning and on days 1 and 3 of pupal stage (5-4, S-1, P-1, and P-3,
respectively), extracted and separated by SDS-PAGE (10%). The gels were blotted to PVDF membranes, which were immersed in labeled CI-8 at 4 OC for 2 days and autoradiographed. Arrow indicates the putative CI-8 receptor (tentatively named FB-rec8).
CI-8M, a pupal component closely related to CI-8
Zymogram analysis shown in Figs. 2-4 and 2-19 exhibited additional bands near CI-8. Hemolymph and the extract of gFB from day 2 pupae again gave a similar band as shown in Fig. 2-20A. This one observed below CI-8 was immunologically cross-reactive \Vith anti-CI-8 (Fig. 2-20B). Thus, the
83
new CI band would be a component closely related to CI-8 and was tentatively named CI -8M.
Pursuit of the origin of CI-8M by injection of radio-labeled CI-8S into larvae Labeled CI-8S was injected into anirnals and, after
24
hr, hemolymph and gFB were examined for Cis by autoradiography after native PAGE (Fig.2-21).
When injected on day4
of the 5th instar, labeled CI-8S appeared only in hemolymph but not in gFB. When the injection was done on day2
ofspinning, CI-8S began to be detected also in gFB. Furthermore, a new band appeared at the position of CI-8M. 'fhis band was detected also in hemolymph and gFB of pupae (male and female) injected on day 3. Interestingly, both CI- 8S and CI-8M were virtually absent in gFB of pupae injected on day 5. Thus, after incorporated into the protein granules of gFB, CI-8 would undergo some modification to become CI-8M, which finally disappeared from the fat body.
Trial to detect CI -8M on SDS-PAGE
To characterize further CI-8M, the above extracts of gFB from animals injected with labeled CI-8S on day
2
of spinning or on day 3 after pupation were immunoprecipitated for CI-8S and subjected to SDS-PAGE followed by autoradiography (Fig.2-22).
In addition to the band of CI-8S, two bands undetected in the hemolymph were revealed. One had a higher molecular weight of about 32,000 and the other migrated as fast as the dye marker (about14,000).
Although conclusive evidence is lacking, it is highly plausible that the former represents CI-8M and both tnay be limited degradation products of CI- 8S.SP2
1 2 3
gFB blood PG
4 5
Cl-8
Fig.
2-19.
Analysis of the contents of granules in the pupal gFB.From day
3
male pupae gFB was prepared, and protein granules were isolated from this, extracted and separated by nativePAGE (10%).
Then the gels were subjected to zymogram analysis for CI activity (Lane4)
or to protein staining with Coomassie Brilliant Blue (Lane3).
Lanes 1 and 5, zymogram patterns for gFB extract and hemolymph (day4
of the 5th instar larvae), respectively;Lane
2,
protein staining for hemolymph (ditto).85
(A) (B)
Bl gFB 81 gFB
Fig. 2-20. Detection of an additional CI band in hemolymph and gFB extract from male day 2 pupae.
These specimens were subjected to native PAGE (10%) and the gels were analyzed by zymogram for CI activity (A) or by Western blotting with anti-CI-8 serum (B). Below the position of CI-8 (arrow), a new band (tentatively named CI -8M) was seen.
86
5-4
1 2
1-8 ---..
S-2
1 2
P-3 (m)
1 2
Fig.
2-21.
Origin and fate ofCI-8M.
P-3 (f)
1 2 1
P-5 2
·.
CI-8S
labeled withl35S
]methionine was injected into hemocoel of animals on day 4 of the 5th instar (5-4), on day2
of spinning(S-2),
or on day 3 or 5 after pupation (P-3 and P-5, respectively). After kept at room temperature for 24 hr, hemolymph and gFB were collected.
The hemolymph specimens were subjected to native PAGE (7.5%) and labeled proteins were visualized by autoradiography (Lanes
1).
Also the gFB specimens were extracted and processed similarly (Lanes2).
