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Properties synthesis and accumulation of storage proteins in Pieris rapae L.

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Archives of Insect Biochemistry and Physiology 10:215-228 (1 989)
Properties, Synthesis, and Accumulation of
Storage Proteins in Pieris rapae 1.
Hak Ryul Kim, Sook J. Seo, and Richard T. Mayer
Department of Biology, Korea University, Seoul, Korea (H.R.K., S.J.S.); Horticultural Research
Laboratoy,Agricultural Research Service, U.S. Department of Agriculture, Orlando, F L
(R.T.M.)
Two kinds of storage proteins (SP-I, SP-2) were confirmed in hemolymph and
fat body of Pieris rapae during metamorphosis. Both proteins were present in
high concentrations in the hemolymph during the last larval instar. Hemolymph
concentrations of SP-1 and SP-2 dropped after pupation as the proteins were
being deposited i n fat bodies. SP-2 is present in a larger amount than SP-1.
Detailed studies on storage proteins determined their properties, mode of
synthesis, and accumulation in the fat body.
SP-1 has a molecular weight of 500,000 and consists of one type of subunit
(M, 77,000), while SP-2 has a molecular weight of 460,000 and i s composed
of two types of subunits (M, 80,000 and 69,000). The pi values of SP-I and
SP-2 were determined to be 6.97 and 7.06, respectively.
Fat body cells from I-day-old fifth instar larvae synthesized storage proteins
in large amounts, whereas those from late prepupae exhibited high protein
sequestration. Proteins taken u p in fat body accumulated in dense granules
during the pupal stage but sharply decreased at the adult stage.
Morphological changes in the fat body tissues were observed during the
larval-pupal transformation; the nuclei of fat body cells became irregularly
shaped, and the boundaries between cells seemed to be obscure. Synthesis,
storage, or degradation of storage proteins in fat body during development
is closely associated with morphological changes in the tissues.
Key words: hemolymph, fat body, storage granules
Acknowledgments: This work was supported by Basic Science Research Support Fund from the
Ministry of Education, Republic of Korea.
Received September 15,1988; accepted December 19,1988.
Address reprint requests to Dr. R. T. Mayer, U.S. Horticultural Research Laboratory, 2120 Camden Road, Orlando, FL 32803.
Sook J. Seo is now at Department of Biology, Cyeongsang National University, Jinju 660-701,
Korea.
Use of a company or product name by the U.S. Department of agriculture does not imply
approval or recommendation of the product to the exclusion of others that may also be suitable. Some of these data were presented at a symposium of the 10th International Congress of
the Korean Society of Science and Technology.
0 1989 Alan R. Liss, Inc.
216
Kim et al.
INTRODUCTION
The major proteins of larval hemolymph in holometabolous insects have been
intensively studied since calliphorin was described in Calliphora erythrocephala
[l].One of the more dramatic examples of developmental regulation is provided by an abundant set of hemolymph proteins collectively called storage
proteins [2]. Storage proteins are synthesized by larval fat body cells and
secreted into hemolymph where they reach high concentrations during the
last instar [3]. Between the end of the feeding stage and pupation they are
sequestered selectively by the fat body cells and accumulate in dense granules [4,5]. Proteins with these properties have been described in many dipteran and lepidopteran insects [6]; however, little information is available on
storage proteins of Pieris r a p e (L.). We report here on the characterization,
synthesis, and accumulation in fat body of storage proteins in the cabbage
white butterfly, P. r a p e L.
MATERIALS AND METHODS
Insects
Cabbage worms, P. r a p e L., were from a colony maintained within the Department of Biology, Korea University, and were reared on kale plants at 27 2
1°Cand 80 & 5%relative humidity with a photoperiod of 16hlight and 8 h dark.
Preparation of Protein Extracts for Electrophoresis
Hemolymph was collected into a small, chilled test tube after puncturing
larvae and pupae in the abdominal region with a fine needle. A few crystals
of phenylthiourea were added to the hemolymph to prevent melanization.
Collected hemolymph was centrifuged at 5,000s for 20 min at 4°C to remove
cellular debris and stored at - 70°C until used.