The marks m and f for P-3 specimens indicate male and female, respectively.
87
81 gFB
5-2
P-3 (f) P-3
(m)Fig. 2-22. Trial to detect CI-8M on SDS-PAGE.
� front
CI-8S labeled with [35S]methionine was injected into hemocoel of animals on day
2
of spinning (S-2) or on day 3 after pupation (P-3). After kept�t
room temperature for24
hr, gFB was collected, extracted and1mmunoprecipitated with the anti-CI-8 serum. The precipitates were dissolved and separated by SDS-PAGE
(12.5%),
and labeled proteins were visualized by autoradiography. The marks m and f for P-3 specimens indicate male and female, respectively. Only males were used for S-2. Bl, hemolymph specimen (on day 2 of spinning) run in parallel as a marker. Arrow indicates the position of CI-8S.Discussion
The insect fat body is derived from the mesoderm and functions not only as a storage organ for lipid, glycogen etc., but also a major synthetic organ for humoral proteins. This chapter confirmed that the fat body was responsible for the synthesis of humoral CI-8 before the spinning stage. In in vitro culture system of the fat body from early 5th in star larvae, label was. found only in the medium, suggesting that the synthesized CI-8 molecules are rapidly secreted into hemolymph.
It is reasonable that the synthetic activity for CI-8 in the fat body per individual increased during the 5th instar, although the activity in female did not seem to increase when compared between days 2 and 4 (see the sex
difference in Fig. 2-2). The incorporation of radioactive precursor does not always mirror the absolute synthetic rate, since the extent of labeling largely depends upon the in vivo aspects of precursor pool. A large fraction of
e
5s]methionine used in this experiment for labeling may be incorporated into SP-1, which is a methionine-rich female-specific protein (Doira, 1968; Tojo et al., 1980; Banno et al., 1993) and actively synthesized in the fat body during the early 5th in star (Tojo et al., 1981 ). Further experiments, wherein the quality and quantity of precursor are varied, will be needed.The effects of hormones on the synthesis of insect humoral proteins have widely been investigated (e.g., Bownes, 1982; Dhadialla and Wyatt, 1983).
Also there are reports on the hormonal modification of CI synthesis in B. mori (Eguchi eta!., 1986b; Matsui and Eguchi, 1991 ). In these experiments,
however, the time span of hormone treatment was too long (2 days) and the results might handle with secondary effects. In the present study, the
inhibitory effects of 20-hydroxyecdysone on CI-8 synthesis were manifested in much shorter time periods of 3 hr in both in vitro and in vivo experiments.
These results are consistent with those in M. sexta larvae (Kanost et al., 1995) where the addition of 20-hydroxyecdysone into hemocoel rapidly reduced the amount of newly synthesized serpin and the expression of the relevant gene.
The synthetic activity of the fat body was decreased suddenly at the onset of spinning. This decrease was more remarkable when the values for labeling were recalculated to a wet weight basis of the tissue (data not shown). From this time on, CI-8 was no more synthesized and the hemolymph concentration of CI-8 decreased.
In contrast to the hemolymph data on the developmental changes in concentration (single peak at the onset of spinning), the fat body titer of CI-8 exhibited two peaks (see Fig. 2-5). The first peak was on days 4 or 5
(depending on sex) of the 5th instar, much earlier than the maximum period for hemolymph, supporting the notion that the fat body synthesizes CI-8 and secretes it into the hemolymph. The second peak was on day 3 of the pupal stage. At this time no synthesis of CI-8 occurs, and the increase must be ascribed to the process under which hemolymph CI-8 was sequestered and accumulated as a storage protein. This problem will be discussed again in what follows.
Larval epidermal cells were stained by immunohistochemistry using anti CI-8 antiserum (see Fig. 2-7). Integument Cis are of interest from the
viewpoint of self-protection from invading microorganisms. However,
epidermal cells also reacted with a normal serum (see Fig. 2-7), obscuring the possibility of actual presence of CI -8. The integument of B. mori was
reported to have inhibitors against proteases from Aspergillus melleus and Beauveria bassiana but not against trypsin and a-chymotrypsin (Yoshida e t al., 1990). Some of inhibitors against the fungal protease exist also in the hemolymph (Yamashita and Eguchi, 1987). One of these inhibitors, FPI-F,
was isolated and determined its amino acid sequence (Eguchi, 1994 ). It consisted of 55 amino acid residues, had a molecular weight of 6,100 and included 8 cysteine residues, indicating that it is a Kunitz type inhibitor.
However, there is no significant similarity between FPI-F and CI-13 or SCI-III (Shinohara, 1993; Sasaki, 1978).
As described above, the insect fat body seems to change in overall function from synthesis to storage at the end of larval stadium (Tojo et al., 1981). The results in the present chapter demonstrated that the fat body tissues which synthesizes CI-8 at the feeding stage has different localization in body cavity from that which incorporate and store CI-8 after the spinning stage.
The author proposes that the fat body will be discriminated into eFB
(peripheral, close to epidermis) synthesizing CI-8 at feeding period and gFB (perivisceral, close to gut) sequestering CI-8 after spinning. In other words, the CI-8 synthesis and sequestration by the fat body could be discriminated by its localization. The two types of fat body tissues would play different roles in addition to those related to CI -8. To the author's knowledge, such
discrimination of fat body in terms of actual physiological function is scarce except for some achievements which will be cited below.
CI-8 detected on the cryostat sections of the larval fat body is likely to be a newly synthesized inhibitor, since its signal was distributed all over the cytoplasms in various types of the fat body. However, at later stages the signal for CI-8 was detected only in the granules (of gFB). The fat body becomes uniform at the mid-pupal stage, and CI -8 was detected again at all locations of fat body, suggesting the possibility that eFB has disappeared until this stage.
The morphological changes of B. mori fat body have been recorded in detail (Waku and Sumimoto, 1968). At larval stages, the cells (adipocytes) of the fat body located around the gut (gFB according to the present terminology) are smaller in individual or bunch size than those located near epidermis
91
(eFB). The gFB cells divide actively during the 4th molt and are still
unmature and small at the early 5th instar. After the onset of spinning, gFB and eFB drastically change their forms and become distinguishable from each other. A lot of protein granules and fat droplets are observed in the gFB cells, while few of these were observable in eFB, suggesting that gFB plays an
important role in uptake and recycle of proteins. At this stage the cells
surrounded by the same basement membrane (branch) seem to be uniform. At the mid-pupal stage, adipocytes generally become uniform similarly to the gFB cells in young pupae. The basement membranes disappear and the cells
separate from each other; this degradation is more notable around the midgut than other regions.
These early observations of fat body support the notion that eFB and gFB have different roles. In this connection, comparison of CI-8 synthesis between eFB and gFB in the present tissue culture is of interest. As described above CI-8 was mainly synthesized in eFB. In the present experiment,
separation of eFB and gFB would be incomplete, and probably gFB does not synthesize CI -8. Furthermore, as shown in Fig. 2-13, gFB seemed not to synthesize other humoral proteins. Thus at the larval stages, eFB may be the synthetic organ of humoral proteins, and gFB might probably under less functional states. Traditionally, the fat body is considered to be a single
multifunctional organ and frequently compared to the mammalian liver and to the adipose tissue (Dean eta/., 1985). Recently, the regional heterogeneity of fat body has been pointed out in the corn earworm, Heliothis zea
(Haunerland et al., 1990), wherein gFB expands rapidly and turns brightly blue after the onset of spinning, because gFB takes into the cytoplasm very high density lipoprotein colored brilliantly blue due to non-covalently bound biliverdin. On the other hand, eFB dose not, remaining white. At the ultimate larval instar of H. zea, the fat body may become heterogeneous and separates
92
into two structurally and functionally different tissues, which are easily
distinguished by the appearance and/or location. It is inferred that eFB is the major fat body at all larval instars and disappears during the pupal stadium.