Fat body was dissected from larvae and pupae in cold Ringer's solution (128
mM NaC1, 1.8 mM CaC12, 1.3 mM KCl), blotted on weighing paper, and the
wet weights were recorded. Fat bodies (100 mg) were homogenized in Ringer's solution (0.5 ml) and centrifuged at 10,OOOg for 20 min at 4"C, and the
supernatant was stored at - 70°C until used.
Electrophoresis and Purification
Slab gel electrophoresis (160 mm x 170 mm x 2 mm) was conducted on a
2.5% stacking gel and 5%polyacrylamide slab gel at 30 mA for 4 h at 4°C according to Davis [7]. Disc gel electrophoresis was carried out on a 5% polyacrylamide separation gel and 2.5% stacking gel (tube size, 130 mm x 5.5 mm ID)
at 2 mA per tube for 3 h at 4°C. Ten microliters of hemolymph and 15 pl of
supernatant from the fat body homogenate (100 mgl0.5 ml Ringer's solution)
were each mixed with an equal volume of 0.1 M Tris-HC1buffer, pH 6.8, containing 20% sucrose and 0.005% bromphenol blue. After electrophoresis, protein bands were stained in 0.1% Coomassie brilliant blue R-250. Slab SDS-PAGE*
(160 mm x 170 mm x 2 mm) was performed at room temperature using a 3%
*Abbreviationsused: DAB = 3,3-diaminobenzidine; PAGE = polyacrylamidegel electrophoresis;
PBS = phosphate buffered saline; (140 mM NaCI, 2.7 mM KCI, 1.5 rnM KH2P04, 8.1 mM Na2HP04,
pH 7.2; SDS = sodium dodecyl sulfate; SP-1 = storage protein-I; SP-2 = storage protein-2.
Pieris rapae Storage Proteins
21 7
stacking gel and 10% separation gel containing 0.1% SDS [8]. Purified SP-1
(10 pl) and SP-2 (10 p1) were mixed with 20 pl of sample buffer (4% SDS, 10%
2-mercaptoethanol, 0.006% bromphenol blue, 20% sucrose, 50 mM Tris-HC1,
pH 8.3) in a microcentrifuge tube, heated for 2 min at lOO"C, and then applied
to the gel.
For the purification of storageprotein large slabgels (170mm x 180mm x 3 mm)
were used. Electrophoresis was conducted using Tris-glycine buffer without
SDS or 2-mercaptoethanol.After electrophoresis, the storage protein band was
excised with a razor blade, eluted, dialysed against 50 mM Tris-glycine buffer
(pH 8.3), and concentrated by freeze drying. Protein content was determined
by the method of Lowry et al. [9].
Determination of Molecular Weight
The molecular weight of native storage protein was determined as described
by Hedrick and Smith [lo]. Marker proteins (Sigma Chemical Co., St. Louis)
were carbonic anhydrase (M, 29,000), chicken egg albumin (M, 45,000),
bovine serum albumin (monomer, M, 66,000; dimer, M, 132,000), and urease (dimer, M, 240,000; tetramer, M, 480,000).
Molecular weights of storage protein subunits were measured by the method
of Laemmli [8] using a 10% SDS slab gel. Marker proteins (Sigma)were trypsin inhibitor (M, 20,100), carbonic anhydrase (M, 29,000), ovalbumin (M,
45,000), bovine serum albumin (M, 66,000), phosphorylase B (M, 97,400),
P-galactosidase (M, 116,000), and myosin (M, 205,000).
Isoelectric Focusing
Isoelectric focusing was conducted on 5% polyacrylamide gels using 1%
ampholytes (pH 3-10) as described by Wrigley [ll]. After electrophoresis, the
gel was stained with a solution containing 27% isopropanol, 10% acetic acid,
0.05% Coomassiebrilliant blue, and 0.5% CuS04for 1h. Destaining was accomplished using a solution of 12%isopropanol, 7% acetic acid, and 0.5% CuS04.
Preparation of SP-2 Antiserum
Purified SP-2 (200 pg) in 0.5 ml saline solution was mixed with an equal volume of Freund's complete adjuvant and injected subcutaneously into a rabbit.