On the other hand, gFB may increase in the last larval instar and incorporate hemolymph proteins including CI-8 and SPs to make protein granules.
Although some ultrastructural changes occur in gFB prior to adult ecdysis, only this tissue persists through the entire pupal stadium and is the precursor of adult fat body (Haunerland et al., 1990; Wang and Haunerland, 1992).
The present study had an advantage in that it utilized a single molecular marker, CI-8. Further analysis about the incorporation mechanisms including receptor-mediated endocytosis through the gFB membranes will be promising when this molecule is used as a marker, and the author tried to detect a CI-8 receptor in the latter half of this chapter.
To utilize in the receptor detection and other experiments a recombinant CI-8 preparation was devised as a convenient tool. The produced CI-8, named CI-8S, ha� no notable characteristic differences as far as tested when compared to natural CI-8 except that CI-8S migrated slightly faster on native PAGE than CI-8 probably due to differences of some posttranscriptional modifications, e.g., glycosylation (cf. Inoue and Suzuki, 1993). Thus, it was possible to use CI-8S in place of CI-8. Also it was easy to prepare a radio-labeled CI-8S;
otherwise it would need a very tedious work.
The ligand assay for the receptor in the membrane fraction of gFB revealed a protein band of about 70,000 interacting with CI-8S. This protein was detected on day 2 of spinning and early pupal stages but not at the feeding stage and at the onset of spinning. Receptor proteins for SPs were detected in other insects than B. mori (Ueno et al., 1983; Tsuchida and Wells, 1990;
Burmester and Scheller, 1992; Wang and Haunerland, 1993,1994). The molecular weight of these proteins were about 120,000 except for the
93
molecules of the blue blowfly, Calliphora vic ina, about 65,000, 96,000 and 130,000. Although there was a precursor of receptor for Sarcophaga
peregrina SP in the fat body from last instar larvae (Ueno and Natori 1984), the precursor of CI-8 receptor was not detected in the present experiment.
CI-8 accumulates in the granules like SPs. It was not clear whether CI-8 and SPs were in the same granules or not. During the study about the fate of CI-8 after incorporated into the fat body, a new CI band under CI-8 (named CI-8M) was detected, which was immunologically cross-reactive with CI-8 and may be its derivative modified in the granules of gFB. When radio-labeled Cl -8S injected into the hemocoel, again a new labeled band located under CI -8 was detected in the extract of gFB (but neither in the hemolymph nor in eFB), supporting the idea that CI-8 undergoes modification to give CI-8M. The relative intensity of the CI-8M band became strong as the development
proceeded. CI-8M and CI-8 no longer remained in gFB in day 5 pupae. This was supported by the results of fluctuation pattern of fat body CI-8 assayed by ELISA (see Fig. 2-5). Upon SDS-PAGE a band that may be ascribable to CI- 8M was detected at the position of 32,000, suggesting that CI-8 is cut to reduce the size by 10,000. The incorporation experiment i1nplied that this
degradation occurs within 24 hr after sequestration.
From the fat body at the early 5th instar, a eDNA for CI-8 has been prepared (Tomoi et al., 1995). Also from the larval fat body was obtained a eDNA for the serpin type inhibitor sw-Achy (Narumi et al., 1993), which, probably a counterpart of CI-8, has a reactive site near the C-terminal (Sasaki,
1985). The eDNA sequence of CI-8 was very similar to that of sw-Achy, and thus the reaction site of CI-8 may also be near the C-terminal. If CI-8 was cut at a position close to the C-terminal, the resultant fragment CI-8M would not have CI activity. At present the author supposes that CI-8 is cut at a position about 10,000 down-stream from the N-terminal in gFB.
94
In summary, the results in this chapter indicated that CI-8 was synthesized in eFB and secreted into the hemolymph. After the onset of spinning, CI-8 was incorporated into gFB by receptor-mediated endocytosis, and then CI-8 underwent limited-degradation in the granules of gFB.
95