Three injections at 2-week intervals were made, and a booster injection was
given 1week after the final injection. Blood was collected 1week after the booster injection, allowed to clot at 4°C overnight, and then centrifuged at 10,OOOg
for 10 min. The supernatant was removed and stored at - 70°C until used.
The specificity of antisera was determined by Ouchterlony [12] doublediffusion tests using 1%agarose (Sigma) prepared in 0.15 M potassium phosphate, pH 7.4, that contained 0.1% sodium azide.
Incubation of Fat Body
Fat body tissues (100 mg) were washed in Ringer's solution two or three
times and incubated in 100 p1 of Grace's insect medium containing 20 pCi of
L-[3,4,5-3H(N)]-leucinefor 5 h in a shaking incubator at 28°C. Following the
incubation, fat body tissues were homogenized in Ringer's solution (0.3 ml)
and centrifuged at 10,OOOg for 20 min at 4"C, and the supernatant was subsequently electrophoresed, stained, destained, and fluorographed.
218
Kimet al.
In order to examine sequestration of storage protein into fat body tissues,
fat body from 1-day-old fifth instar larvae was cultured, homogenized, and
centrifuged as described above. Free amino acids in the supernatant (100 p1)
were removed using a centrifuged desalting column [131. Sephadex G-50 was
packed and equilibrated in 50 mm phosphate buffer (pH 7.5) in a 1-ml disposable syringe. After centrifugation at 1,600g for 4 min in a bench centrifuge,
the effluent from the syringe was collected and used as the labeled protein
source. The radioactivity recovered in the protein fractions was determined
by direct scintillation counting of aqueous samples dispersed in 5.0 ml Aquascint (ICN, Irvine, CA) with a 40% counting efficiency. Fat bodies (50 mg) of
various ages (3-day-old fifth instar larvae to 3-day-old pupae) were incubated
in Grace's medium containing 3H-labeledproteins (1,000 dpd100 p1 medium)
for 5 h in a shaking incubator at 28°C. Following the incubation, the fat bodies
were washed in Ringer's solution three times and cut into 5-km-thick sections
using a cryostat at - 40°C for autoradiography.
Tissue Preparation for Light Microscopy
The fat bodies were fixed in glutaraldehyde (5%with 50 mMcacodylate buffer,
pH 7.4) at - 4°C for 18 h and washed. Five-micron-thick serial sections were
cut using a cryostat at - 40°C and stored at - 20°C until used. Fat body sections
were immunohistochemically stained by the method of Hsu et al. [14]. Tissue
sections were hydrated in distilled water and washed with PBS. After washing, the sections were treated with methanolic hydrogen peroxide followed by
5% normal rabbit serum to block the endogenous peroxidase activity and to
reduce the nonspecific background staining. The primary antibody (anti SP-2,
100 11.) was applied over the section and allowed to incubate at 4°C for 24 h in a
water-saturated atmosphere. Afterward, sections were washed in three changes
of PBS (15 min each) and incubated for 1h at room temperature with the secondary antibody (biotinylated anti-rabbit IgG, Vectastain, Vector Laboratories,
Burlingame, C A PK4001). Subsequently, tissue sections were rinsed for 15min
in three changes of PBS and then incubated with a 1:lOO dilution of the avidin
biotin peroxidase complex (Vectastain, PK4001) for 1 h. Sections were then
placed in 200 ml of 0.075% fresh, filtered solution of DAB tetrahydrochloride
in 50 mM Tris buffer, pH 7.6. The DAB treatment was accompanied by constant stirring. The sections were suspended in the DAB solution for 5 min, after
which 0.003% hydrogen peroxide was added, and incubation was continued
for an additional 15 min. Coverslips were placed on all slides in preparation
for light microscopy. Antigen-antibody binding sites were visualized as the
brown product of the reaction with DAB.
Autoradiographic Procedure
Dry-mount autoradiography was combined with immunohistochemistry,
thereby allowing both storage protein accumulation and the storage protein
uptake site to be confirmed in the same preparation. After the immunohistochemical staining procedure, the tissue sections were dehydrated in an ethanol
series, air dried, and coated with Kodak nuclear emulsion NTB 2. After exposure
for 30 days in light-proof desiccator slide boxes at 4"C, the autoradiograms
were transferred to freshly prepared Kodak D-19 developer and developed at
Pieris rapae Storage Proteins
21 g
15°C for 4 min. After briefly rinsing the slides in tap water (15"C), the autoradiograms were incubated for 5 minin Kodak fixer at 15°C. Rinsing in tap water at
18°Cwas followed by a 5 min wash in 10 mM PBS, pH 7.5, at room temperature.
RESULTS
Protein Profiles in Hemolymph and Fat Body During Development
Aliquots of fat body extracts and hemolymph were electrophoresed to determine quantitative changes of storage proteins during the metamorphosis of
the last larval instar to the pupal stage. Two storage proteins occur in hemolymph. Figure 1 shows the electrophoretic pattern of hemolymph proteins;
the upper band on the gel (Fig. 1A) was designated SP-1, and the lower band
was designated SP-2. Storage proteins in the hemolymph are present in high
concentrationsuntil the early prepupal stage, at which time they decrease gradually and completely disappear at 5 days after pupation. Conversely, the same
storage proteins in fat body were present in trace amounts until the early
prepupal stages, after which they accumulated in large amounts (Fig. 1B).
Purification of Storage Proteins
Hemolymph from 3-day-old fifth instar larvae was subjected to nondenaturing
PAGE, the storage protein bands were excised with sharp razor blades, and
proteins were extracted and concentrated (SP1,20mg/ml; SP2,15 mg/ml).When
the purified proteins were analyzed by disc gel electrophoresis, the same mobility of storage proteins was observed in the hemolymph and fat body (Fig. 2).
Hemolymph and fat body of various ages were used to confirm the pure antibody for SP-2 antigen. This analysis was conducted in 1%agarose gel using
the Ouchterlony double-diffusion test (Fig. 3). SP-2 antibody yielded single
precipitation line with purified SP-2,3-day-oId fifth instar larval hemolymph,
prepupal hemolymph, and 5-day-old pupal fat body but no line with 5-day-old
pupal hemolymph, indicating pure antibody for only SP-2.
Physiochemical Characteristics of SP-1 and SP-2
SP-1 was estimated to have a M, of 500,000 and composed of one subunit
with a M, of 77,000 (Fig. 4). The M, of SP-2 was found to be 460,000 and
composed of two kinds of subunits with M,s of 80,000 and 69,000, respectively (Fig. 4).
Isoelectric focusing of the storage proteins was conducted on 5% polyacrylamide gel including ampholytes. SP-1 and SP-2 were determined to have PI
values of 6.97 and 7.06, respectively.
Biosynthesis of Storage Proteins
Fat bodies from fourth and newly ecdysed fifth instar larvae were incubated
and fluorographed as described in Materials and Methods; a negative reaction was observed (data not shown). Fat body was incubated at 1-day intervals from 1-day-old fifth instar larvae to 2-day-old pupae. High incorporation
of radioactivity into storage proteins occurred in fat body from 1-day-old fifth
instar larvae, with low incorporation in 2- and 3-day-old fifth instar larvae. No
incorporation was found during the pupal stage (Fig. 5).
220
Kimetal.
5L1 5L3 PPE PPL PO PI p3 p5
A
SPl
SP2
+
B
I
+
Fig. 1. Electrophoretic patterns of hemolymph and fat body in f? rapae during the metamorphosis. A Hemolyrnph (10 PI).B: Fat body (15 PI). 5L1, I-day-old fifth instar larvae; 5L3,3-day-old
fifth instar larvae; PPE, early prepupae; PPL, late prepupae; PO, newly ecdysed pupae; PI, 1day-old pupae; P3'3-day-oId pupae; P5,s-day-old pupae.
Morphological Changes of Fat Bodies During Development
Fat body tissue from different developmental stages was examined with a
light microscope to determine the relationship between the morphological
changes and physiological function. Nuclear and morphological changes in
Pieris rapae Storage Proteins
HL
FB
SP1
sP2
221
SPl+SP2
+
Fig. 2. PAGE of hernolymph, fat body extract, and purified storage proteins. HL, male prepupal hemolyrnph (10 PI); FB, male prepupal fat body (15pl), SPI, 30 pl; SP2,30 pl; SP1 + SP2,20
pl
+ 10pl.
Fig. 3. Ouchterlony double-diffusion analysis of the storage protein. The center well contained serum against storage protein-2 (20 PI). Wells 1 and 4 contained the purified storage protein-2 (20 PI). Wells 2 and 3 contained soluble proteins from 5-day-old pupal fat body (20 pI) and
hernolyrnph (15 PI), respectively. Well 5 contained prepupal hernolyrnph (15 PI). Well 6 contained 3-day-old fifth instar larval hemolyrnph (15 pl).
fat body tissues were observed after staining in hematoxylin only. Sections of
fat body from 1-day-old fifth instar larvae showed normal cell morphology with
distinct nuclei (Fig. 6: 5L1). Two to 3 days later, the nuclei became irregularly
shaped, and small basophilic granules appeared around them (Fig. 6: 5L3). In
pupae, the boundaries between the cytoplasm and the nucleus seemed to be
222
Kimetal.
SP1 SP2
205 K
116K
97.4K
80 K
66K
69 K
77K
45K
29 K
20.1 K
Fig. 4. Determination of molecular weight of storage protein subunits by electrophoresis in
10% polyacrylamide gel containingO.l% SDS by the method of Laemmli [7].Purified SP1 (10 1.1)
and SP2 (10 1.1.)
Protein markers used were myosin (205,000), p-galactosidase (116,000), phosphorylase (97,400), bovine serum albumin (66,000), ovalbumin (45,000), carbonic anhydrase
(29,000), and trypsin inhibitor (20,100).
obscured, possibly by granules, and the cytoplasm increasingly filled with storage granules (Fig. 6: PO). At the end of the pupal instar, the fat body was
filled with many more granules (Fig. 6: P5).
Analysis of SP-2 Granules
Granules containing storage protein were analyzed in sections of fat body
by immunohistochemical staining with SP-2 antiserum. Granules containing
SP-2 were not detected in sections of fat body from 2-day-old fifth instar larvae
(Fig. 7 5L2). Storage protein granules appeared soon after the prepupal stage
(Fig. 7 PPI) and gradually increased in number toward the end of pupal life
(Fig. 7: PO, P5). The number and size of the storage protein granules reached
a maximum in 5-day-old pupae (Fig. 7: P5) but drastically decreased in the
adult stage (Fig. 7: A3).
SP-2 Uptake by Fat Body
Incubation of fat body obtained from larvae and pupae of various ages with
3H- proteins was performed for 5 h. The highest uptake was attained during
the late prepupal stage (Fig. 8). As shown in Figure 8, silver deposits (indicated as black specks) were dispersed throughout the fat body tissue. Some
grains were overlapped by darker shading (arrows)which indicated the presence
of the SP-2 antibody; other silver deposits had no shading (triangles) which
Pieris rapae storage Proteins
5L1
5L2
5L3
PPI
PO
223
p2
Fig. 5. Incorporation of [3H]-leucine into proteins by P rapae fat body at different stages. The
fat bodies obtained from larvae and pupae of several ages were incubated in the presence of 20
pCi of [3H]-leucine for 5 h at 28°C and submitted to electrophoresis and fluorography as described in “Materials and Methods.” Stages as in Figure 1.
indicated the absence of SP-2. Protein uptake by the fat body appears to be
nonspecific as judged by these experiments.
DISCUSSION
The synthesis of storage proteins used in the formation of adult tissues occurs
during the larval stages of the insect [1,3,6]. This is a distinct event in holometabolic insects, which do not feed during the pupal instar [l]. Storage proteins are synthesized by fat body cells, released into the hemolymph during
the last larval instar, and taken up by the fat body of prepupae and pupae
[15-191. In the present work, two storage proteins (SP-l,SP-2) have been identified in the hemolymph of last instar larvae of P. rupue. The concentrations of
SP-1 and SP-2 in the hemolymph and the fat body of P. rupue change drastically during metamorphosis; these data coincide with the report of Tojo et al.
[4] on the hemolymph storage protein in H. cecropia.
The molecular weights of storage proteins have been reported to range from
235,000 for Galleria melonella [20] to 720,000 for Calpodes ethlius [21]. Storage
proteins were usually hexamers or trimers [2,18,22,23]. Most storage proteins
have one or two types of subunits [4,24-261 with molecular weights from 72,000
for Rhynchosciuru umericunu [26] to 92,000 for Munducu sextu [24]. Storage proteins of I? rapae appear to follow this pattern. Molecular weights of SP-1 and
SP-2 of P. rupue were determined to be 500,000 and 460,000, respectively. SP-1
consists of one type of subunit with molecular weight of 77,000, whereas SP-2
224
Kimetal.
Fig. 6. Light photomicrographs of fat body from /? rapae at different stages. 5L1, I-day-old
fifth instar larvae; 5L3, 3-day-old fifth instar larvae; PO, newly ecdysed pupae; P5, 5-day-old
pupae; 5LI-P0, nuclei (arrows); P5, storage granules (arrows). Hernatoxylin staining. X 400.
consists of two types of subunits with molecular weights of 80,000 and 69,000.
This suggests that the storage proteins of P. r a p e are hexamers.
The storage proteins of P. r a p e have PIS that are almost neutral. The pH of
P. r a p e hemolymph is 6.9, which is a little higher than those reported for other
insect species [27-291. In general, insect storage proteins are acidic and dissociate into subunits at more alkaline pHs [6]. Munn et al. [22] indicated that
the pH of the hemolymph of C. erythrocephulu is about 6 during the larval instar
but increases to ca. 7 during the pupal instar. Calliphorin, the storage protein
of C. erythrocephulu, is used during the pupal instar for the synthesis of adult
tissues, suggesting some physiological significance for the dissociation of
calliphorin at about pH 6.5. However, other storage proteins, e.g., protein-6
of R. urnericunu [26] and manducin of M.sextu [24], do not dissociate at pHs as
high as 9.0, indicating the diversity of storage proteins in the various species
of insects.
Biosynthesis of storage proteins by fat body has been reported previously
[2,21,25,26]. Bianchi et al. [30]reported that fat body from Muscu domesticu
synthesized storage protein during the last larval instar (feeding stage), ceased
the synthesis at the prepupal stage, and accumulated storage proteins after
the prepupal stages. The synthesis of P. r a p e storage proteins by the fat body
was also confirmed in last instar larvae.
Pieris rapae Storage Proteins
225
Fig. 7. lrnrnunohistochernical staining of F! r a p e fat body at different stages with SP-2 antiserum. Cont, control not treated with SP-2 antiserum, 5-day-old pupae; 5L2, 2-day-old fifth
instar larvae; PPI, late prepupae; PO, newly ecdysed pupae; P5, 5-day-old pupae; A3, 3-dayold adult; arrows, storage protein granules. x 400.
Tojo et al. [4]reported that during the larval-pupal transformation rough
endoplasmic reticula and mitochondria decreased in number in fat body cells,
while the cytoplasm increasingly filled with lipid and protein granules. A comparable morphological difference was observed in the fat body cells after hematoxylin staining during the metamorphosis of I? rape. At the beginning of the
pupal instar, nuclear shape change and storage granule accumulation suggest
226
Kirnetal.
Fig. 8. lmmunohistochemically stained autoradiograms of late prepupal fat body indicating
uptake of 13H] SP-2 (arrows) by the fat body. Other [3H-]proteins were also taken up (triangles).
A: x 400; B: X 1,000.
that fat body cell function might be switched from a biosynthetic role in larvae to a storage role after pupation. As shown in Figure 7, storage granules
seem to contain many different proteins or others in addition to storage
protein-2. Some authors reported that storage granules contain protein, lipid,
and urate [4,31,32].
It has been suggested that the fat body cells in Calliphoru larvae synthesize
and export proteins into the hemolymph and also take up protein from the
hemolymph 1331. We have used an autoradiographic-immunohistochemical
experiment to confirm the uptake of storage proteins into the fat body in P.
rupue. Fat body cells from late prepupae of P. r a p e had the highest storage
Pieris rapae Storage Proteins
227
protein uptake. Our observations, together with those of others on Lepidoptera, indicate that the synthesis and deposition in the fat body of a class of
storage protein is a general process in insect metamorphosis.
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